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. Author manuscript; available in PMC: 2020 Nov 30.
Published in final edited form as: Bioorg Med Chem Lett. 2019 Jan 25;29(6):778–781. doi: 10.1016/j.bmcl.2019.01.033

Radiosynthesis and evaluation of [11C]CMP, a high affinity GSK3 ligand

Jaya Prabhakaran a,b,*,e, Kiran Kumar Solingapuram Sai c,e, Anirudh Sattiraju d, Akiva Mintz d, J John Mann a,b,d, JS Dileep Kumar a
PMCID: PMC7703381  NIHMSID: NIHMS1646212  PMID: 30709652

Abstract

Dysfunction of GSK3 is implicated in the etiology of many brain, inflammatory, cardiac diseases, and cancer. PET imaging would enable in vivo detection and quantification of GSK3 and can impact the choice of therapy, allow non-invasive monitoring of disease progression and treatment effects. In this report, the synthesis and evaluation of a high affinity GSK3 ligand, [11C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide, ([11C]CMP, (3), (IC50 = 3.4 nM, LogP = 1.1) is described. [11C]CMP was synthesized in 25 ± 5% yield by radiomethylating the corresponding phenolate using [11C]CH3I. The radioligand exhibited modest uptake in U251 human glioblastoma cell lines with ~50% specific binding. MicroPET studies in rats indicated negligible blood–brain barrier (BBB) penetration of [11C]CMP, despite its high affinity and suitable logP value for BBB penetration. However, administration of cyclosporine prior to [11C]CMP injection showed significant improvement in brain radioactivity uptake and the tracer binding. This finding indicates that [11C]CMP might be a P-gp efflux substrate and therefore has some limitations for routine in vivo PET evaluations in brain.

Keywords: PET, GSK3, Radiotracer, Brain

Glycogen synthase kinase 3 (GSK3) is an important member of serine/threonine kinase family of protein kinases. GSK3 plays a significant role as a signaling mediator and it deals with more than 100 substrates in most cells.1–3 GSK3 functions to regulate cell metabolism, cell survival, proliferation, neural development and neurotransmission.4–6 Among the three isoforms of GSK3 in human, GSK3α and GSK3β share 98% sequence similarity, whereas GSKβ2 is an alternative splice variant of GSK3β.1–3 Human post-mortem studies reveal the presence of high concentration of GSK3β in cortical regions, locus coeruleus, hippocampus and amygdala and the lowest in caudate and putamen of the brain.7–10 A large body of literature support the hypothesis that inhibition of GSK3 represents a therapeutically relevant target for metabolic disorders, neuropsychiatric diseases, neurodegenerative disorders, cardiac diseases, and cancer.11–19 Small molecule inhibitors of GSK3 are currently under development for a broad range of central nervous system (CNS) disorders including bipolar disorder, depression, diabetes, schizophrenia and Alzheimer’s disease. Non-invasive and in vivo detection of the changes in GSK3 expression using PET imaging can impact the choice of therapy, enable monitoring the progress of treatment, and can be a tool to accelerate new drug development through occupancy measurement studies. However, at present there is no validated PET ligands are available for in vivo imaging of GSK3. We recently reported the synthesis and in vivo evaluation of the GSK3 PET ligand, [11C]A1070722, and demonstrated that the tracer showed low binding in vervet monkey brain.20 Hence, we continued the evaluation of suitable GSK3 ligands to identify an optimum PET tracer with higher target to non-target ratio for the robust in vivo quantification of GSK3 in brain. Herein, we report the automated radiochemical synthesis and evaluation of a high affinity GSK3 ligand [11C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide ([11C]CMP) (IC50 = 3.4 nM) and logP 1.1.21

Synthesis of nonradioactive CMP (3) was achieved by the coupling of 2-(cyclopropanecarboxamido)isonicotinic acid (1) and 4-methoxypyridin-3-amine (2) in 85% yield (Scheme 1) using O-(1,2-Dihydro-2-oxo-1-pyridyl)-N,N,N′-N′-tetramethyluronium tetrafluoroborate (TPTU) and N,N-diisoproylethylamine (DIEA) in dimethylformamide (DMF).22 Desmethyl-CMP (5) was obtained by coupling of 4-hydroxy-pyridine amine (4) with isonicotinic acid, 1, in 70% yield (Scheme 1).23 The radiochemical synthesis of [11C]CMP was optimized and automated on a GE-FX2MeI/FX2M radiochemistry module by alkylating desmethyl-CMP precursor with [11C]MeI in in presence of sodium hydroxide (Scheme 1). [11C]CMP was produced in high radiochemical purity (> 98%) and molar activity (1.8 ± 0.3 Ci/mmol) with a 25 ± 5% radiochemical yield, decay corrected to EOS (n = 12).24

Scheme 1.

