Synthesis optimization of pittsburgh compound B by the captive ...

Synthesis Optimization of Pittsburgh Compound B by the Captive Solvent Method G. Clemente, V. Alves and A.J. Abrunhosa

A.J. Abrunhosa

ICNAS – Instituto de Ciências Nucleares Aplicadas à Saúde Universidade de Coimbra, Coimbra, Portugal [email protected], [email protected], [email protected] (PI)

IBILI – Instituto Biomédico de Investigação da Luz e Imagem, Faculdade de Medicina Coimbra, Portugal [email protected]

Abstract—Carbon-11 is a positron emitting nuclide that has been used extensively to label compounds destined for molecular targets in the brain. Pittsburgh compound B ([11C]PiB) is a benzothiazole derivative of thioflavin T that is used to image beta-amyloid deposits in Alzheimer’s disease patients with Positron Emission Tomography (PET). In this paper we report on the optimization of a fully automated synthesis, purification and reformulation of [11C]PiB suitable for use in human PET studies. [11C]PiB was prepared from 2-(4’aminophenyl)-6-hydroxybenzothiazole by [11C]-methylation with methyl triflate reacting in an high-performance liquid chromatography (HPLC) loop, purified and reformulated by solid phase extraction. The specific activity of [11C]PiB was 25 ± 10 GBq/µmol, and radiochemical purity was better than 95%. Index Terms—carbon-11; [11C]Pittsburg Compound B; [ C]Methyl Triflate; loop methylation; positron emission tomogralphy; neurological diseases. 11

I.

Carbon-11 is one of the most widely used radioisotopes in PET radiochemistry due to the ubiquity of the element in organic structures that allow it to be incorporated into biomolecules of interest without significant loss of biological activity, in contrast, for example, with the negative effect in the bioactivity of a promising compound after labeling with 18 F. However, parallel reactions with the environmental [12C]CO2 and a half-life period of approximately 20 minutes are some of the reasons that made the synthesis of high specific activity carbon-11 radiotracers a real challenge. The development of automated radiosynthesis modules using the captive solvent method has facilitated the production of radiotracers labeled with [11C] with rapid reactions (typically a maximum of 5 minutes) and low amount of side products that can be quickly purified by HPLC and reformulated for preclinical and clinical use.. Below we describe the optimization of these procedures for the production of [11C]PiB with a specific activit suitable for in vivo imaging of amyloid plaque deposition in Alzheimer's disease [2] and the methodological aspects in its synthesis, purification and reformulation.

INTRODUCTION

Positron Emission Tomography (PET) tracers available today provide valuable information regarding many aspects of neurobiological processes that can help us to understand the underlying causes on many brain diseases. Many of these molecules, labeled with short lived nuclides auch as 11C and 18 F,, can be used to monitor certain neural mechanisms during the course of dementia providing valuable tools for seeking early diagnosis, evaluate the response to novel therapies and aid the development of the next generation of psychoactive drugs. The challenge of brain PET imaging is to develop radiotracers that penetrate the highly selective blood-brain barrier. For this they must have a low molecular weight (less than 600 Dalton), neutral or low ionization at physiological pH, and present low percentage of plasma protein binding. It is also important that they have high specific activity and that they are not metabolized to active compounds that could mask the specific signal of the parent compound [1].

II.

GOALS

In this paper we propose a method for the fully automated synthesis of carbon-11 radiotracers as well as their reformulation to a saline and non-pyrogenic solution suitable for human in vivo studies, starting from cyclotron-produced [11C]CO2. The optimization of the radiosynthesis in the automatic module is a critical procedure as this has a high impact on chemical and radiochemical yields and especially on specific activity. The use of [11C]PiB is now well established in the literature in order to help in the differential diagnosis between Alzheimer’s disease and other types of neurodegeneration and therefore can have an impact in the quality of life of affected patients.

III.

