Automated GMP Synthesis of [18F]ICMT-11 for In Vivo Imaging of Caspase-3 Activity
Introduction
The ability to detect and quantify biological processes in vivo is the primary objective of Positron Emission Tomography (PET). Apoptosis, or Programmed Cell Death, is an energy conserved process in which the contents of a cell are broken down and engulfed by macrophages prior to re-use by other cells. Excessive apoptosis is implicated in neurodegenerative diseases (e.g. Alzheimer's), ischemia and transplant rejection [1]. By contrast, insufficient apoptosis is defined as one of the hallmarks of cancer [2], [3]. It therefore follows that strategies for imaging apoptosis in vivo would be of considerable interest both in the clinic, for patient diagnosis and evaluation of response to treatment [4], and as a tool to aid evaluation of new therapeutics, both clinically and pre-clinically.
Previously, apoptosis imaging using PET has concentrated on Annexin V radiolabelled probes [5], [6], [7]. These proteins interact with phosphatidylserine residues, in the presence of calcium ions, externalised on the cell surface during apoptosis. However, this strategy suffers from several drawbacks, chiefly an inability to distinguish between apoptosis and necrosis, leading to reduced specificity in vivo. An alternative approach involves imaging caspase activity. Caspases are expressed as inactive zymogens in normal cells but cleave to their active form in response to cellular stress signals. There are two classes of caspase involved in the apoptotic process, caspases 2, 8, 9, 10 are initiator caspases that cleave the effector caspases 3, 6, 7 into their active forms. These effector caspases are then responsible for the process of cellular breakdown by cleavage of key cellular structural and repair proteins such as PARP [8].
Recently, isatin-5-sulfonamides have been proposed as suitable probes for radiolabelling and in vivo detection of caspase-3 activity [9], [10], [11]. In vivo monitoring of caspase-3 activity using a radiolabelled isatin was initially studied in a liver model of apoptosis. In this model cycloheximide treated rats showed higher liver uptake of [18F]WC-II-89 and [11C]WC-98 than control treated rats [12]. More recently, an 18F-click labelled isatin derivative ([18F]ICMT11), has been developed using 2-[18F]fluoroethyl azide [13], [14]. In a different, more clinically relevant model, 38C13 tumour bearing mice were treated with cyclophosphamide, demonstrating increased uptake of [18F]ICMT-11 relative to control treated mice. [18F]ICMT-11 has become established as the lead radiotracer for PET imaging of caspase-3 activity [15], [16]. The radiolabelling of [18F]ICMT-11 has been adapted for automation by using an acetal protected tosylate precursor and the radiosynthesis has been transferred to a cassette based automated platform (FASTlab).
Section snippets
Methods
The synthesis of protected isatin alkyne 1 ((S)-1-{[1′-[1-(2-propynyl)]-(1′2′-dihydro-2′-oxospiro(1,3-dioxane-2,3′-[3H]indol)-5′-sulfonyl}-2-(2,4-difluorophenoxymethyl)-pyrrolidine) is described elsewhere [13], [14]. All other reagents and solvents were purchased from either VWR International (Lutterworth, United Kingdom) or Sigma-Aldrich (Gillingham, United Kingdom) in the highest available purity and were used without further purification. Sodium chloride for injection 0.9% w/v and water for
Radiosynthesis of ICMT-11
The original synthetic stratagem for [18F]ICMT-11 (Scheme 1, I) utilised a copper catalysed “Click” reaction involving 2-[18F]fluoroethylazide with an alkyne precursor 5 [13]. This synthesis resulted in a modest specific activity of 1.2 GBq/μMol and a stable isatin analogue impurity at a concentration of 14 μg/mL. In an effort to improve the specific activity of [18F]ICMT-11 and reduce the concentration of stable isatin impurities we investigated a modified click radiochemistry strategy (Scheme 1
Conclusion
A simplified, more robust methodology for the synthesis of the apoptosis imaging agent, (S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluorophenoxymethyl)-pyrrolidine-1-sulfonyl)isatin ([18F]ICMT-11) has been achieved by the SN2 displacement of a tosylate leaving group with [18F]fluoride ion resulting in an increased purity and specific activity. A fully automated GMP synthesis of [18F]ICMT-11 suitable for clinical use has been developed providing in excess of 4 GBq of [18
Acknowledgments
The authors would like to acknowledge Andrew Black (MDx Fundamental Radiochemistry Team, GE Healthcare, Amersham, U.K.) for HPLC-MS/MS data and analysis and Dave Turton (Hammersmith Imanet, GE Healthcare, London, U.K.) for Cation HPLC analysis.
