Preparation of cyclohexene isotopologues and stereoisotopomers from benzene.

The hydrogen isotopes deuterium (D) and tritium (T) have become essential tools in chemistry, biology and medicine1. Beyond their widespread use in spectroscopy, mass spectrometry and mechanistic and pharmacokinetic studies, there has been considerable interest in incorporating deuterium into drug molecules1. Deutetrabenazine, a deuterated drug that is promising for the treatment of Huntington's disease2, was recently approved by the United States' Food and Drug Administration. The deuterium kinetic isotope effect, which compares the rate of a chemical reaction for a compound with that for its deuterated counterpart, can be substantial1,3,4. The strategic replacement of hydrogen with deuterium can affect both the rate of metabolism and the distribution of metabolites for a compound5, improving the efficacy and safety of a drug. The pharmacokinetics of a deuterated compound depends on the location(s) of deuterium. Although methods are available for deuterium incorporation at both early and late stages of the synthesis of a drug6,7, these processes are often unselective and the stereoisotopic purity can be difficult to measure7,8. Here we describe the preparation of stereoselectively deuterated building blocks for pharmaceutical research. As a proof of concept, we demonstrate a four-step conversion of benzene to cyclohexene with varying degrees of deuterium incorporation, via binding to a tungsten complex. Using different combinations of deuterated and proteated acid and hydride reagents, the deuterated positions on the cyclohexene ring can be controlled precisely. In total, 52 unique stereoisotopomers of cyclohexene are available, in the form of ten different isotopologues. This concept can be extended to prepare discrete stereoisotopomers of functionalized cyclohexenes. Such systematic methods for the preparation of pharmacologically active compounds as discrete stereoisotopomers could improve the pharmacological and toxicological properties of drugs and provide mechanistic information related to their distribution and metabolism in the body.

Synthesis of Tetrabenazine and Its Derivatives, Pursuing Efficiency and Selectivity.

Tetrabenazine is a US Food and Drug Administration (FDA)-approved drug that exhibits a dopamine depleting effect and is used for the treatment of chorea in Huntington's disease. Mechanistically, tetrabenazine binds and inhibits vesicular monoamine transporter type 2, which is responsible for importing neurotransmitters from the cytosol to the vesicles in neuronal cells. This transportation contributes to the release of neurotransmitters inside the cell to the synaptic cleft, resulting in dopaminergic signal transmission. The highly potent inhibitory activity of tetrabenazine has led to its advanced applications and in-depth investigation of prodrug design and metabolite drug discovery. In addition, the synthesis of enantiomerically pure tetrabenazine has been pursued. After a series of research studies, tetrabenazine derivatives such as valbenazine and deutetrabenazine have been approved by the US FDA. In addition, radioisotopically labeled tetrabenazine permits the early diagnosis of Parkinson's disease, which is difficult to treat during the later stages of this disease. These applications were made possible by the synthetic efforts aimed toward the efficient and asymmetric synthesis of tetrabenazine. In this review, various syntheses of tetrabenazine and its derivatives have been summarized.

Synthesis of (±)-Tetrabenazine by Visible Light Photoredox Catalysis.

(±)-Tetrabenazine was synthesized in six steps from commercially available compounds. The key cyclization substrate was assembled rapidly via Baylis-Hillman and aza-Michael reactions. Annulation of the final ring was achieved through visible light photocatalysis, wherein carbon-carbon bond formation was driven by the oxidation of a tertiary amine. Solvent played a critical role in the photoredox cyclization outcome, whereas methanol led to a mixed ketal, acetonitrile/water (10:1) gave direct cyclization to (±)-tetrabenazine and occurred more rapidly.

Brain uptake of a non-radioactive pseudo-carrier and its effect on the biodistribution of [(18)F]AV-133 in mouse brain.