Scheme 1.

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Radiosynthesis of [11C]CMP.

After the successful radiosynthesis and automation, we examined the cell uptake of [11C]CMP in glioblastoma (GBM) U251 cells at 5, 30 and 60 min in triplicate, as proof of concept to demonstrate whether GSK3 can be a biomarker for GBM. Although some recent reports indicate that there are variations in growth characteristics of GBM U251 cells, they are widely used for in vitro experiments as well as to generate in vivo xenogeneic mouse (subcutaneous and intracranial) models of GBM.25–27 The radioligand exhibited equilibrium binding at 60 min incubation time in glioblastoma (GBM) U251 cells with modest uptake and approximately 50% specific binding while co-incubating with 10 μM of unlabeled CMP (Fig. 1).28

Fig. 1.

Fig. 1.

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Uptake of [11C]CMP in GBM-U251 cells. Values are reported as the mean ± SD from three independent experiments.

Dynamic microPET acquisition of [11C]CMP was then performed in anesthetized male Sprague-Dawley rats (250–300 g) (n = 3) with Trifoil μPET for 40 min with our established protocols.29–31 The imaging experiments showed no significant uptake of radioactivity in brain indicating negligible blood brain barrier (BBB) penetration of [11C]CMP (Fig. 2A), despite its high affinity and adequate lipophilicity (LogP = 1.1) for passive brain entry. Therefore, we examined the effect of the efflux transporter, P-gp, on the BBB permeability of CMP in rodents. P-gp inhibitor cyclosporine A32,33 (50 mg/kg, i.v,) was administered to the rats 60 min prior to injection of the radiotracer and microPET scanning was performed. Images showed improvement of radioactive uptake in brain after cyclosporine treatment (Fig. 2B, n = 2). Subsequently, the specificity of [11C]CMP binding was examined by blocking with cyclosporine 60 min and 5 mg/kg non-radioactive CMP (i.v) 30 min prior to radioligand administration. We found that [11C]CMP binding was blocked with the administration of unlabeled CMP (Fig. 2C, n = 1).

Fig. 2.

Fig. 2.

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MicroPET images of [11C]CMP in rats (n = 3) A. Baseline; B: Effect of cyclosporine; C. Cyclosporin and unlabeled CMP blocking, Ist column: coronal; 2nd column: transaxial; 3rd column: sagittal), circle represent brain region).

Time activity curves (TACs) also indicate no significant brain uptake of [11C]CMP under baseline condition, whereas, more than three times higher uptake was obtained after pretreatment with cyclosporine (Fig. 3). We have also noticed a higher uptake of the tracer outside the brain after cyclosporine treatment. Brain and periphery activity of [11C]CMP was blocked with unlabeled CMP (Fig. 3).

Fig. 3.

Fig. 3.

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Time activity curves of a representative baseline image (blue) and cyclosporine pretreated in rat brain (green) (n = 2).

The above experiments indicate that [11C]CMP may be a P-gp efflux substrate, and therefore it is not useful for central imaging of GSK3 in rodents. Further studies would prove whether the tracer exhibits enhanced brain uptake in larger species including monkeys, and would evaluate its utility as a PET imaging agent for GSK3 in periphery. However; CMP, with its favorable pharmacological and molecular profiles such as high GSK3 affinity, and availability of suitable sites for radiolabeling might be a candidate for structure affinity relationship (SAR) studies in order to identify next generation PET tracers with desirable properties for imaging GSK3 in CNS.

Acknowledgement

This work was supported by National Institute of Mental Health (NIMH), USA Grant MH112037 (J.P.).

