Synthesis and Biological Evaluation of Novel 99mTc-Labeled

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Synthesis and biological evaluation of novel 99mTclabeled palbociclib derivatives targeting cyclin-dependent kinase 4/6 (CDK4/6) as potential cancer imaging agents Xiaoqing Song, Qianqian Gan, Xuran Zhang, and Junbo Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00540 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Molecular Pharmaceutics

Synthesis and biological evaluation of novel 99mTc-labeled palbociclib derivatives targeting cyclin-dependent kinase 4/6 (CDK4/6) as potential cancer imaging agents Xiaoqing Song†, Qianqian Gan†, Xuran Zhang, Junbo Zhang* Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, 100875, P. R. China. Abstract Cancer results from cell proliferation that exceeds normal growth control. There are various specific proteins that control and regulate the cell cycle, such as cyclin-dependent kinases (CDKs), cyclins and retinoblastoma protein (pRb). The aberration of the cyclin D-CDK4/6-INK4-pRb pathway occurs frequently in cancers; thus, CDK4/6 is an attractive target for the development of radiopharmaceuticals for tumor imaging. In this study, we chose palbociclib which was approved by FDA for treating ER+/HER2- advanced breast cancer as the target vector and the isonitrile group which can coordinate strongly with the [99mTc(CO)3]+ core as the bifunctional chelator, to develop four novel 99mTc-labeled radiotracers for tumor imaging. The ligands (L2, L3, L4 and L5) were synthesized by reacting palbociclib with isocyanide-containing active esters and then radiolabeling with [99mTc(CO)3]+ core to produce radiotracers (99mTc-L2,

99mTc-L3, 99mTc-L4

and

99mTc-L5)

with high

radiochemical purity (>95%) and good stability in vitro. The structures of the 99mTc complexes were identified by preparation and characterization of the corresponding stable rhenium complexes. Partition coefficient results indicated that these complexes were lipophilic. A kinase inhibition assay demonstrated the high affinity of the stable Re complexes for CDK4. A cell study showed that all four complexes had substantial uptake by MCF-7 cells and could be significantly inhibited by palbociclib and non-radiolabeled ligand, indicating a CDK4/6-specific uptake mechanism. Biodistribution studies in nude mice bearing MCF-7 tumors showed that the complexes had obvious accumulation in tumors at 2 h post-injection. 99mTc-L2 exhibited the highest tumor uptake and tumor/blood ratio, while 99mTc-L4 showed the highest tumor/muscle ratio. The microSPECT/CT study showed that complex

99mTc-L4

had visible uptake at the tumor site, and the accumulation was clearly

reduced in the image after pretreatment with palbociclib, further indicating CDK4/6 specificity. All the results showed that the 99mTc-labeled complexes in this work have the potential for tumor imaging. Key words: palbociclib  CDK4/6  cancer  99mTc  imaging ACS Paragon Plus Environment

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Introduction Cancer results from cell proliferation that exceeds normal growth control.1, 2 Cell proliferation is a complex, well-controlled process regulated by a molecular procedure called the cell cycle. The cell cycle consists of four phases: S phase (DNA replication), M phase (mitosis), and two gaps (G1 and G2) between S and M. There is also a quiescence state named G0 in nonproliferating cells.3, 4 There are various specific proteins that control and regulate the cell cycle, such as cyclin-dependent kinases (CDKs), cyclins and retinoblastoma protein (pRb). CDKs are a family of serine/threonine kinases that can be divided into two subfamilies (CDKs 1-6, 11, 14-18 for regulating the cell cycle and CDKs 7-13, 19, 20 for transcription). The activation of CDKs is controlled by the association with their regulatory subunits (cyclins) and phosphorylated inhibitory proteins.5 CDK4 and closely related CDK6 play a vital role in the transition from G1 to S when coordinating with cyclin D by phosphorylating pRb, which suppresses the G1-to-S transition with an unphosphorylated state.6-8 The aberration of the cyclin D-CDK4/6-INK4-pRb pathway occurs frequently in cancer; thus, the inhibition of CDK4/6 becomes an attractive topic for the treatment of cancer.9 In recent years, many CDK4/6 inhibitors have been developed for the treatment of cancer, especially breast cancer, with sound effectiveness and few side effects.10-13 Palbociclib was approved by FDA in 2015 to treat hormone receptor-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) advanced breast cancers in combination with letrozole. In addition to pharmacotherapeutic uses, CDK4/6 has also drawn the attention of researchers as a target for the development of radiopharmaceuticals for imaging and functional characterization of tumorigenesis. Lena Koehler et al radiosynthesized 124I-labeled