EXPERIMENTAL

A. Materials and Apparatus All chemicals and solvents were obtained from commercials suppliers with Ph. Eur. Grade, when available,

and used without further purification. The reagents, lithium aluminium hydride (0.1M solution in dried tetrahydrofuran) and hydriodic acid (aqueous solution, >55% redistilled) were obtained from ABX (Raderberg, Germany). Silver triflate (better than 99.95% trace metals basis) was obtained from Sigma-Aldrich. The precursor (6-OH-BTA-0), PiB reference standard (6OH-BTA-1) and the O-methyl possible impurity (6-MeOBTA-0) were also obtained from ABX (Germany). The solvents, anhydrous acetonitrile, acetone were obtained from VWR. B. Production of [11C]Methyl Iodide and [11C]Methyl Triflate Production of [11C]Methyl Iodide ([11C]CH3I) and 11 [ C]Methyl Triflate ([11C]MeOTf) was made using a MeI-Plus System Module (Bioscan Inc. Washington DC, USA). Initially, the Carbon-11 was produced in the form of [11C]Carbon dioxide ([11C]CO2) with a Cyclone 18/9 cyclotron (IBA) by the 14 N(α,p)11C nuclear reaction. In our procedure, the [11C]CH3I was obtained by the so-called ‘wet’ method based on the reduction of [11C]CO2 with Lithium Aluminium Hydride (LiAlH4) in tetrahydrofuran (0.1M). After the evaporation of tetrahydrofuran (THF), hydriodic acid (HI) was added to produce [11C]CH3I [4] Methyl iodide is then destilled at a temperature of 115-120ºC in a stream of nitrogen through an ascarite/P2O5 trap before passing through the triflate column to form [11C]MeOTf. To reduce the amount of precursor used in

the loop reaction, the [11C]CH3I was converted to [11C]MeOTf, because this is a more reactive methylation agent (less volatile and thus more easily trapped in small volumes of solvent) at room temperatures [5]. So, the [11C]MeOTf is generated by the reaction of [11C]CH3I with 1g of silver triflate and 2g of graphite powder mixed together in a glass column and heated during the process a high temperatures (170-180ºC). MeOTf is carried under a constant flow (10 ml/min.) of Nitrogen (Figure 1). C. Production of [11C]PiB (Pittsburg Compound B)¨in a captive solvent loop This process is based on the ‘loop method’ developed by Wilson et al.[6]. The automated methylation labeling occurs in a loop system (AutoLoop system, Bioscan Inc. Washington DC, USA). [11C]MeOTf produced as described above was trapped in a 2 ml stainless-steel HPLC loop (AutoLoop) precharged with a solution of 6-OH-BTA-0 (1 mg) in acetone/acetonitrile 1/1 (100µl). After trapping, the [11C]MeOTf is left to react with the precursor for 1 min at room temperature. After, the reaction products are purified by a semipreparative HPLC system consisting of a pump (K-501, Knauer, Germany), a PhenomenexLuna C18(2) 5µ column (250 x 10 mm), a mobile phase (CH3CN/H20 (40/60) + 0.1N Ammonium formate; flow rate = 9ml/min), a UV detector set at 254 nm (K-200, Knauer, Germany) and a radiation detector present on reformulation system (ReForm-Plus system, Bioscan Inc. Washington DC, USA). The desired fraction BIOSCAN. Autoloop System

LiAlH4

HI

N2

BISOCAN. MeI System

Figure 1. Schematic diagram of the automated Carbon-11 labeling system.