References

  • 1.Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther. 2015;148:114–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ali A, Hoeflich KP, Woodgett JR. Glycogen synthase kinase-3: properties, functions, and regulation. Chem Rev. 2001;101:2527–2540. [DOI] [PubMed] [Google Scholar]
  • 3.Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001;2:769–776. [DOI] [PubMed] [Google Scholar]
  • 4.Salcedo-Tello P, Ortiz-Matamoros A, Arias C. GSK3 function in the brain during development, neuronal plasticity, and neurodegeneration. Int J Alzheimers Dis. 2011;2011:189728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Luo J The role of GSK3beta in the development of the central nervous system. Front Biol. 2012;7:212–220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kim W-Y, Snider WD. Functions of GSK-3 signaling in development of the nervous system. Front Mol Neurosci. 2011;4:44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Pandey GN, Dwivedi Y, Rizavi HS, et al. GSK-3beta gene expression in human postmortem brain: regional distribution, effects of age and suicide. Neurochem Res. 2009;34:274–285. [DOI] [PubMed] [Google Scholar]
  • 8.Karege F, Perroud N, Burkhardt S, et al. Protein levels of beta-catenin and activation state of glycogen synthase kinase-3beta in major depression. A study with postmortem prefrontal cortex. J Affective Disord. 2012;136:185–188. [DOI] [PubMed] [Google Scholar]
  • 9.Karege F, Perroud N, Burkhardt S, et al. Alteration in kinase activity but not in protein levels of protein kinase B and glycogen synthase kinase-3beta in ventral prefrontal cortex of depressed suicide victims. Biol Psychiatry. 2007;61:240–245. [DOI] [PubMed] [Google Scholar]
  • 10.Lesort M, Greendorfer A, Stockmeier C, Johnson GV, Jope RS. Glycogen synthase kinase-3beta, beta-catenin, and tau in postmortem bipolar brain. J Neural Transm. 1999;106:1217–1222. [DOI] [PubMed] [Google Scholar]
  • 11.Duda P, Wiśniewski J, Wójtowicz T, et al. Targeting GSK3 signaling as a potential therapy of neurodegenerative diseases and aging. Expert Opin. Ther. Targets 2018;22:833–848. [DOI] [PubMed] [Google Scholar]
  • 12.Dandekar MP, Valvassori SS, Dal-Pont GC, Quevedo J. Glycogen synthase kinase-3β as a putative therapeutic target for bipolar disorder. Curr Drug Metab. 2018;19:663–673. [DOI] [PubMed] [Google Scholar]
  • 13.Takahashi-Yanaga F Roles of glycogen synthase kinase-3 (GSK-3) in cardiac development and heart disease. J UOEH. 2018;40:147–156. [DOI] [PubMed] [Google Scholar]
  • 14.Huang A, Patel S, McAlpine CS, Werstuck GH. The role of endoplasmic reticulum stress-glycogen synthase kinase-3 signaling in atherogenesis. Int J Mol Sci. 2018;19:1607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mathuram TL, Reece LM, Cherian KM. GSK-3 inhibitors: a double-edged sword? – an update on tideglusib. Drug Res. 2018;68:436–443. [DOI] [PubMed] [Google Scholar]
  • 16.Mancinelli R, Carpino G, Petrungaro S, Mammola CL, Tomaipitinca L, et al. Multifaceted roles of GSK-3 in cancer and autophagy-related diseases. Oxid Med Cell Longev. 2017;2017:4629495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Nagini S, Sophia J, Mishra R. Glycogen synthase kinases: moonlighting proteins with theranostic potential in cancer. Semin Cancer Biol. 2018;S1044–579X:30179–30187. [DOI] [PubMed] [Google Scholar]
  • 18.Saraswati AP, Ali Hussaini SM, Krishna NH, Babu BN, Kamal A. Glycogen synthase kinase-3 and its inhibitors: potential target for various therapeutic conditions. Eur J Med Chem. 2018;144:843–858. [DOI] [PubMed] [Google Scholar]
  • 19.Maqbool M, Hoda N. GSK3 inhibitors in the therapeutic development of diabetes, cancer and neurodegeneration: past present and future. Curr Pharm Des. 2017;23:4332–4350. [DOI] [PubMed] [Google Scholar]
  • 20.Prabhakaran J, Zanderigo F, Sai KKS, et al. Radiosynthesis and in vivo evaluation of [11C]A1070722, a high affinity GSK-3 PETTracer in primate Brain. ACS Chem Neurosci. 2017;8:1697–1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Luo G, Chen L, Burton CR, et al. Discovery of isonicotinamides as highly selective, brain penetrable, and orally active glycogen synthase kinase-3 inhibitors. J Med Chem. 2016;59:1041–1051. [DOI] [PubMed] [Google Scholar]