CDK4/6 inhibitors ([124I]CKIA and [124I]CKIB) and evaluated their potential as tumor imaging agents.14, 15

However, the results showed that [124I]CKIA and [124I]CKIB had low tumor uptake from small animal PET and autoradiography studies. The increasing thyroid uptake from 5 min to 60 min of [124I]CKIA and [124I]CKIB indicated deiodination in vivo, which showed the limitation of these compounds as tumor imaging agents. To the best of our knowledge, there have been no 99mTc-labeled CDK4/6 inhibitors reported in recent years for use in tumor imaging.

99mTc

is the most important radionuclide for SPECT imaging in the clinic due to its excellent physical properties

(t1/2 = 6 h, Eγ = 140 keV) and easy availability from a 99Mo/99mTc generator. In addition, the quality of SPECT images has ACS Paragon Plus Environment

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improved dramatically since the hardware and image reconstruction methods have shown great progression. Therefore, the development of

99mTc-labeled

99mTc-labeled

radiotracers is of great significance. To develop

bifunctional chelator is needed to link to the biomolecules and to coordinate with

99mTc.

radiopharmaceuticals, a

An isonitrile group can act as a

favorable bifunctional chelator for the design of 99mTc-labeled radiopharmaceuticals because it can coordinate strongly with [99mTc(CO)3(H2O)3]+ core to form a stable complex.16, 17 Therefore, in this study, we chose palbociclib as the target vector and the isonitrile group as the bifunctional chelator and changed the length of the carbon chain between them to develop novel 99mTc-labeled

radiotracers, and we evaluated their potential as tumor imaging agents.

Experimental section Materials All chemicals were purchased from commercial sources and used without further purification. Palbociclib was obtained from Beijing Lunarsun Pharmaceutical Co. Ltd. NMR spectra were obtained on 400 MHz and 600 MHz NMR spectrometers. Mass spectra (MS) were recorded on an AB SCIEX TripleTOFTM 5600 spectrometer (AB Sciex, Concord, Canada). The 99Mo/99mTc generator was from the Chinese Institute of Atomic Energy (CIAE). High-performance liquid chromatography (HPLC) analyses were performed on a Waters 600 binary HPLC pump equipped with a Raytest Gabi radioactivity detector and a Waters 2487 UV absorbance dual λ detector (Milford, MA, USA). The HPLC conditions were as follows: system 1 used a reverse-phase column (Kromasil C18, 250 × 4.6 mm) at a flow rate of 1 mL/min (A, H2O with 0.1% TFA; B, CH3CN with 0.1% TFA; Gradient: 0-2 min 10% B; 2-20 min 10%-90% B; 20-28 min 90% B; 28-40 min 90%-10% B); system 2 used a semipreparative column (Kromasil C18, 250 × 10 mm) at a flow rate of 4 mL/min (A, H2O with 0.1% TFA; B, CH3CN with 0.1% TFA; Gradient: 0-2 min 10% B, 2-20 min 40% B, 20-40 min 60% B, 40-50 min 90% B). Imaging studies were performed on a Triumph SPECT/CT scanner (TriFoil Imaging, California, USA). Chemical synthesis The synthesis of active esters (3a-3d) was according to the literature reported previously17 and the detailed procedure was shown in the Supplementary Information. General procedure for the synthesis of L2-L5 ACS Paragon Plus Environment