INTRODUCTION: 9-[(18)F]Fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133) is a new PET imaging agent targeting vesicular monoamine transporter type II (VMAT2). To shorten the preparation of [(18)F]AV-133 and to make it more widely available, a simple and rapid purification method using solid-phase extraction (SPE) instead of high-pressure liquid chromatography (HPLC) was developed. The SPE method produced doses containing the non-radioactive pseudo-carrier 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). The objectives of this study were to evaluate the brain uptake of AV-149 by UPLC-MS/MS and its effect on the biodistribution of [(18)F]AV-133 in the brains of mice. METHODS: The mice were injected with a bolus including [(18)F]AV-133 and different doses of AV-149. Brain tissue and blood samples were harvested. The effect of different amounts of AV-149 on [(18)F]AV-133 was evaluated by quantifying the brain distribution of radiolabelled tracer [(18)F]AV-133. The concentrations of AV-149 in the brain and plasma were analyzed using a UPLC-MS/MS method. RESULTS: The concentrations of AV-149 in the brain and plasma exhibited a good linear relationship with the doses. The receptor occupancy curve was fit, and the calculated ED50 value was 8.165mg/kg. The brain biodistribution and regional selectivity of [(18)F]AV-133 had no obvious differences at AV-149 doses lower than 0.1mg/kg. With increasing doses of AV-149, the brain biodistribution of [(18)F]AV-133 changed significantly. CONCLUSION: The results are important to further support that the improved radiolabelling procedure of [(18)F]AV-133 using an SPE method may be suitable for routine clinical application.

Synthesis and biological evaluation of 10-(11) C-dihydrotetrabenazine as a vesicular monoamine transporter 2 radioligand.

In this study, we synthesized a new carbon-11-labeled radiotracer, 10-(11) C-dihydrotetrabenazine (10-(11) C-DTBZ), and evaluated its potential as a vesicular monoamine transporter 2 (VMAT2) radioligand. The radiolabeled precursor 10-O-desmethyl-dihydrotetrabenazine (10-O-desmethyl-DTBZ) was prepared with a six-step reaction using 3-methoxy-4-benzyloxybenzaldehyde as starting material. 10-(11) C-DTBZ was synthesized by heating 1.0 mg of 10-hydroxy precursor and (11) C-methyl iodide in the presence of 0.3 mL of dimethyl sulfoxide and 4.0 µL of 3 N KOH at room temperature for 3 min. After purification by solid phase extraction using an alumina Sep-Pak cartridge, the final 10-(11) C-DTBZ product was obtained with a radiochemical purity of >99% and an uncorrected radiochemical yield of 18-26% (end of bombardment (EOB), n = 6). The overall synthesis time was approximately 20 min from the EOB to release of the product for quality control. Using small-animal positron emission tomography (microPET), the striatum of normal rats was found to exhibit symmetrical labeling (STR /STL = 0.98 ± 0.05, n = 3) and the highest uptake of radioactivity (striatum/cerebellum, ST/CB = 2.89 ± 0.31 at 30-60 min, n = 3). In contrast, rats with 6-hydroxydopamine unilateral lesions yielded asymmetrical striatal images with a higher 10-(11) C-DTBZ concentration on the unlesioned side (STunlesioned /CB = 2.53 ± 0.18, at 30-60 min, n = 3) compared with the lesioned side (STlesioned /CB = 1.26 ± 0.10, n = 3). These results suggest that 10-(11) C-DTBZ may represent a promising PET radiotracer for imaging VMAT2.

A ring-closing metathesis-based approach to the synthesis of (+)-tetrabenazine.

A modular stereoselective synthesis of the vesicular monoamine transport inhibitors (+)-tetrabenazine ((+)-1) and (+)-α-dihydrotetrabenazine ((+)-2) has been developed. The approach is based on amine 4 and acid 5 as the key building blocks, which were elaborated into macrolactam 3 by amide coupling and a subsequent highly E-selective RCM reaction. Macrolactam 3 could be converted into tetrabenazine in three known steps.

Simple, rapid and reliable preparation of [¹¹C]-(+)-α-DTBZ of high quality for routine applications.

[¹¹C]-(+)-α-DTBZ has been used as a marker of dopaminergic terminal densities in human striatum and expressed in islet beta cells in the pancreas. We aimed to establish a fully automated and simple procedure for the synthesis of [¹¹C]-(+)-α-DTBZ for routine applications. [¹¹C]-(+)-α-DTBZ was synthesized from a 9-hydroxy precursor in acetone and potassium hydroxide with [¹¹C]-methyl triflate and was purified by solid phase extraction using a Vac tC-18 cartridge. Radiochemical yields based on [¹¹C]-methyl triflate (corrected for decay) were 82.3% ± 3.6%, with a specific radioactivity of 60 GBq/µmol. Time elapsed was less than 20 min from end of bombardment to release of the product for quality control.

