Synthesis of ApoSense compound [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732) by nucleophilic ring opening of an aziridine precursor

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Abstract

Introduction

The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies.

Methods

We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18-labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile:dimethylsulfoxide with tetrabutyl ammonium fluoride proceeds through an aziridine intermediate (4A) to afford two regioisomers: 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [18F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6 N hydrochloric acid.

Results

Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by high-performance liquid chromatography. The total radiochemical yield was 15%±3% (uncorrected, n=18) from the aziridine precursor in a 70-min synthesis time with a radiochemical purity >99%.

Conclusion

Fluorine-18-labeled ApoSense compound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.

Introduction

Apoptosis or programmed cell death is a normal biological phenomenon of multicellular organisms. Apoptosis produces cell fragments called apoptotic bodies that are engulfed by healthy surrounding cells and tissues [1], [2], without local inflammation from leakage of cell contents. Abnormal apoptosis plays a role in an extensive variety of diseases. Molecular imaging of this process in vivo is a potentially powerful tool for early diagnosis of disease [3], [4], [5], [6] and monitoring the efficiency of treatments with apoptosis-inducing anticancer drugs [7], [8], [9], [10], [11]. Additionally, imaging of apoptosis may assist the early evaluation of organ transplant rejection [12], [13], [14].

There are several changes during the early stages of apoptosis which can be imaged by different kinds of probe [15], [16], [17], [18]. Phosphatidylserine (PS) is a phospholipid component, usually present on the inner leaflet, normally confined to the cytoplasmic face of cell membranes by an enzyme called flippase [19], [20]. When a cell undergoes apoptotic cell death, PS is no longer restricted to the cytosolic part of the membrane but becomes exposed on the surface of the cell. Annexin V, a 35.8-kDa protein, has nanomolar affinity for PS [21]. Imaging of apoptosis using annexin V attached to either a fluorescent probe or a radionuclide has been studied extensively [21], [22], [23], [24], [25]. However, certain drawbacks such as nonspecific binding, poor signal/noise ratio and slow clearance from the nontargeted tissue limit further application of this imaging agent in clinical studies [26]. Moreover, annexin V is a relatively large protein, requiring complex procedures for synthesis and detectable marker attachment. Therefore, several other proteins, peptides, small molecules and nanoparticles have been investigated for the detection of apoptosis [17], [27], [28], [29], [30], [31], [32].

ApoSense compounds are a family of small molecules which can specifically identify apoptotic cells. These compounds accumulate within the cytoplasm [17], [33] of apoptotic cells from the early stages of the death process, unlike annexin V, which binds to the PS head groups exposed on the surface. Molecular imaging and therapy developer Aposense is currently in Phase 2 clinical trials of its apoptosis molecular imaging agent, fluorine-18-labeled ML-10 (NCT00791063, NCT00696943, NCT00805636) (Fig. 1).

The performance of other members of the ApoSense family (fluorescent compounds DCC, NST-732, NST-729, tritium-labeled ML-9) has been reported in various animal models [5], [33], [34], [35], [36], [37], [38]. Recent reports demonstrated the performance of fluorine-18-labeled dansylhydrazone (DFNSH) in detecting paclitaxel-induced cancer cell death and ketamine-induced neuronal apoptosis [39], [40]. Fluorine-18 labeling of dansyl-group-containing NST732 has been proposed previously by Ziv et al. [36]. In the present study, we are reporting the first synthesis of [18F]NST732 from the easy-to-prepare aziridine precursor.

Section snippets

Materials

Methyl 2-aminobutyrate hydrochloride was purchased from TCI America (Portland, OR, USA) and used as received. Tetrabutylammonium hydrogen carbonate (0.075 M) for radiolabeling work was obtained from ABX (Radeberg, Germany). Whole human serum was obtained from MP Biomedicals, LLC (Solon, OH, USA). All other commercially available organic precursors and dry solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received unless otherwise stated. Tetrahydro-7-methoxy-3,7,7a

Synthesis of precursor and standard compounds

To prepare [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732), we synthesized the following precursors: the tosylate precursor, methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-((tosyloxy)methyl)butanoate (4) and the aziridine precursor, methyl 1-((5-(dimethylamino)naphthalen-1-yl)sulfonyl)-2-ethylaziridine-2-carboxylate (4A).

Synthesis of the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-((tosyloxy)methyl)butanoate

Conclusion

We have synthesized two different precursors and performed fluorine-18 labeling reactions with [18F]TBAF to prepare [18F]NST732. An uncorrected radiochemical yield of 15%±3% (uncorrected, n=18) was obtained using aziridine precursor within a 70-min synthesis time. The described procedure would be easily adaptable to the commercially available automatic synthesis units. Additionally, fluorination of the aziridine precursor also produced a regioisomer of [18F]NST732 which could also represent a

Acknowledgments

The authors would like to thank Christine Enders for her excellent technical assistance. This study was funded by National Institutes of Health through its 2004 Roadmap for Medical Research Initiative.

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