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Palbociclib (447 mg, 1 mmol) and triethylamine (160 μL, 1.15 mmol) were dissolved in 20 mL of dichloromethane in a 50 mL round-bottom flask and stirred on ice bath for 10 min. Then, compound 3a (or 3b-3d, 1 mmol) was added, and the mixture was stirred at room temperature for 3 h. The solvent was then removed under reduced pressure, and the residue was purified via silica gel column chromatography (dichloromethane/methanol = 20/1) to afford compounds L2-L5 as yellow powders. 6-acetyl-8-cyclopentyl-2-((5-(4-(3-isocyanopropanoyl)piperazin-1-yl)pyridin-2-yl)amino)-5-methylpyrido[2,3d]pyrimidin-7(8H)-one (L2): Yield, 90%. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 2.7 Hz, 1H), 7.33 (d, J = 9.1 Hz, 1H), 5.85 (p, J = 8.9 Hz, 1H), 3.84-3.73 (m, 3H), 3.63 (dt, J = 11.8, 5.0 Hz, 3H), 3.16 (dt, J = 17.1, 4.9 Hz, 4H), 2.80 (t, J = 7.0 Hz, 1H), 2.61 (t, J = 5.1 Hz, 1H), 2.52 (s, 3H), 2.35-2.29 (m, 5H), 2.04 (p, J = 7.5 Hz, 2H), 1.92-1.81 (m, 2H), 1.72-1.58 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 202.76, 170.05, 167.06, 161.48, 158.12, 157.37, 155.61, 145.95, 143.01, 141.88, 137.14, 130.86, 126.98, 113.76, 107.78, 54.26, 50.06, 49.75, 45.29, 41.68, 33.02, 31.61, 28.15, 14.04. HRMS (m/z): calculated for C28H33N8O3 [M+H]+ 529.2670, found 529.2676. IR (KBr)/cm-1:2150.6 (-NC). 6-acetyl-8-cyclopentyl-2-((5-(4-(4-isocyanobutanoyl)piperazin-1-yl)pyridin-2-yl)amino)-5-methylpyrido[2,3d]pyrimidin-7(8H)-one (L3) Yield, 49%. 1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 8.16 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 2.8 Hz, 1H), 7.29 (dd, J = 9.1, 2.9 Hz, 1H), 5.85 (p, J = 8.6 Hz, 1H), 3.78 (t, J = 4.9 Hz, 2H), 3.66 (t, J = 4.5 Hz, 2H), 3.53 (t, J = 6.2 Hz, 2H), 3.14 (dt, J = 14.5, 4.9 Hz, 4H), 2.54 (t, J = 6.8 Hz, 2H), 2.50 (s, 3H), 2.38-2.29 (m, 5H), 2.03 (m, 4H), 1.921.79 (m, 2H), 1.74-1.60 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 202.71, 169.62, 161.44, 158.17, 157.47, 155.61, 145.90, 143.03, 141.97, 137.17, 130.65, 126.59, 113.55, 107.50, 54.33, 49.92, 49.75, 45.23, 41.55, 31.61, 28.89, 28.11, 25.77, 24.37, 14.00. HRMS (m/z): calculated for C29H35N8O3 [M+H]+ 543.2826, found 543.2823. IR (KBr)/cm-1:2146.7 (-NC). 6-acetyl-8-cyclopentyl-2-((5-(4-(5-isocyanopentanoyl)piperazin-1-yl)pyridin-2-yl)amino)-5-methylpyrido[2,3d]pyrimidin-7(8H)-one (L4): Yield, 89%. 1H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.90 (s, 1H), 7.15 (d, J = 10.9 Hz, 1H), 5.77 (p, J = 8.6 Hz, 1H), 3.70 (s, 2H), 3.57 (s, 2H), 3.38-3.37 (m, 2H), 3.13-2.96 (m, 4H), 2.42 (s, 3H), 2.37 (t, J = 6.6 Hz, 2H), 2.29 (m, 2H), 2.24 (s, 3H), 1.98 (s, 2H), 1.78 (s, 2H), 1.77-1.67 (m, 4H), 1.63 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 202.66, 170.66, 161.37, 158.05, 157.60, 155.47, 145.58, 142.79, 142.15, 136.88, 130.11, 125.87, 113.08, 106.94, 54.41, 49.64, 49.46, 45.28, 41.39, 32.04, 31.58, 28.73, 28.02, 25.65, 21.97, 13.87. HRMS (m/z): calculated for ACS Paragon Plus Environment

