The Synthesis and Initial Evaluation of MerTK Targeted PET Agents

MerTK (Mer tyrosine kinase), a receptor tyrosine kinase, is ectopically or aberrantly expressed in numerous human hematologic and solid malignancies. Although a variety of MerTK targeting therapies are being developed to enhance outcomes for patients with various cancers, the sensitivity of tumors to MerTK suppression may not be uniform due to the heterogeneity of solid tumors and different tumor stages. In this report, we develop a series of radiolabeled agents as potential MerTK PET (positron emission tomography) agents. In our initial in vivo evaluation, [18F]-MerTK-6 showed prominent uptake rate (4.79 ± 0.24%ID/g) in B16F10 tumor-bearing mice. The tumor to muscle ratio reached 1.86 and 3.09 at 0.5 and 2 h post-injection, respectively. In summary, [18F]-MerTK-6 is a promising PET agent for MerTK imaging and is worth further evaluation in future studies.


Introduction
MerTK, a receptor tyrosine kinase of the TAM (TYRO3, AXL, and MERTK) family, is over-expressed or ectopically expressed in a wide variety of cancers [1,2], including acute lymphoblastic leukemia (ALL) [3], non-small cell lung cancer (NSCLC) [4], melanoma [5], prostate cancer [6], glioblastoma [7], etc. In fact, MerTK mediates the activation of several canonical oncogenic signaling pathways in cancer cells [8,9]. In addition, due to the important physiological role of MerTK in the innate immune system, MerTK inhibitors may potentially reduce tumor growth by changing the immunosuppressive environment and stimulating antitumor immunity [10,11]. Indeed, based on the important functions of MerTK, many MerTK targeted therapies are in development to enhance outcomes for patients with a variety of types of cancers, and a few are in clinical trials [12]. Despite the enthusiasm, tumor sensitivity to MerTK suppression may not be uniform due to the heterogeneity of solid tumors and different disease stages (for example, primary v. metastatic disease) [13,14]. Clearly, there is an urgent need to better predict which cancer patients are likely to respond to such novel interventions, as well as monitor the therapeutic responses. Although the drug metabolism study based on Mass analysis could provide information on biodistribution and metabolism of small pharmaceutical molecules in vivo [15], PET is a non-invasive imaging technology that can quantitatively evaluate biological targets or biochemical processes in vivo [16][17][18][19]. Nevertheless, research on MerTK targeted PET agent are very limited [20]. Therefore, the aim of this research is to develop radio-labeled agents that will allow us to directly measure MerTK expression and distribution during different disease stages, non-invasively and repetitively.
We have been committed to the development of novel therapeutics against MerTK for an extended period and have developed several small-molecule MerTK inhibitors with great potency and different selectivity profiles [21][22][23][24][25]. UNC5293 is a new MerTKspecific inhibitor developed recently at UNC, which is extremely potent against MerTK (Ki is 0.19 nM) and very selective against the kinome (Ambit selectivity score S 50 = 0.041 at 100 nM) [25]. Since target specificity is one of the key requirements of PET agents, the discovery of UNC5293A provides us with a solid foundation for developing MerTK PET ligands.
In this research, we developed a series of potential MerTK PET agents based on the core of UNC5293 (UNC6429/UNC5650) and evaluated their use in B16F10 tumor-bearing mice.