BIOSCAN. ReFORMSystem

([11C]PiB was collected with a retention time between 7-8 minutes (radiometric and UV detector). D. Reformulation and filtration of the final product Reformulation was carried out on a ReForm-Plus system (Bioscan Inc. Washington DC, USA), by diluting the collected product with water (10 ml), trapping in a solid phase extraction (SPE) column (Waters, Sep-Pak® Light C18), eluting with 0.6 ml ethanol (ABX, Germany), and then eluting with saline solution (9 ml). These last two steps were performed with filtration through a 0.22 µm filter (Millex-GS, Millipore). Synthesis and reformulation of [11C]PiB took approximately 25 - 30 min. In the end of each cycle, a cleaning procedure is run, which removes all radioactivity and residual chemicals in the modules. This cleaning process consists of passing ethanol, acetone and ether for the MeI system and water, ether and acetone for the Autoloop system. A final drying step with nitrogen was performed in both modules. The system was then prepared for next synthesis. E. Quality Control Radiochemical purity of [11C]PiB was measured with an Agilent 1200 Series HPLC system (254nm UV detector) with a radiometric detector (RaytestGmbh, Straubenhardt, Germany). GC was also used to monitor residual solvents using an Agilent Zorbax Eclipse XDB-C18 analytical column (150x4.6 mm, 5µ) eluted with a mixture of CH3CN/H2O (40/60) + 0.1N ammonium formate at 4 ml/min. IV.

RESULTS

The development process aimed to obtain the highest possible amount radioactivity, in order for it to be used in human studies. All experiments were carried out long irradiation times (~30min.; integrated current = 12.8 µAh). A set of 15 synthesis of [11C]PiB was performed under the experimental conditions described above. At the end of synthesis (EOS), 1.6 ± 0.6 GBq of pure [11C]PiB was obtained with a total synthesis time of 25-30 min. Quality control results revealed a radiochemical purity above 95% (Figure 2). The residual ethanol and acetonitrile in the solution was controlled with a gas chromatographer (Agilent 6850 Series II). pH and radionuclide purity were also tested always showing a range of 5.5-7.5 and a half-life of 20.38±5%, respectively. Average specific activity at EOS was 25 ± 10 GBq/µmol.

PiB

[11C]PiB

Figure 2. Chromatogram of the final [11C]PiB solution ready for injection. Top, ultraviolet (UV), absorbance at 254 nm; and bottom, radioactivity. The retention time of [11C]PiB was 2 min. Radiochemical purity is over 95%.

V.

DISCUSSION

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[ C]Methyl iodide can be produced by two distinct methods: the ´gas phase’ and the so-calleed wet method. In the former, cyclotron-produced [11C]CH4 is converted to [11C]CH3I by free radical iodination vapor at high temperatures in the gas phase [7-9]. Although this method provides higher specific activities, it has a disadvantage of providing lower radiochemical yields [9].On the other side, the ‘wet’ method, used by our group, provides better radiochemical yields and ensures high activity for human PET studies. Although with the procedures described above we ensures a reproducible method for synthesis of [11C]PiB, we continue the optimization of all steps in synthesis and reformulation for a faster production and higher specific activity and radiochemical yields. A. Conversion of [11C]Methyl iodide into [11C]Methyl triflate In all experiments performed, the production of the byproduct [11C]CH3OH was direct associated with residual water present on the lines and columns. Therefore, before the first synthesis on every day, all lines, including the triflate oven, were thoroughly dry with nitrogen flow. B. Trapping and reacting of [11C]Methyl triflate on loop Gómez-Vallejo and Llop[9], describe the influence of precursor solvent in trapping the [11C]MeOTf. In our tests, the use of acetone/acetonitrile (1/1) instead of Methyl Ethyl Ketone (MEK) improved the trapping efficiency of [11C]Methyl triflate in the loop. All [11C]MeOTf was trapped for 2 minutes, and reacted with 6-OH-BTA-0 precursor for 1 min (Figure 3).

VI. A

B

CONCLUSIONS

We reported on the optimization of a fully automated synthesis of [11C]Pittsburg Compound B suitable for human studies totally reproducible with good specific activities and satisfactory radiochemical yields.

C

With this system, and some minor modifications, many other [11C]-labeled compounds can be optimized to be produced in good quality for human studies.

