An F-labeled poly(ADP-ribose) polymerase ... - ACS Publications

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An F-labeled poly(ADP-ribose) polymerase positron emission tomography imaging agent. Filip Zmuda, Adele Blair, Maria Clara Liuzzi, Gaurav Malviya, Anthony J. Chalmers, David Lewis, Andrew Sutherland, and Sally L. Pimlott J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00138 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Journal of Medicinal Chemistry

An 18F-labeled poly(ADP-ribose) polymerase positron emission tomography imaging agent. Filip Zmuda,*,†,‡ Adele Blair,† Maria Clara Liuzzi,†,± Gaurav Malviya,¥ Anthony J. Chalmers,◊ David Lewis,¥ Andrew Sutherland,† and Sally L. Pimlott# †

WestCHEM, School of Chemistry, The Joseph Black Building, University of Glasgow,

Glasgow G12 8QQ, UK. ‡

Wolfson Whol Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow,

Glasgow G61 1QH, UK. ±

School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow,

Glasgow G12 8QQ, UK. ¥

Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK.

◊

Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences,

University of Glasgow, Glasgow G12 8QQ, UK. #

West of Scotland PET Centre, Greater Glasgow and Clyde NHS Trust, Glasgow G12 0YN, UK.

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ABSTRACT.

Poly(ADP-ribose) polymerase (PARP) is involved in repair of DNA breaks and is overexpressed in a wide variety of tumors, making PARP an attractive biomarker for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. Consequently, over the last decade there has been a drive to develop nuclear imaging agents targeting PARP. Here, we report the discovery of a PET tracer that is based on the potent PARP inhibitor olaparib (1). Our lead PET tracer candidate [18F]20 was synthesized and evaluated as a potential PARP PET radiotracer in mice bearing subcutaneous glioblastoma xenografts using ex vivo biodistribution and PET/MR imaging techniques. Results showed that [18F]20 could be produced in a good radioactivity yield and exhibited specific PARP binding allowing visualisation of PARP overexpressing tumors.

[18F]20 is therefore a potential candidate

radiotracer for in vivo PARP PET imaging.


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INTRODUCTION. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that exhibits a broad range of functions and is involved in transcription, mitosis, apoptosis, and DNA damage repair.1–2 PARP inhibition has been investigated as a therapeutic approach to treat cancers by either synthetic lethality, where tumor cells deficient in a type of DNA repair termed homologous recombination are sensitized to PARP inhibition, or chemoradiosensitization, where PARP inhibition sensitizes tumor cells to conventional chemo- or radiotherapy. To date, olaparib (Lynparza), niraparib (Zejula), and rucaparib (Rubraca), are the only PARP inhibitors to receive approval for clinical use in the US or Europe.3–4 Olaparib (1) (Figure 1) was the first agent in its class to receive such approval. In the EU it is currently indicated for the treatment of BRCAmutated (homologous recombination deficient) ovarian, fallopian-tube and peritoneal cancers,4 where it has been shown to increase progression-free5 and overall6 survival. In the US, 1 can also be used for treatment of BRCA-mutated metastatic breast cancer7, and as a maintenance therapy for patients with platinum-sensitive recurrent epithelial ovarian, fallopian-tube, or primary peritoneal cancer irrespective of BRCA mutations.8 In both cases, 1 was once again shown to increase progression-free survival.9–10 Olaparib 1 is also being investigated as a radio- and chemosensitizer for the treatment of solid cancers, including gliomas. However, adding PARP inhibitors to cytotoxic chemotherapy agents has been shown to exacerbate bone marrow toxicity in humans, hindering the establishment of effective PARP inhibitor and chemotherapy dosage regimens with acceptable safety profiles.11 In the case of brain tumors, matters are further complicated as 1 suffers from poor blood brain barrier (BBB) permeability and delivery of the drug to the tumor is reliant on 3 ACS Paragon Plus Environment