Chemistry
As shown in Scheme 1, UNC6429 and UNC5650 were synthesized using a three-step sequence. Generally, the starting material 1 was heated with an appropriate primary amine (commercially available and enantiomerically pure) in a sealed tube under basic conditions for 3 days to complete the S N Ar replacement reaction. After purification, the resulting intermediate 2 underwent a Suzuki coupling reaction, followed by deprotection of the Boc group with hydrogen chloride to afford us with intermediate 3. Finally, UNC6429 and UNC5650 were prepared by hydrogenation of the double bond using palladium on carbon with overall yields of 33% and 62%, respectively.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 12 targets or biochemical processes in vivo [16][17][18][19]. Nevertheless, research on MerTK targeted PET agent are very limited [20]. Therefore, the aim of this research is to develop radio-labeled agents that will allow us to directly measure MerTK expression and distribution during different disease stages, non-invasively and repetitively. We have been committed to the development of novel therapeutics against MerTK for an extended period and have developed several small-molecule MerTK inhibitors with great potency and different selectivity profiles [21][22][23][24][25]. UNC5293 is a new MerTK-specific inhibitor developed recently at UNC, which is extremely potent against MerTK (Ki is 0.19 nM) and very selective against the kinome (Ambit selectivity score S50 = 0.041 at 100 nM) [25]. Since target specificity is one of the key requirements of PET agents, the discovery of UNC5293A provides us with a solid foundation for developing MerTK PET ligands.
In this research, we developed a series of potential MerTK PET agents based on the core of UNC5293 (UNC6429/UNC5650) and evaluated their use in B16F10 tumor-bearing mice.

Chemistry
As shown in Scheme 1, UNC6429 and UNC5650 were synthesized using a three-step sequence. Generally, the starting material 1 was heated with an appropriate primary amine (commercially available and enantiomerically pure) in a sealed tube under basic conditions for 3 days to complete the SNAr replacement reaction. After purification, the resulting intermediate 2 underwent a Suzuki coupling reaction, followed by deprotection of the Boc group with hydrogen chloride to afford us with intermediate 3. Finally, UNC6429 and UNC5650 were prepared by hydrogenation of the double bond using palladium on carbon with overall yields of 33% and 62%, respectively.  The inhibitory activities of standards towards MerTK, Axl, Tyro3 and Flt3 were determined in our in-house microcapillary electrophoresis (MCE) assays [25]. As presented in Table 1, the primary targets of these compounds are all MerTK. The inhibitory activities of standards towards MerTK, Axl, Tyro3 and Flt3 were determined in our in-house microcapillary electrophoresis (MCE) assays [25]. As presented in Table 1, the primary targets of these compounds are all MerTK.  The inhibitory activities of standards towards MerTK, Axl, Tyro3 and Flt3 were determined in our in-house microcapillary electrophoresis (MCE) assays [25]. As presented in Table 1, the primary targets of these compounds are all MerTK.  The inhibitory activities of standards towards MerTK, Axl, Tyro3 and Flt3 were determined in our in-house microcapillary electrophoresis (MCE) assays [25]. As presented in Table 1, the primary targets of these compounds are all MerTK. a Values are the mean of two or more independent assays.

Radiochemistry
With the precursors and standards in hand, we explored their radiolabeling with easily available positron nuclides: carbon-11, Gallium-68, and Fluorine-18. C-11 labeled MerTK-1 and MerTK-2 were obtained with lower yields due to the difficulty in HPLC purification (the precursor and the product had close retention times). The short half-life of 11 C (t1/2 = 20.4 min) added more challenges: only one HPLC purification could be done for each reaction. The IC50 value of MerTK-1 and MerTK-2 against MerTK were determined to be 4.2 nM and 61 nM, respectively (Table 1). Good selectivity over Axl, Tyro3 and Flt3 was observed. The 68 Ga (half-life of 67.6 min and up to 1.89 MeV positron energy) could label MerTK-3 and MerTK-4 efficiently; however, the initial pilot study in mice did not provide promising results (<1%ID/g tumor uptakes were observed). Therefore, we did not measure their binding affinity and focused on developing fluorine-18 labeled PET agents for MerTK imaging due to its relatively long half-life (109.8 min) and high resolution (up to 0.64 MeV positron energy) on the PET imaging.
As shown in Scheme 2, the fluorine-18 labeling on UNC 5650 and UNC6429 were carried out using a two-step sequence. were purified on radio-HPLC and followed by reformulation. The identity of the final product was confirmed by coinjection with the standard compound in HPLC. The IC50 value of MerTK-5 and MerTK-6 were determined to be 15 nM and 37 nM against MerTK, respectively, with good selectivity over Axl, Tyro3 and Flt3 (Table 1). Although MerTK-5 had a higher binding affinity 13 4100 180 >30,000 a Values are the mean of two or more independent assays.