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C30H37N8O3 [M+H]+ 557.2983, found 557.2988. IR (KBr)/cm-1:2146.7 (-NC). 6-acetyl-8-cyclopentyl-2-((5-(4-(6-isocyanohexanoyl)piperazin-1-yl)pyridin-2-yl)amino)-5-methylpyrido[2,3d]pyrimidin-7(8H)-one (L5): Yield, 61%. 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.28 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 2.8 Hz, 1H), 7.40 (dd, J = 9.2, 3.0 Hz, 1H), 5.85 (p, J = 8.9 Hz, 1H), 3.85-3.77 (m, 2H), 3.70-3.62 (m, 2H), 3.45-3.37 (m, 2H), 3.16 (m, 4H), 2.54 (s, 3H), 2.44-2.38 (m, 2H), 2.36 (s, 3H), 2.35-2.25 (m, 2H), 2.12-2.01 (m, 2H), 1.93-1.83 (m, 2H), 1.71 (m, 6H), 1.53 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 202.67, 171.07, 161.45, 158.12, 157.32, 155.62, 145.79, 143.22, 141.82, 137.10, 130.91, 126.85, 113.71, 107.86, 54.19, 50.17, 49.85, 41.43, 32.87, 31.60, 28.99, 28.15, 26.23, 25.83, 24.27, 14.05. HRMS (m/z): calculated for C31H39N8O3 [M+H]+ 571.3139, found 571.3144. IR (KBr)/cm-1:2146.7 (-NC). Preparation of Re-Ln (n=2, 3, 4, 5) Bromopentacarbonylrhenium(I) ([Re(CO)5Br]) (40 mg, 0.1 mmol) was added to water (2 mL) in a flask, and the mixture was heated to reflux for 24 h. Next, 1 mL (0.5 mmol) of Ln in DMF was added, and the solution was stirred at 90 °C for 6 h. After completion of the reaction, the mixture was filtered through a 0.22 μm microfiltration membrane and then purified by HPLC (system 2) to obtain Re-Ln (n=2, 3, 4, 5). Re-L2: Yield, 13%. 1H NMR (600 MHz, CD3OD) δ 9.03 (s, 1H), 8.15 (dd, J = 9.7, 2.8 Hz, 1H), 7.87 (d, J = 2.9 Hz, 1H), 7.53 (d, J = 9.6 Hz, 1H), 5.97-5.91 (m, 1H), 4.19 (t, J = 6.1 Hz, 2H), 3.83-3.78 (m, 2H), 3.76-3.74 (m, 2H), 3.34-3.33 (m, 2H), 3.29-3.28 (m, 2H), 3.08 (t, J = 6.2 Hz, 3H), 2.45 (s, 3H), 2.37 (s, 3H), 2.30-2.22 (m, 2H), 2.10-2.01 (m, 2H), 1.89-1.84 (m, 2H), 1.67-1.65 (m, 2H). 13C NMR (150 MHz, CD3OD) δ 202.40, 168.02, 161.12, 156.85, 155.83, 155.43, 143.06, 142.56, 141.46, 135.20, 132.91, 127.40, 121.10, 116.46, 110.37, 54.24, 44.42, 43.55, 41.01, 40.49, 36.85, 31.82, 30.06, 27.70, 25.36, 16.22, 12.84. HRMS (m/z): calculated for C87H97N24O12Re [M+H]2+ 928.3636, found 928.3633. IR (KBr)/cm-1: 2056.1, 1990.5 (-Re(CO)). Re-L3: Yield, 10%. 1H NMR (600 MHz, CD3OD) δ 9.04 (d, J = 5.1 Hz, 1H), 8.15 (dq, J = 9.7, 3.0 Hz, 1H), 7.86 (q, J = 3.0 Hz, 1H), 7.53 (dd, J = 9.3, 5.0 Hz, 1H), 5.96 (dt, J = 17.4, 6.0 Hz, 1H), 4.03 (q, J = 6.5 Hz, 2H), 3.79-3.73 (m, 4H), 3.28-3.25 (m, 2H), 2.64 (q, J = 6.5 Hz, 2H), 2.45 (s, 3H), 2.38 (s, 3H), 2.29-2.25 (m, 2H), 2.17-2.14 (m, 2H), 2.08-2.04 (m, 2H), 1.891.86 (m, 2H), 1.68-1.63 (m, 3H), 1.30-1.25 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 202.40, 170.60, 161.13, 156.89, 155.84, ACS Paragon Plus Environment