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BBB disruption.12 The degree of BBB disruption in brain tumors is very variable13–15; this could affect tumor penetration by 1 and hence reduce the clinical effectiveness of PARP inhibitor therapy. Furthermore, in vivo animal studies have revealed that prolonged treatment with 1 can result in increased tumor P-glycoprotein efflux transporter expression, and subsequent drug resistance.16 The above mentioned issues highlight the challenges that are associated with PARP inhibitor therapy in the context of synthetic lethality as well as chemo- and radiosensitization. Nuclear imaging of an appropriately radiolabelled PARP inhibitor could be used to overcome these challenges. In combination with a suitable blockade study protocol, nuclear imaging could indirectly establish the distribution and retention of PARP inhibitors in tumors and normal tissues, and subsequently identify therapeutic dosage regimens for which the combination of PARP inhibitors and cytotoxic agents exerts maximal tumor and minimal bone marrow cytotoxicity. Furthermore, a radiolabelled PARP probe could be used to indirectly ascertain the occupancy, retention, and target engagement of 1 in brain tumor tissue. This type of approach has been successfully applied in pre-clinical models of small-cell lung17 and epithelial ovarian cancer.18 In a clinical setting, this could be used to identify patients that are unlikely to respond to PARP inhibitor therapy due to weak target engagement as a consequence of poor drug tumor uptake or resistance caused by efflux transporter overexpression. Previously, we reported the synthesis and characterization of a [123I]-labelled compound with potential for single-photon emission computed tomography (SPECT) imaging of PARP.19 Despite the established nature of the SPECT imaging modality, the use of positron emission tomography (PET) is rapidly expanding and, in many cases, has become the preferred nuclear 4 ACS Paragon Plus Environment

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imaging modality in the clinic. This can be attributed to the superior spatial resolution,20 quantification and sensitivity21 of PET compared with SPECT. It is, therefore, not surprising that many of the current nuclear imaging agents for PARP were designed with PET imaging in mind (Figure 2).22–23 Importantly, the rationale for developing PARP PET imaging agents has been recently solidified by [18F]4, which was shown to be capable of non-invasively ascertaining PARP-1 expression in epithelial ovarian tumors in humans.18,24 Here, we report the synthesis of a small library of fluorinated analogs of the clinical PARP inhibitor 1 with potential for PARP PET imaging and the in vitro characterization of these compounds. The lead analog 20 was synthesized in its radiofluorinated version and evaluated as a potential PARP PET radiotracer in mice bearing subcutaneous glioblastoma xenografts using ex vivo biodistribution and PET/magnetic resonance (MR) imaging techniques. RESULTS AND DISCUSSION.

Chemistry and In Vitro Characterization. Due to poor accessibility of the central ring fluorine atom of 1 for radiolabelling25, focus was directed at synthesizing analogs of 1 bearing distal fluorinated moieties that were more likely to be amenable to radiofluorination methods. Initially, six fluorinated PARP inhibitors (8– 13) containing the characteristic phthalazinone scaffold were synthesized through amide or Nalkyl coupling of commercially available benzoic carboxylic acids or benzyl halides with piperazine 7, the synthesis of which we described previously19 (Table 1). The structures of compounds 8–13 were confirmed in part by NMR spectroscopic analysis, which showed that the majority of these exist as a mixture of amide rotamers. The design of these analogs was partly 5 ACS Paragon Plus Environment


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driven by previous reports,25–27 which have shown that structural modifications can be performed in the cyclopropane bearing region of 1 without having a marked effect on PARP inhibition. To confirm this, cell-free PARP-1 IC50 assays were performed on compounds 8–13, and the results of these experiments were compared against the cell-free IC50 of 1 (Table 1). Compared with 1, all compounds showed improved PARP-1 potency, except for compound 13, which had an overlapping cell-free IC50 95% confidence interval. Compounds 8–13 were also evaluated for their lipophilic and plasma protein binding properties, defined by log Poct and percentage plasma protein binding (%PPB) parameters (Table 1). All six analogs exhibited greater log Poct and %PPB values when compared with 1. This may be attributed to the addition of aromatic and methyl moieties, which have the potential to increase lipophilicity and can, in turn, result in an increased %PPB due to the hydrophobic nature of plasma protein interactions.28 From the perspective of nuclear imaging, radiotracers with high log Poct (>3.0)29 and %PPB (>95%)30 values can be associated with poor passive diffusion across biological membranes and vascular retention, which can in turn result in a poor target to background signal ratio. The physiochemical parameters of 8–12 were all found to be within the optimal range for radiotracer development. However, following analysis of the established in vitro parameters and potential radiochemical accessibility, compound 8 was identified as an initial lead candidate for further advancement in this research program. In order to establish the potency of 8 against PARP in living cells, cellular IC50 assays were performed using primary (G7) and secondary (T98G) human glioblastoma cell lines (Table 2) and previously described methodology.19 Compound 8 exhibited low nanomolar IC50 values that were in line with those observed for 1 in both cell lines, suggesting that 8 was able to effectively penetrate cellular membranes and reach PARP localized within cellular nuclei. 6 ACS Paragon Plus Environment