This work was funded by Cancer Research UK–Engineering and Physical Sciences Research Council grant (in association with the Medical Research Council and Department of Health (England)) grant C2536/A10337. E.O.A.'s laboratory receives core funding from
References (26)
- et al.
The hallmarks of cancer
Cell
(2000) - et al.
Hallmarks of cancer: the next generation
Cell
(2011) - et al.
Synthesis, radiolabeling, and in vivo evaluation of an F-18-labeled isatin analog for imaging caspase-3 activation in apoptosis
Bioorg Med Chem Lett
(2006) - et al.
Improved radiosynthesis of the apoptosis marker 18F-ICMT11 including biological evaluation
Bioorg Med Chem Lett
(2011) - et al.
Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay
Bioorg Med Chem Lett
(2011) - et al.
A color spot test for the detection of Kryptofix 2.2.2 in F-18 FDG preparations
Nucl Med Biol
(1997) - et al.
Determination of residual Kryptofix 2.2.2 levels in F-18-labeled radiopharmaceuticals for human use
Appl Radiat Isot
(2007) - et al.
Nguyen Q-d, Perumal M, et al. Radiosynthesis and pre-clinical evaluation of (18)F fluoro-1,2-(2)H(4) choline
Nucl Med Biol
(2011) Apoptosis-based therapies
Nat Rev Drug Discov
(2002)Imaging of apoptosis
J Nucl Med
(2008)
Imaging of apoptosis (programmed cell death) with Tc-99m annexin V
J Nucl Med
In vitro selectivity, in vivo biodistribution and tumour uptake of annexin V radiolabelled with a positron emitting radioisotope
Br J Cancer
Site-specific labeling of annexin V with F-18 for apoptosis imaging
Bioconjugate Chem
Cited by (34)
Controlled and diastereoselective synthesis of α-(3-hydroxy-2 oxoindolin-3-yl)-β-aminopropanoates
2020, TetrahedronCitation Excerpt :The selective formation of the single diastereomer 5 could be rationalized by invoking the plausible reaction mechanism Scheme 4. The common intermediate of seven-member ring d with syn and anti faces are generated from the enolate c by the addition of amino nucleophile in adduct 1a [8]. The product stereoselectivity and syn addition of proton can be facilitated through the steric arrangement of t-isobsa and aryl moiety of intermediate d.
Synthesis of isatin based N<sup>1</sup>-alkylated 3-β-C-glycoconjugated-oxopropylidene oxindoles as potent antiplasmodial agents
2019, European Journal of Medicinal ChemistryCitation Excerpt :A few of the chalcone derivatives including one of our sugar derived chalcone, have demonstrated potent antimalarial activities [22], (Fig. 1). Our recent work showed that glycohybridised isatin hydrazones are potent antiplasmodial agents [23]. Thus, keeping all these facts in mind and in continuation of our previous efforts in the development of novel antimalarials [22d,24], we embarked upon a new series of isatin glycoconjugates and evaluated them for their antiplasmodial activity.
Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers
2017, Bioconjugate ChemistrySynthesis and evaluation of a [<sup>18</sup>F]BODIPY-labeled caspase-inhibitor
2017, Bioorganic and Medicinal ChemistryCitation Excerpt :Apart from other approaches, small molecules based on isatin have been extensively studied for in vitro and in vivo applications.42–51 18F-radiolabeling of isatins was already successful using different approaches.52–63 Synthesis of BODIPY 9 started from p-hydroxybenzaldehyde (2) by two substitution reactions via bromoethoxy compound 3 and 4-(2-azidoethoxy)-benzaldehyde (4) using literature-known procedures.64,65
Asymmetric synthesis of chiral 3,3-disubstituted oxindoles using isatin as starting material
2015, Tetrahedron AsymmetryCitation Excerpt :Substituted isatin analogues constitute valuable building blocks for potential pharmaceuticals with a wide range of biological properties such as antimicrobial,12 antitumor,13–16 antitubercular,17,18 antimalaria,19 anti-HIV,20 and antibacterial.21 Furthermore, isatin is a core fragment of many drugs.22–24 Recently, we reviewed the advances in the use of isatin in the synthesis of various heterocyclic compounds via 1,3-dipolar cycloaddition reactions.25
Design, synthesis and initial characterisation of a radiolabelled [<sup>18</sup>F]pyrimidoindolone probe for detecting activated caspase-3/7
2015, Organic and Biomolecular Chemistry
- 1
These authors contributed equally.
- 2
Present address: Post-Graduate Medical Institute, University of Hull, Cottingham Road, Hull, HU6 7RX, UK.