A concise synthesis of tetrabenazine: an intramolecular aza-Prins-type cyclization via oxidative C-H activation.

A concise synthesis of tetrabenazine and dihydrotetrabenazine is described. The key feature of this synthesis is the intramolecular aza-Prins-type cyclization of an amino allylsilane via oxidative C-H activation.

Synthesis and biological evaluation of 3-alkyl-dihydrotetrabenazine derivatives as vesicular monoamine transporter-2 (VMAT2) ligands.

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [(125)I]-iodovinyl-TBZ or [(18)F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 (K(i)=5.98 nM) and may be a useful lead compound for future structure-activity studies.

Preparation and evaluation of tetrabenazine enantiomers and all eight stereoisomers of dihydrotetrabenazine as VMAT2 inhibitors.

Tetrabenazine (TBZ) ((±)-1) and dihydrotetrabenazines (DHTBZ) are potent inhibitors of VMAT2. Herein, a practical chemical resolution of (±)-1 and stereoselective synthesis of all eight DHTBZ stereoisomers are described. The result of VMAT2 binding assay revealed that (+)-1 (Ki=4.47 nM) was 8000-fold more potent than (-)-1 (Ki=36,400 nM). Among all eight DHTBZ stereoisomers, (2R,3R,11bR)-DHTBZ ((+)-2: Ki=3.96 nM) showed the greatest affinity for VMAT2. The (3R,11bR)-configuration appeared to play a key role for VMAT2 binding. In summary, (+)-1, (+)-2, and their derivatives warrant further studies in order to develop more potent and safer drugs for the treatment of chorea associated with Huntington's disease and other hyperkinetic disorders.

A concise total synthesis of (+)-tetrabenazine and (+)-α-dihydrotetrabenazine.

Highly concise asymmetric total syntheses of (+)-tetrabenazine (1), a drug for the treatment of chorea associated with Huntington's disease, and of (+)-α-dihydrotetrabenazine (2), an active metabolite of 1, have been accomplished. Our synthetic route features a trans-selective enol etherification, followed by an unprecedented cation-dependent aza-Claisen rearrangement to establish the carbon framework and two stereogenic centers of tetrabenazine. The syntheses consist of seven steps (34 % overall yield) for (+)-2 and eight steps (22 % overall yield) for (+)-1.

An improved radiosynthesis of [18F]AV-133: a PET imaging agent for vesicular monoamine transporter 2.

INTRODUCTION: Recently, a PET tracer, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system has been reported. It is currently under Phase II clinical trials to establish its usefulness in the diagnosis of neurodegenerative diseases including dementia with Lewy bodies and Parkinson's disease. The radiolabeling of [(18)F]AV-133, nucleophilic fluorination reaction and potential effects of pseudo-carrier were evaluated by in vivo biodistribution. METHODS: The preparation of [(18)F]AV-133 was evaluated under different conditions, specifically by employing different precursors (-OTs or -Br as the leaving group at the 9-propoxy position), reagents (K222/K(2)CO(3) vs. tributylammonium bicarbonate) and solvents (acetonitrile vs. DMSO), reaction temperature and reaction time. With optimized conditions from these experiments, radiosynthesis and purification with solid-phase extraction (SPE) of [(18)F]AV-133 were performed by an automated nucleophilic [(18)F]fluorination module. In vivo biodistribution in mice on [(18)F]AV-133 purified by either HPLC (no-carrier-added) or the SPE method (containing a pseudo-carrier) was performed and the results compared. RESULTS: Under a mild fluorination condition (heating at 115 degrees C for 5 min in dimethyl sulfoxide), [(18)F]AV-133 was obtained in a high yield using either -OTs or -Br as the leaving group. However, the -OTs precursor gave better radiochemical yields (>70%, thin layer chromatography analysis) compared to those of the -Br precursor. The optimized reaction conditions were successfully implemented to an automated nucleophilic fluorination module. Labeling and purification of [(18)F]AV133 were readily achieved via this automatic module in good radiochemical yield of 21-41% (n=10) in 40 min. The radiochemical purity was larger than 95%. Biodistribution of SPE-purified product (containing a pseudo-carrier) in mice showed a high striatum/cerebellum ratio (4.18+/-0.51), which was comparable to that of HPLC-purified [(18)F]AV-133 (4.51+/-0.10). CONCLUSIONS: The formation of [(18)F]AV-133 was evaluated under different labeling conditions. These improved labeling conditions and SPE purification were successfully implemented into an automated synthesis module. This offers a short preparation time (about 40 min), simplicity in operation and ready applicability for routine clinical operation.