Radiochemistry
With the precursors and standards in hand, we explored their radiolabeling with easily available positron nuclides: carbon-11, Gallium-68, and Fluorine-18. C-11 labeled MerTK-1 and MerTK-2 were obtained with lower yields due to the difficulty in HPLC purification (the precursor and the product had close retention times). The short half-life of 11 C (t1/2 = 20.4 min) added more challenges: only one HPLC purification could be done for each reaction. The IC50 value of MerTK-1 and MerTK-2 against MerTK were determined to be 4.2 nM and 61 nM, respectively (Table 1). Good selectivity over Axl, Tyro3 and Flt3 was observed. The 68 Ga (half-life of 67.6 min and up to 1.89 MeV positron energy) could label MerTK-3 and MerTK-4 efficiently; however, the initial pilot study in mice did not provide promising results (<1%ID/g tumor uptakes were observed). Therefore, we did not measure their binding affinity and focused on developing fluorine-18 labeled PET agents for MerTK imaging due to its relatively long half-life (109.8 min) and high resolution (up to 0.64 MeV positron energy) on the PET imaging.
As shown in Scheme 2, the fluorine-18 labeling on UNC 5650 and UNC6429 were carried out using a two-step sequence. were purified on radio-HPLC and followed by reformulation. The identity of the final product was confirmed by coinjection with the standard compound in HPLC. The IC50 value of MerTK-5 and MerTK-6 were determined to be 15 nM and 37 nM against MerTK, respectively, with good selectivity over Axl, Tyro3 and Flt3 (Table 1). Although MerTK-5 had a higher binding affinity 15 1100 190 1000

MerTK-6
Molecules 2022, 27 a Values are the mean of two or more independent assays.

Radiochemistry
With the precursors and standards in hand, we explored their radiolabeling with easily available positron nuclides: carbon-11, Gallium-68, and Fluorine-18. C-11 labeled MerTK-1 and MerTK-2 were obtained with lower yields due to the difficulty in HPLC purification (the precursor and the product had close retention times). The short half-life of 11 C (t1/2 = 20.4 min) added more challenges: only one HPLC purification could be done for each reaction. The IC50 value of MerTK-1 and MerTK-2 against MerTK were determined to be 4.2 nM and 61 nM, respectively (Table 1). Good selectivity over Axl, Tyro3 and Flt3 was observed. The 68 Ga (half-life of 67.6 min and up to 1.89 MeV positron energy) could label MerTK-3 and MerTK-4 efficiently; however, the initial pilot study in mice did not provide promising results (<1%ID/g tumor uptakes were observed). Therefore, we did not measure their binding affinity and focused on developing fluorine-18 labeled PET agents for MerTK imaging due to its relatively long half-life (109.8 min) and high resolution (up to 0.64 MeV positron energy) on the PET imaging.
As shown in Scheme 2, the fluorine-18 labeling on UNC 5650 and UNC6429 were carried out using a two-step sequence. were purified on radio-HPLC and followed by reformulation. The identity of the final product was confirmed by coinjection with the standard compound in HPLC. The IC50 value of MerTK-5 and MerTK-6 were determined to be 15 nM and 37 nM against MerTK, respectively, with good selectivity over Axl, Tyro3 and Flt3 (Table 1). Although MerTK-5 had a higher binding affinity 37 2100 120 5500 a Values are the mean of two or more independent assays.