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155.44, 143.10, 142.60, 141.48, 135.10, 132.90, 127.36, 121.17, 116.41, 110.36, 54.23, 44.59, 44.08, 40.97, 36.85, 30.05, 29.04, 27.70, 25.37, 24.42, 16.21, 12.83. HRMS (m/z): calculated for C90H103N24O12 Re [M+H]2+ 949.3871, found 949.3871. IR (KBr)/cm-1: 2058.1, 1992.5 (-Re(CO)). Re-L4: Yield, 15%. 1H NMR (600 MHz, CD3OD) δ 9.05 (s, 1H), 8.17 (dd, J = 9.8, 2.6 Hz, 1H), 7.88 (s, 1H), 7.54 (d, J = 9.5, Hz, 1H), 5.97 (p, J = 8.8 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.79-3.76 (m, 4H), 3.27-3.26 (m, 2H), 2.59 (t, J = 7.1 Hz, 2H), 2.48 (s, 3H), 2.40 (s, 3H), 2.26-2.32 (m, 2H), 2.11-2.06 (m, 2H), 1.92-1.86 (m, 4H), 1.84-1.79 (m, 2H), 1.71-1.67 (m, 2H), 1.29 (s, 2H). 13C NMR (150 MHz, CD3OD) δ 203.08, 172.43, 161.84, 157.60, 156.57, 156.15, 143.84, 143.32, 142.19, 135.67, 133.63, 130.23, 122.07, 117.09, 111.07, 54.97, 45.31, 45.17, 41.62, 32.10, 30.77, 28.83, 28.43, 27.49, 23.10, 22.38, 13.56. HRMS (m/z): calculated for C93H109N24O12Re [M+H]2+ 970.4108, found 970.4109. IR (KBr)/cm-1: 2061.9, 1986.7 (-Re(CO)). Re-L5: Yield, 12%. 1H NMR (600 MHz, CD3OD) δ 9.05 (d, J = 3.4 Hz, 1H), 8.20-8.12 (m, 1H), 7.86 (d, J = 3.9 Hz, 1H), 7.53 (dd, J = 9.9, 3.9 Hz, 1H), 5.99-5.93 (m, 1H), 3.99-3.96 (m, 2H), 3.78-3.73 (m, 4H), 3.25-3.24 (m, 2H), 2.52-2.50 (m, 2H), 2.47 (s, 3H), 2.39 (s, 3H), 2.30-2.24 (m, 2H), 2.08-2.04 (m, 2H), 1.89-1.85 (m, 4H), 1.72-1.64 (m, 5H), 1.57-1.51 (m, 2H), 1.31-1.26 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 202.39, 172.22, 161.13, 156.90, 155.87, 155.42, 143.15, 142.59, 141.47, 135.19, 132.93, 121.08, 116.41, 116.18, 110.40, 54.24, 44.71, 44.41, 40.88, 32.14, 30.05, 28.37, 27.70, 25.84, 25.38, 23.96, 12.83. HRMS (m/z): calculated for C96H115N24O12Re [M+H]2+ 991.4341, found 991.4337. IR (KBr)/cm-1: 2061.9, 1994.4 (-Re(CO)). CDK kinase inhibition assay The inhibition study of CDK4 and CDK2 was measured for the rhenium analogs (Re-Ln, n = 2-5) along with palbociclib as positive control compound by LANCE Ultra kinase assays kit. The experiment was performed in 384-well assay plate. The kinase reaction was conducted by mixing CDK4/cyclin D1 or CDK2/cyclin A complex, LANCE Ultra ULight-4E-BP1peptide substrate, ATP and different concentration inhibitors in reaction buffer. The mixture was incubated at room temperature for 90 min. EDTA was added to stop the kinase reaction and then the Europium-anti-phosphor specific antibody was added in detection buffer. The mixture was incubated at room temperature for 60 min. The plate was reading by EnVision Reader. The IC50 values were calculated by XLfit 5 software. ACS Paragon Plus Environment