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivatives as probes for imaging vesicular monoamine transporter-2.

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4-6, targeting the vesicular monoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates prepared from rat striatal tissue homogenates with both [(125)I]-iodovinyl-TBZ and [(3)H]DTBZ. There was a good correlation (r(2)=0.925) between the affinities of the different compounds for [(125)I]-iodovinyl-TBZ and [(3)H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter (K(i)=8.68+/-1.26 nM and 7.01+/-0.07 nM, respectively), which may be a good lead compound for further structural modification to develop useful probes for VMAT2.

Asymmetric synthesis of tetrabenazine and dihydrotetrabenazine.

The enantioselective synthesis of (+)-tetrabenazine (TBZ) and (+)-dihydrotetrabenazine (DTBZ), agents of significant interest for therapeutic and molecular imaging applications, has been completed in 21% (TBZ) and 16% (DTBZ) overall yield and in >97% ee from the starting dihydroisoquinoline. The synthesis utilizes Sodeoka's palladium-catalyzed asymmetric malonate addition to set the initial stereocenter followed by a number of diastereoselective transformations to incorporate the remaining asymmetric centers.

[Quick and simple synthesis of (11)C-(+)-alpha-dihydrotetrabenazine to be used as a PET radioligand of vesicular monoamine transporters]. / Síntesis rápida y simplificada de C-(+)-alpha-dihidrotetrabenazina para su utilización como radioligando PET de los transportadores vesiculares de monoaminas.

UNLABELLED: Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,3,4,6,7-hexahydro-11bH-benzo[a]-quinolizine, DTBZ) has become the ideal radioligand for the presynaptic vesicular monoamine transporter VMAT2 based on its high binding affinity and optimal lipophilicity. OBJECTIVE: To develop an automatic procedure for labelling DTBZ with carbon-11, which has been shown to be a highly effective marker for in vivo studies of neuronal losses in animal models with Parkinson's disease using positron emission tomography (PET). MATERIALS AND METHODS: We have developed a new fully automated synthesis procedure to obtain 11C-(+)DTBZ quickly and simply through labelling the precursor -(+)desmethyldihy-drotetrabenazine- at room temperature in the presence of dimethyl sulfoxide (DMSO) and potassium hydroxide (KOH), using 11CH3I as primary precursor. The final purification was carried out by solid phase extraction using commercially available cartridges and the residual solvents (DMSO and ethyl ether) were eliminated by evaporation. RESULTS: The whole procedure was automated, and after 54 syntheses, an average production of 1.94 GBq of sterile, pyrogen-free 11C-(+)DTBZ with a radiochemical purity > 99 % was obtained with 5 minutes irradiation and 6 minutes of synthesis after 11CH3I production. 11C-(+)DTBZ binding to presynaptic dopamine nerve terminals has been demonstrated by MicroPET studies in Wistar rats and M. Fascicularis monkeys. CONCLUSIONS: This new synthesis procedure is quick and simple, due to optimised techniques, which have allowed elimination of residual solvents based on their polarity for the final purification. It is also applicable to other automatic syntheses for obtaining compounds labelled by methylation reactions.

Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels.

Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.

A simple synthesis of [11C]dihydrotetrabenazine (DTBZ).