Radiochemistry
With the precursors and standards in hand, we explored their radiolabeling with easily available positron nuclides: carbon-11, Gallium-68, and Fluorine-18. C-11 labeled MerTK-1 and MerTK-2 were obtained with lower yields due to the difficulty in HPLC purification (the precursor and the product had close retention times). The short half-life of 11 C (t 1/2 = 20.4 min) added more challenges: only one HPLC purification could be done for each reaction. The IC 50 value of MerTK-1 and MerTK-2 against MerTK were determined to be 4.2 nM and 61 nM, respectively (Table 1). Good selectivity over Axl, Tyro3 and Flt3 was observed. The 68 Ga (half-life of 67.6 min and up to 1.89 MeV positron energy) could label MerTK-3 and MerTK-4 efficiently; however, the initial pilot study in mice did not provide promising results (<1%ID/g tumor uptakes were observed). Therefore, we did not measure their binding affinity and focused on developing fluorine-18 labeled PET agents for MerTK imaging due to its relatively long half-life (109.8 min) and high resolution (up to 0.64 MeV positron energy) on the PET imaging.
As shown in Scheme 2, the fluorine-18 labeling on UNC 5650 and UNC6429 were carried out using a two-step sequence.  (Table 1). Although MerTK-5 had a higher binding affinity towards MerTK, the initial PET study suggested that MerTK-6 had more prominent tumor uptake and contrast. Therefore, we focused on MerTK-6 in the initial evaluation. The HPLC spectra in Figure 1 illustrate the purification and quality control of [ 18 F]-MerTK-6.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 12 towards MerTK, the initial PET study suggested that MerTK-6 had more prominent tumor uptake and contrast. Therefore, we focused on MerTK-6 in the initial evaluation. The HPLC spectra in Figure 1 illustrate the purification and quality control of [ 18 F]-MerTK-6.

Evaluation of the LogP
In order to evaluate the hydrophilicity and lipophilicity of this fluorine-18 labeled agent [ 18 F]-MerTK-6, we measured the 1-octanol/water partition coefficient (LogP) of [ 18 F]-MerTK-6. The resulting fractions were counted using a gamma counter. The reaction was repeated three times. The logP values of [ 18 F]-MerTK-6 (1.56 ± 0.02) showed that it was moderately lipophilic, indicating that it had good cell membrane permeability and tumor cell uptake potential.

Evaluation of the LogP
In order to evaluate the hydrophilicity and lipophilicity of this fluorine-18 labeled agent [ 18 F]-MerTK-6, we measured the 1-octanol/water partition coefficient (LogP) of [ 18 F]-MerTK-6. The resulting fractions were counted using a gamma counter. The reaction was repeated three times. The logP values of [ 18 F]-MerTK-6 (1.56 ± 0.02) showed that it was moderately lipophilic, indicating that it had good cell membrane permeability and tumor cell uptake potential.

Chemistry
Microwave reactions were carried out using a CEM Discover-S reactor with a vertically focused IR external temperature sensor and an Explorer 72 autosampler. The dynamic mode was used to set up the desired temperature and hold time with the following fixed parameters: PreStirring, 1 min; Pressure, 200 psi; Power, 200 W; PowerMax, off; Stirring, high. Flash chromatography was carried out on Teledyne ISCO Combi Flash ® Rf 200 with pre-packed silica gel disposable columns. Preparative HPLC (Agilent Technologies 1260 Infinity, Santa Clara, CA, U.S.A) was performed with UV detection at 220 or 254 nm. Samples were injected onto a 75 × 30 mm, 5 μm, C18(2) column at room temperature. The flow rate was 30 mL/min. Various linear gradients were used with solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile). Analytical HPLC was performed with a prominence diode array detector (Shimadzu SPD-M20A, Kyoto, Japan). Samples were injected onto a 3.6 μm PEPTIDE XB-C18 100 Å, 150 × 4.6 mm LC column at room temperature. The flow rate was 1.0 mL/min. Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F254, and 0.25 mm pre-coated TLC plates. The TLC plates were visualized using UV254 and phosphomolybdic acid with charring. All 1 H NMR spectra were obtained with a 400 MHz spectrometer (Agilent VnmrJ, Santa Clara, CA, U.S.A) using CDCl3 (7.26 ppm), or CD3OD (2.05 ppm) as an internal reference. Signals are reported as m (multiplet), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), and bs (broad singlet); and coupling constants are reported in hertz (Hz). The 13 C NMR spectra were obtained with a 100 MHz spectrometer (Agilent VnmrJ, Santa Clara, CA, U.S.A) using CDCl3 (77.2 ppm), or CD3OD (49.0 ppm) as the internal standard. LC/MS (Agilent Technologies 1260 Infinity II, Santa Clara, CA, U.S.A) was performed using an analytical instrument with the UV detector set to 220 nm, 254 nm, and 280 nm, and a single quadrupole mass spectrometer using an electrospray ionization (ESI) source. Samples were injected (2 μL) onto a 4.6 × 50 mm, 1.8 μm, C18 column at room temperature. A linear gradient from 10% to 100% B (0.1% acetic acid in MeOH) in 5.0 min was followed by pumping 100% B for another 2 or 4 min with A being H2O + 0.1% acetic acid. The flow rate was 1.0 mL/min. The purity of all final compounds (>95%) was determined by LC-MS.