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Radiosynthesis The intermediate [99mTc(CO)3(H2O)3]+ was prepared according to the method published previously.18 The pH of the intermediate was adjusted to 5~6 using 1.0 mol/L HCl, and then 0.5 mg of ligand (L2-L5) in DMF was added. The mixture was heated at 100 °C for 30 min. The 99mTc-labeled complexes were obtained after purification through HPLC (system 1). Stability studies Purified

99mTc-Ln

(n=2, 3, 4, 5) was kept at room temperature for 4 h or incubated in mouse serum at 37 °C for 4 h. The

radiochemical purity was assessed by HPLC (system 1). Determination of the partition coefficient To evaluate the lipophilicity of the 99mTc-labeled complexes, the partition coefficient between 1-octanol and phosphate buffer (0.025 mol/L, pH 7.4) was determined according to the method described previously.19 The final partition coefficient value was expressed as log P ± SD. Metabolic study in vivo The in vivo metabolic study of 99mTc-L4 was assessed as the example of 99mTc-derivatives. A Balb/c mouse was intravenously injected with 4.44 MBq of

99mTc-L4.

After 1 h, the mouse was sacrificed by decapitation. Urine, blood and liver samples

were collected and treated with a published method20 with little modification. In brief, the urine sample was directly diluted with 0.2 mL of saline. The blood sample was centrifuged immediately for 5 min at 9,000 rpm. The supernatant was collected and 0.3 mL of acetonitrile was added. The precipitated protein was removed after centrifugation. The liver sample was washed with saline, and then homogenized in 2 mL of mixture of saline and acetonitrile (v/v, 1/1) by a homogenizer at full speed for 5 min. The resulting homogenate was centrifuged for 5 min at 12,000 rpm. Supernatants were passed through a 0.22 μm millipore filter and then analyzed by HPLC with system 1. Cellular studies Cell culture The MCF-7 cell line (human breast adenocarcinoma cell line) was obtained from the Chinese Infrastructure of Cell Line Resource and cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin in a 5% ACS Paragon Plus Environment


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CO2 humidified incubator at 37 °C. Cells were removed from the flasks by incubation in 0.25% trypsin/EDTA (ethylenediaminetetraacetic acid) for passage or for transfer to 24-well assay plates. Uptake assay Cells were seeded in 24-well plates at a density of 2 × 105 cells/well and allowed to attach overnight. The medium was removed, and the cells were washed with 0.5 mL of medium. Afterwards, the cells were incubated with purified 99mTc-labeled complex (7.4 kBq, 0.1 mL) at 37 °C for 15 min, 30 min, 60 min, 90 min or 120 min. The cells were then washed with icecold PBS (containing 0.2% BSA) 3 times, lysed with 0.5 mL of 1 M NaOH and transferred to γ-counter tubes. Radioactivity was measured by a γ-counter (Wizard 2480, PerkinElmer). The bicinchoninic acid (BCA) assay was performed using the BCA protein assay reagent (M&C Gene Technology Ltd., Beijing, China) to measure the protein content. The experiment was repeated three times. Inhibition assay Cells were seeded in 24-well plates at a density of 2 × 105 cells/well and allowed to attach overnight. The medium was removed, and the cells were washed with 0.5 mL of medium. The cells were incubated with palbociclib (60 μM) or nonradiolabeled ligand Ln (60 μM) for 30 min. Then, purified 99mTc-labeled complex (0.2 μCi, 0.1 mL) was added, and the mixture was incubated at 37 °C for 2 h. The treatment of the cells afterward was the same as that in the uptake assay. The inhibition experiment was repeated three times. Biodistribution experiments Approximately 1 × 107 MCF-7 cells were suspended in 0.1 mL of DMEM medium and implanted in the left hind leg of female Balb/c nude mice by subcutaneous injection. When the tumors were 5-10 mm in diameter (7-10 days), 0.1 mL of purified 99mTc-labeled