[11C]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-[11C]methoxy-10 -methoxy-1,2,3,4,6,7,- hexahydro-11bH-bezo[alpha]-quinolizine) ([11C]DTBZ) was synthesized by reacting the 9-hydroxy precursor in DMSO with gas-phase [11C]methyl iodide on a column of alumina impregnated with KOH. The reaction was instantaneous at room temperature. This column was then connected to the inlet of a short column containing basic alumina. Elution with cyclohexane removed radioactive contaminants. The radioactive product was then eluted with a few milliliters ether containing 1% ethanol. The [11C]DTBZ was obtained in isolated yields of > 200 mCi and specific activities > 1600 Ci/mmol.

Absolute configuration of (+)-alpha-dihydrotetrabenazine, an active metabolite of tetrabenazine.

Chiral column liquid chromatography and enantiospecific enzymatic hydrolysis were utilized to separate the enantiomers of alpha- and beta-dihydrotetrabenazine and alpha-9-O-desmethyldihydrotetrabenazine, three benzo[a]quinolizines derived from the amine-depleting drug tetrabenazine. An X-ray crystal structure analysis of (-)-alpha-9-O-desmethyldihydrotetrabenazine gave an absolute structure of that compound as the 2S, 3S, 11bS isomer. Therefore, (-)-alpha-dihydrotetrabenazine also has the 2S, 3S, 11bS absolute configuration. (+)-alpha-Dihydrotetrabenazine, the single biologically active isomer from the metabolic reduction of tetrabenazine, thus has the absolute configuration of 2R, 3R, 11bR. For further in vitro and in vivo studies of the vesicular monoamine transporter, it is now possible to use the single enantiomer of radiolabeled alpha-dihydrotetrabenazine.

In vitro and in vivo studies of benzisoquinoline ligands for the brain synaptic vesicle monoamine transporter.

Tetrabenazine is a high-affinity inhibitor of the vesicular monoamine transporter in mammalian brain. As part of a program to develop in vivo imaging agents for these transporters in human brain, a series of 2-alkylated dihydrotetrabenazine ligands was synthesized and evaluated in vitro and in vivo for binding to the brain vesicular monoamine transporter. Additions of organometallic reagents to tetrabenazine produced 2-methyl, 2-ethyl, 2-n-propyl, 2-isopropyl, and 2-isobutyl derivatives of dihydrotetrabenazine. The stereochemistry and conformation of the addition products were thoroughly verified by two-dimensional NMR techniques. All of these alkyl derivatives displayed in vitro affinity for the vesicular monoamine transporter binding site in rat brain using competitive assays with the radioligand [3H]methoxytetrabenazine. Except for the isopropyl derivative, all compounds when tested at 10 mg/kg iv showed an ability to inhibit in vivo accumulation of the radioligand [11C]methoxytetrabenazine in the mouse brain striatum. Derivatives with small alkyl groups (methyl, ethyl) were more effective than those with large groups (propyl, isobutyl). These studies suggest that large groups in the 2-position of the benzisoquinoline structure will significantly diminish both in vitro and in vivo binding of these compounds to the vesicular monoamine transporter.

Amino- and amido-tetrabenazine derivatives: synthesis and evaluation as potential ligands for the vesicular monoamine transporter.

Tetrabenazine (TBZ) and dihydrotetrabenazine are well known inhibitors of the CNS vesicular monoamine transporter (VMAT), which is responsible for the packaging of monoamine neurotransmitters in presynaptic vesicles. Amino and amido derivatives of tetrabenazine were prepared as potential ligands for the vesicular monoamine transporter. Ultimately, organotin derivatives of promising ligands were prepared for radiolabeling with 125I. The compounds were evaluated for their ability to inhibit the specific binding of a selective radioligand to the transporter in rat striatal homogenates. Of the compounds evaluated, three amine derivatives of TBZ (primary, secondary and tertiary) were found to have modest to high affinity for the transporter, while two amides exhibited low to undectable affinity. The secondary propargyl amine was found to possess the highest affinity (Ki = 7.6 nM) and was chosen for further evaluation. The organotin derivative of this compound was synthesized in order to prepare the corresponding radioiodinated ligand. However, our inability to synthesize and characterize the iodinated amine precluded its evaluation as a potential radioiodinated ligand for the transporter. Alternative approaches for decreasing the lipophilicity of TBZ analogs while maintaining high binding affinity are currently being explored.