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In order to access the [18F]-radiofluorine analogue of 8, standard aromatic nucleophilic substitution chemistry was employed using p-nitrobenzamide precursor 14, which was generated through amide coupling of 7 with commercially available 4-nitrobenzoic acid (Scheme 1). However, optimization of the radiofluorination step proved challenging (see supporting information), and the maximum radiochemical yield (based on HPLC analysis of crude product) achieved was only 19% (Scheme 1). It was proposed that the poor yield was a consequence of a lack of activation of the system for aromatic nucleophilic substitution due to the weak electron withdrawing properties of the amide located para- to the nitro leaving group. Since commencing this work, Carney et al were able to synthesize [18F]8 with an optimized radioactivity yield of 38 ± 2.5% (isolated product) using a multi-step early stage radiofluorination approach that circumvented the aforementioned issue of poor activation.17 Therefore, our attention was redirected towards an alternative target compound, 20, bearing a p-(fluoromethyl)benzamide group. It was proposed that a precursor analogue of 20 would be more amenable to radiofluorination than p-nitrobenzamide 14, thereby allowing for late-stage radiofluorination and radiosynthetic automation. Compound 20 was synthesized by first performing an amide coupling reaction between mono-Boc protected piperazine 15 and commercially available 4(chloromethyl)benzoic acid, giving access to intermediate 16 in 40% yield (Scheme 2). pChlorobenzamide 16 was then subjected to nucleophilic fluorination with tetra-nbutylammonium fluoride (TBAF), and this was followed by acid-mediated cleavage of the Bocprotecting group to give 18 in 86% yield over two steps. Finally, reaction of carboxylic acid 19, previously synthesized within our research group,19 and piperazine 18 under standard amide coupling conditions gave target 20. The log Poct, %PPB, and cell-free and cellular IC50 values



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were established for 20 in the same manner as described for the other analogs (8–13) in this series (Table 3).

As expected, compound 20 exhibited higher log Poct and %PPB values than 1 (Table 3). However, these were still within the optimal range for a nuclear imaging agent. Interestingly, despite 20 having an identical log Poct value to 8, the former compound exhibited a higher degree of %PPB. Furthermore, compound 20 had a cell-free IC50 value that was 10.8- and 3.0-fold less than that acquired for 1 and 8, respectively. Conversely, the cellular PARP inhibitory properties of 20 were comparable to compound 1, and marginally weaker than ascertained for compound 8. Collectively, the optimal physiochemical properties and low nanomolar PARP cell-free and cellular IC50 values supported further investigation of 20 as a potential radiotracer for PARP. In the body, radiotracers can be exposed to a number of metabolic pathways including blood plasma hydrolysis and liver functionalization or conjugation reactions that can have a significant effect on the kinetic properties of the tracer, and subsequently its usefulness in nuclear imaging. With this in mind, the in vitro plasma and liver microsome stability of lead candidate 20 were established by incubating the compound in mouse plasma proteins and human liver microsome enzymes respectively using previously described methodologies.19 Candidate 20 appeared stable in mouse plasma with only minimal decomposition following a 20 hour incubation (Table 3). However, the intrinsic clearance parameter (a predictor of phase I liver metabolism) was approximately 3-fold greater for compound 20 when compared with 1. Despite this, it was proposed that compound 20 would exhibit sufficient tissue retention to allow for nuclear imaging of PARP. This was justified by previously acquired data using a radioiodinated p-iodobenzamide analog that exhibited similar in vitro intrinsic clearance properties to 20, but still displayed a degree of retention in PARP overexpressing tumor tissue.19 8 ACS Paragon Plus Environment