  • 22..Synthesis of CMP (3): A solution of 4-methoxypyridin-3-amin2-(cyclopropanecarboxamido)isonicotinic acid (1, 100 mg, 0.48 mmol) and 4-methoxypyridine-3-amine (2, 60. mg, 0.48 mmol) in dimethylformamide (DMF, 2 mL) was treated with diisopropyl ethylamine (0.35 mL, 2 mmol) and TPTU (297 mg, 1 mmol) for overnight at room temperature. The reaction was checked for completion using thin layer chromatography using 10% methanol in dichloromethane as the solvent system. The mixture was diluted with water and the products were extracted into ethyl acetate (4 × 10 mL). The combined ethyl acetate layers were washed with water (1 × 10 mL), and brine and dried over anhydrous magnesium sulfate. The solvent was evaporated and the product was purified by silica gel column chromatography. CMP was isolated using 2–3% methanol in dichloromethane as a white solid (118 mg, 80%); purity > 98% as determined by HPLC (40:60 acetonitrile 0.1M aq AMF). 1HNMR (400 MHZ, CDCl3). 9.5 (s, 1H), 8.6 (s, 1H), 8.5–8.3 (m, 3H), 8.1 (s, 1H), 7.6 (m, 1H), 6.9 (m, 1H), 4.0 (s, 3H), 1.8 (m, 1H), 1.1–0.8, m, 4H). HRMS calculated for C16H16N4O3, 313.1301; found, 313.1296 (MH+).
  • 23..Synthesis of desmethylCMP (5): As described above, a solution of acid (1), 200 mg (0.96 mmol), 3-aminopyridin-4-ol (4, 119 mg, 1.1 mmol) in acetonitrile (5 mL) was stirred with DIEA (0.52 mL, 1.3 mmol) and TPTU (356 mg, 1.2 mmol) for overnight. Acetonitrile was evaporated and the residue dissolved in ethyl acetate. The reaction mixture was further processed as described above for compound 5 The product was isolated by column chromatography using 5% methanol in dichloromethane as a white solid (238 mg, 80%). 1HNMR (400 MHZ, CD3OD); 9.1 (s, 1H), 8.6 (s, 1H), 8.5 (d, 1H), 7.8 (m, 1H), 7.5 (m, 1H), 6.6 (m, 1H), 1.9 (m, 1H), 1.1–0.9, m, 4H); HRMS calculated for C15H13N4O3, 297.0988; found, 297.0981 (MH+).
  • 24..Radiosynthesis of [11C]CMP ([11C]3): [11C]MeI from FX2MeI module was bubbled to the reaction vial placed in FX2M module containing desmethyl-CMP 5 (~0.5–1.0 mg) in anhydrous DMF (0.5 mL) and 10 μL of aq NaOH solution for ~5 min at room temperature. After the complete transfer of radioactivity, the sealed reaction vial was heated at 80 °C for 5 min. The reaction mixture was quenched with HPLC mobile phase (1.0 mL) and injected onto a reverse-phase semi-preparative C18 Phenomenex ODS (250 × 10 mm, 10 μ) HPLC column with mobile phase solution consisted of 45% acetonitrile, 65% 0.1 M aqueous ammonium formate solution (pH value 6.0–6.5) with UV wavelength at 254 nm and a flow rate of 5.0 mL/min. The radioactive product (Rt = 7–8 min) was collected and diluted with 50 mL deionized water, and passed through C18 SepPak cartridge (WAT036800, Waters, Milford, MA) to trap the radiotracer. Radioactive product was then eluted from the cartridge with absolute ethanol (1.0 mL) and formulated with saline (10% ethanol in saline). The final product [11C]CMP was collected into a sterile vial through a sterile 0.22 μm pyrogen-free filter (Millipore Corp., Billerica, MA) for further animal studies and quality control analysis. Purity and molar activity of [11C]CMP was assessed using an analytical reverse phase Phenomenex ODS C18 analytical HPLC column (250 × 4.6 mm, 5 μ) with a mobile phase (1.0 mL/min, λ = 254 nm) consisted of 50% acetonitrile and 50% 0.1M aqueous ammonium formate pH 6.0–6.5 solution. [11C]CMP showed a retention at 6 min, and authentication of the product was performed with co-injection of the non-radioactive standard CMP, which demonstrated a similar retention times.