Chemistry
Microwave reactions were carried out using a CEM Discover-S reactor with a vertically focused IR external temperature sensor and an Explorer 72 autosampler. The dynamic mode was used to set up the desired temperature and hold time with the following fixed parameters: PreStirring, 1 min; Pressure, 200 psi; Power, 200 W; PowerMax, off; Stirring, high. Flash chromatography was carried out on Teledyne ISCO Combi Flash ® R f 200 with pre-packed silica gel disposable columns. Preparative HPLC (Agilent Technologies 1260 Infinity, Santa Clara, CA, USA) was performed with UV detection at 220 or 254 nm. Samples were injected onto a 75 × 30 mm, 5 µm, C18(2) column at room temperature. The flow rate was 30 mL/min. Various linear gradients were used with solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile). Analytical HPLC was performed with a prominence diode array detector (Shimadzu SPD-M20A, Kyoto, Japan). Samples were injected onto a 3.6 µm PEPTIDE XB-C18 100 Å, 150 × 4.6 mm LC column at room temperature. The flow rate was 1.0 mL/min. Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F 254 , and 0.25 mm pre-coated TLC plates. The TLC plates were visualized using UV 254 and phosphomolybdic acid with charring. All 1 H NMR spectra were obtained with a 400 MHz spectrometer (Agilent VnmrJ, Santa Clara, CA, USA) using CDCl 3 (7.26 ppm), or CD 3 OD (2.05 ppm) as an internal reference. Signals are reported as m (multiplet), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), and bs (broad singlet); and coupling constants are reported in hertz (Hz). The 13 C NMR spectra were obtained with a 100 MHz spectrometer (Agilent VnmrJ, Santa Clara, CA, USA) using CDCl 3 (77.2 ppm), or CD 3 OD (49.0 ppm) as the internal standard. Representative NMR spectrums were provided in Supplementary Material. LC/MS (Agilent Technologies 1260 Infinity II, Santa Clara, CA, USA) was performed using an analytical instrument with the UV detector set to 220 nm, 254 nm, and 280 nm, and a single quadrupole mass spectrometer using an electrospray ionization (ESI) source. Samples were injected (2 µL) onto a 4.6 × 50 mm, 1.8 µm, C18 column at room temperature. A linear gradient from 10% to 100% B (0.1% acetic acid in MeOH) in 5.0 min was followed by pumping 100% B for another 2 or 4 min with A being H 2 O + 0.1% acetic acid. The flow rate was 1.0 mL/min. The purity of all final compounds (>95%) was determined by LC-MS.

Synthesis of UNC5650
General procedure A [25]. A mixture of 1 (3.30 g, 10.0 mmol); (S)-pentan-2-amine (3.48 g, 40.0 mmol); potassium carbonate (5.52 g, 40.0 mmol); and N,N-diisopropylethylamine (7.0 mL, 40.0 mmol) in iPrOH (80 mL) was heated at 120 • C for 3 d. The reaction mixture was extracted between EtOAc (3 × 80 mL) and H 2 O (80 mL). The combined organic layers were washed with brine (50 mL), dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column to afford the desired product 2 as a pale-yellow solid (2.82 g, 74%). 1   A suspension of 3a (383 mg, 1.0 mmol) and palladium on carbon (10% Pd, 380 mg) in MeOH (20 mL) was stirred at rt under hydrogen atmosphere overnight. The resulting mixture was filtered through a pad of Celite and the solvent was removed under reduced pressure. The residue was purified by an ISCO silica gel column to afford the desired product 4 (UNC5650) as a yellow solid (240 mg, 62%). 1