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Initial attempts to generate the radiofluorinated version of 20 involved performing nucleophilic substitution reactions between the

18 –

F nucleophile and the chloromethyl group of

precursor 21, which was obtained by amide coupling of 7 with commercially available 4(chloromethyl)benzoic acid (Scheme 3). However, these attempts were not successful as competing oligomerization reactions between the chloromethyl group of 21 and the phthalazinone core prevented effective radiofluorination. In order to overcome this issue, the phthalazinone core of 21 was Boc-protected to give compound 22.

Precursor 22 was then subjected to a screen of radiofluorination conditions as outlined in Table 4. The radiochemical yield (based on HPLC analysis of crude product) was 30% when tetra-n-butylammonium hydrogen carbonate (TBAHCO3) was used as a phase transfer agent, which was markedly higher when compared with that obtained for Kryptofix™ (K222) (Entries 1 and 2). It has been reported in the literature that the introduction of a sterically-hindered protic alcohol can have a beneficial effect on aliphatic nucleophilic radiofluorination reactions.31–32 By using a 2:1 mixture of t-BuOH and MeCN as the reaction solvent, the radiofluoride incorporation increased from 30% to 48% when compared with the same volume of MeCN alone (Entries 1 and 3). A 30 minute reaction time was established to be optimal based on lower radiochemical yields observed after a shorter reaction time (Entry 4) and the short lived nature of the

18

F

radioisotope (half-life = 109.8 minutes), which prevented longer reaction periods. Radiofluoride incorporation was further improved by increasing the reaction temperature from 100 ºC to 110 ºC, which resulted in a 51% radiochemical yield (Entry 5). Interestingly, doubling the reaction solvent volume had a negative impact on the degree of radiofluorination (Entry 6). Based on these data, the reaction conditions described in entry 5 were deemed optimal.



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In order to access [18F]20, compound [18F]23 was subjected to a Boc-deprotection, which was achieved in 5 minutes and with minimal defluorination taking place by using water as an acid/base catalyst (Scheme 4). The use of hydrochloric acid was also investigated as a deprotecting agent, but its use was associated with marked defluorination (see supporting information). The optimized two-step one-pot radiochemical reaction allowed access to [18F]20 in a radioactivity yield (isolated product) of 9 ± 2% (n = 7) and a molar activity of >4.32 ± 1.46 Ci µmol−1 (n = 3). Importantly, the one-pot nature of the reaction opens up the potential for radiosynthetic automation.

In vivo Characterization. Following successful optimization of the radiochemistry, the behaviour of [18F]20 was investigated in vivo in mice bearing subcutaneous U87MG-Luc2 human glioblastoma tumor xenografts using ex vivo biodistribution and PET/MR imaging techniques. Ex vivo biodistribution of [18F]20 was established at 30, 60, and 120 minutes post intravenous radiotracer administration, and PET data were acquired by performing a 45 minute dynamic scan. These experiments showed that a large proportion of radioactivity was detectable in the liver and small bowel at 30–45 minutes post tracer administration (Figure 3a and b), and mostly concentrated in the cecum matter and solid feces after 120 minutes (Figure 3a). This is in line with our previous findings19 and other literature reports,25,33–34 which showed in vivo hepatobiliary clearance of a range of related radioiodinated and radiofluorinated compounds based on the structure of 1. Interestingly, the mean percentage of injected dose per gram (%ID/g) of femur tissue remained relatively high across all three biodistribution timepoints (i.e. >8.5%) (Figure 3a), which was also confirmed by PET imaging where high skeletal uptake of radioactivity was visible (Figure 10 ACS Paragon Plus Environment

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3b). This is in contrast to observations made by Carney et al, who reported

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