  • 25.Torsvik A, Stieber D, Enger P-Ø, XGolebiewska A, Molven A, Svendsen A, Westermark B, et al. U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells. Cancer Med. 2014;3:812–824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Leblond MM, Gérault AN, Dulmont A-C, et al. Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models. Oncoimmunology. 2016;5:e1056442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li X, Dai J, Tang Y, Li L, Jin G. Quantitative proteomic profiling of tachyplesin I targets in U251 gliomaspheres. Mar Drugs. 2017;15:20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28..Cell-uptake assays were performed using the cluster tray method as reported in the literature. GBM-U251cell lines was obtained from ATCC (VA, USA). Cell lines were cultured at 1×105 cells per 6 well (Corning, NY, USA) for 48 h in a 5% CO2 atmosphere in 10% phosphate buffer culture medium (Gibco, Fishersci, VA). Three days before the uptake assays, aliquots of 2.5 × 104 cells suspended in culture media were added to each well of Costar 6-well plates to achieve log growth phase with approximately 70% confluency at the time of the uptake assay. Additionally, the cells were incubated for an additional 24 h prior to the day of performing the cell assay. The blocking solution of CMP (5.0 μM) was freshly prepared on the same day of cell assay. All assays were performed in triplicate at pH 7.4. To demonstrate the blocking efficacy, CMP was added to the corresponding blocking wells 30 min prior to the addition of [11C]CMP. The assay buffer was formulated with radiotracer by adding 20 μL/mL of [11C]CMP, at a concentration of 2.0 μCi/mL to each block and non-block condition. Three incubation times of 5, 30, 60 min were selected to demonstrate the baseline uptake and blocking. The cell uptake assays were initiated by rinsing the cells with 2 × 2 mL of the phosphate buffer at room temperature. Uptake was allowed to proceed for selected time periods and then rinsed with 1 mL of the ice-cold buffer solution. Residual fluid was removed by pipette, and 200 μL of 0.1% aqueous sodium dodecylsulfate lysis buffer solution was added to each well. The plate was then agitated at room temperature and 1 mL of the lysate was taken from each well for counting. The radioactivity was counted using the Wallac 1480 Wizard gamma counter (Perkin Elmer, Turku, Finland). Additional 20 μL aliquots were taken in triplicate from each well for protein concentration determination using the Pierce bicinchoninic acid protein assay kit method (Rockford, IL). The uptake data in each sample from each well and the standard counts for each condition were expressed as counts per minute (cpm) of activity and are decay corrected for elapsed time. The cpm values of each well were normalized to the amount of radioactivity added to each well and the protein concentration in the well and expressed as percent uptake relative to the control condition. The data were expressed as %ID/mg of protein present in each well with p values ≤ .05 considered statistically significant.
  • 29.Solingapuram Sai KK, Prabhakaran J, Ramanathan G, et al. Radiosynthesis and evaluation of [11C]HD-800, a high affinity brain penetrant pet tracer for imaging microtubules. ACS Med Chem Lett. 2018;9:452–456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kumar JSD, Solingapuram Sai KK, Prabhakaran J, et al. Radiosynthesis and in vivo evaluation of [11C]MPC-6827, the first brain penetrant microtubule PET ligand. J Med Chem. 2018;61:2118–2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Solingapuram Sai KK, Prabhakaran J, Sattiraju A, Mann JJ, Mintz A, Kumar JSD. Radiosynthesis and evaluation of IGF1R PET ligand [11C]GSK1838705A. Bioorg Med Chem Lett. 2017;27:2895–2897. [DOI] [PubMed] [Google Scholar]
  • 32.Muzi M, Mankoff DA, Link JM, et al. Imaging of cyclosporine inhibition of P-glycoprotein activity using 11C-verapamil in the brain: studies of Healthy Humans. J Nucl Med. 2009;50:1267–1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Damont A, Goutal S, Auvity S, et al. Imaging the impact of cyclosporin A and dipyridamole on P-glycoprotein (ABCB1) function at the blood-brain barrier: A [(11)C]-N-desmethyl-loperamide PET study in nonhuman primates. Eur J Pharm Sci. 2016;91:98–104. [DOI] [PubMed] [Google Scholar]

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