Synthesis of UNC6429
The title compound UNC6429 was synthesized according to the general procedure A as a yellow solid (240 mg, 0.523 mmol). 1  General procedure C. The synthesis of MerTK was modified from literature method [25]. To a solution of UNC5650 (10.0 mg, 21.8 µmol) and 2-fluoroethyl 4-toluenesulfonate (3.7 µL, 22 µmol) in acetonitrile (2.2 mL) was added sodium iodide (1.6 mg, 11 µmol), and sodium carbonate (10.4 mg, 98.2 µmol). The reaction mixture was heated at 65 • C for 18 h and concentrated in vacuo. The residue was purified by normal phase chromatography (dichloromethane/methanol gradient) to afford the desired compound MerTK-5 as a pale-yellow oil, which was freeze dried to give an orange solid (4.0 mg, 9.3 µmol) in 43% yield. 1 (Table 2), and ATP at the Km for each enzyme ( Table 2). All reactions were terminated by addition of 20 µL of 70 mM EDTA. After an 180 min incubation, phosphorylated and unphosphorylated substrate peptides (Table 2) were separated in buffer supplemented with 1 x CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software.

Radiochemistry
General procedure D.

Evaluation of LogP
The LogP value of the [ 18 F]-MerTK-6 was calculated by the gamma particle counts of samples in the aqueous phase or 1-octanol phase by Automatic Gamma Counter 2480-0010 (PerkinElmer Instruments Inc., Waltham, MA, USA).
The [ 18 F]-MerTK-6 was collected after HPLC purification. After reformulation (pH value around 7.4), 20 µL [ 18 F]-MerTK-6 sample in saline was added to the mixture of 1 mL Mili-Q®water and 1 mL 1-octanol in a 5 mL Eppendorf tube. The tube was shacked thoroughly and then let stand still for 5 min. Then the 100 µL 1-octanol phase and 100 µL aqueous phase were subjected to a gamma counter separately and the gamma counts were recorded (n = 3). The LogP value was then calculated and expressed as a mean value ± standard derivation.

Mouse Model
All animal studies were reviewed and approved by The University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. The B16F10 tumor cell was obtained from the LCCC tissue culture facility (the University of North Carolina at Chapel Hill, Chapel Hill, NC, USA). The B16F10 tumor-bearing nude mouse model was prepared as described previously [28]. Briefly, B16/F10 cells were subcutaneously injected on the right flank of C57BL/6 female mice (Jackson Laboratory). The tumor volume was measured daily. When the tumor size reached 100 mm 3 , the mice were used for PET imaging studies.

PET Imaging
B16F10 tumor-bearing mice (n = 3/group) were intravenously injected via the tail vein with tracers. At 30 min and 120 min post-injection, a 10-min static emission scan was acquired with a SuperArgus small-animal PET/CT scanner. The regions of interests (ROIs) were drawn over the tumor and other organs and calculated as %ID/g. The mean uptake and standard deviation were calculated.

Conclusions
In this study, we synthesized several MerTK targeted PET agents based on the core structure of MerTK-specific inhibitor UNC5293. Of them, [ 18 F]-MerTK-6 showed a significant uptake rate (4.79 ± 0.24%ID/g) in B16F10 tumor-bearing mice. At 0.5 and 2 h after injection, the tumor to muscle ratio reached 1.86 and 3.09, respectively. In summary, [ 18 F]-MerTK-6 is a promising PET agent for MerTK imaging and worthy of further evaluation in future studies. There are a few MerTK inhibitors entered into clinical trials recently, such as MRX-2843 [3], INCB081776 [29], and RXDX-106 [30]. The MerTK-target PET imaging tracer would potentially help evaluating target engagement and adjusting treatment plan for individual patient.
Supplementary Materials: The following are available online. Figures S1-S7: 1 H NMR spectra for standard compounds.