VII. PLANNED DEVELOPMENTS Figure 3. Radiometric detector profile for [ 11C]methyl triflate trapped in the loop during [11C]PiB synthesis using acetone/acetonitrile (1/1) as a solvent. (A) Start trapping; (B) Start React; (C) Transfer to HPLC for purification.

We also found, like Verdurand et al. [10], that the ideal amount of precursor to inject into the loop is 1 mg. Using the acetone/acetonitrile mixture as a solvent, the injection of 1mg dissolved on 80µl of solvent gives a good radiochemical yields and reduces the costs with precursor.

In the future we intend to optimize the synthesis and reformulation of other tracers for the central nervous system including [11C]PK11195, [11C]flumazenil, [11C]raclopride and other new [11C]-labeled molecules to support the pre-clinical and clinical programs at ICNAS.

REFERENCES [1] Lee Y-S., Radiopharmaceuticals for Molecular Imaging. The Open Nuclear Medicine Journal , 2010, vol. 2, pp. 178-85.

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C. HPLC Purification of [ C]PiB In the HPLC purification step, the [11C]PiB peak was successful separated and collected into a reformulation system (ReFORM-Plus, Figure 1).

[2] Tolboom N., Yaqub M., van der Flier WM., Boellaard R., Luurtsema G., Windhorst AD., "Detection of Alzheimer Pathology In Vivo Using Both 11C-PIB and 18F-FDDNP PET", J Nucl Med, 2009, vol. 50, pp. 191197. [3] Kadir A and Nordberg A.,

"Target-Specific PET Probes for

Neurodegenerative Disorders Related to Dementia", J Nucl Med, 2010; vol. 51, pp. 1418-1430.

A B [11C]PiB

[4] Dannals RF and Langstrom B. A simple one-pot apparatus for the production of carbon-11 labeled methyl iodide, 1985.

[11C]CH3OH

[5] Jewett DM., "A simple synthesis of [11C]methyl triflate.", Int J Rad Appl O-[11C]methylbenzothiazole

Instrum [A], 1992, vol. 43, pp. 1383-1385. [6] Wilson AA., Garcia A., Chestakova A., Kung H., and Houle S., "A rapid one-step radiosynthesis of the β-amyloid imaging radiotracer N-methyl[11C]2-(4͛-methylaminophenyl)-6-hydroxybenzothiazole ([11C]-6-OHBTA-1)", Journal of Labelled Compounds and Radiopharmaceuticals, 2004; vol. 47, pp. 679-682.

Precursor

[7] Andersson J, Truong P, and Halldin C., "In-target produced [11C]methane: Increased specific radioactivity.", Applied Radiation and Isotopes, 2009, vol. 67, pp. 106-110. PiB

[8] Wuest F, Berndt M, and Kniess T. "Carbon-11 Labeling Chemistry Based upon [11C]Methyl Iodide.", PET Chemistry. In: PA Schubiger, L Lehmann, and M Friebe editors: Springer Berlin Heidelberg, 2007, pp. 183-213. [9] Gómez-Vallejo V and Llop J. "Fully automated and reproducible radiosynthesis

Figure 4. Preparative chromatogram of [11C]PiB. Bottom, ultraviolet (UV), absorbance at 254 nm; and top, radioactivity. The retention times for [11C]CH3OH, [11C]PiB and O-[11C]methylbenzothiazole are 1.7, 7.3 and 14.6 min, respectively. (A) Start collect; (B) Stop collect.

of

high

specific

activity

[11C]raclopride

and

[11C]Pittsburgh compound-B using the combination of two commercial synthesizers", Nuclear Medicine Communications, 2011, vol. 32, pp. 1011-1017. [10] Verdurand M, Bort G, Tadino V, Bonnefoi F, Le Bars D, and Zimmer L. "Automated radiosynthesis of the Pittsburg compound-B using a commercial synthesizer. Nuclear Medicine Communications", 2008, vol. 29, pp. 920-926.