radiotracer (74 kBq) was injected intravenously via the tail vein. The mice were sacrificed by decapitation 2 h

post-injection. Tumor, blood and other normal tissues of interest were collected and weighed, and their radioactivity was measured via a γ-counter. The results were expressed as the percent uptake of the injected dose per gram of tissue (% ID/g). For the inhibition study, the mice were pretreated with 100 μg palbociclib for 30 min and then 0.1 mL of purified 99mTc-L4 was injected intravenously. The mice were sacrificed by decapitation 1 h post-injection and the radioactivity of tissues was ACS Paragon Plus Environment

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measured. All animal studies were carried out in compliance with the Regulations on Laboratory Animals of Beijing Municipality. Micro-SPECT/CT imaging Purified

99mTc-L4

(7.4 MBq, 0.1 mL) was injected intravenously into a Balb/c nude mouse bearing an MCF-7 tumor. The

mouse was anesthetized with 1.5% isoflurane in air at 500 mL/min, and at 30 min, 60 min and 120 min, a SPECT/CT scan was acquired. Two days later, to confirm the in vivo CDK4/6-specific binding, 0.1 mL of palbociclib (1 mg/mL) and 0.1 mL of purified 99mTc-L4 (7.4 MBq) were injected intravenously into the same mouse, and a SPECT/CT scan was acquired 1 h post-injection by means of the same method. The SPECT acquisition (Peak 140 keV, 20% width, no rotation, MMP 930 collimator) was first carried out for 20 min, followed by a CT scan (512 views, 2 × 2 binding, 75 kV, exposure time 230 ms) for 4 min. The SPECT/CT images were acquired using the HiSPECT software and the vivoquant 2.5 software.

Results and Discussion Synthesis and radiosynthesis The pyrido-[2,3-d]pyrimidin-7-ones ring of palbociclib was proved to be critical for specificity and activity, for there were two hydrogen bonds between the N3 and N2-H and the ATP binding pocket. The nitrogen of the aminopyridine side chain and acetyl group were also important to form additional H-bonds for the specificity of palbociclib. It seemed that the piperazine group was modifiable due to similar binding affinity of replacing the piperazine with a variety of heterocyclic groups21, 22. Furthermore, a compound modified at the tail piperazine ring had lower IC50 value toward MCF-7 than palbociclib did23. Therefore, we chose this position to conjugate with a bifunctional chelator for

99mTc

labeling. Isonitrile has strong

coordination ability toward 99mTc. Besides, Mizuno et al.17 developed a convenient method to isocyanide-containing active esters. The ligands (L2-L5) were designed based on the concept of the “coupling method” and synthesized by reacting palbociclib with isocyanide-containing active esters. The reaction route of ligands (L2-L5) and metal complexes is shown in Scheme 1. Isocyanide-containing active esters (3a-3d) were obtained through three steps according to literature reported previously17, 24 and then treated with palbociclib in the presence of triethylamine to produce ligands (L2-L5), respectively. The ligands were characterized by IR, 1H NMR, 13C NMR and HRMS. 99mTc-labeled complexes were formed by reacting the corresponding ACS Paragon Plus Environment


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ligand with the [99mTc(CO)3(H2O)3]+ intermediate at pH 5~6 at 100 °C for 30 min and then purified via radio-HPLC. The radiochemical yields of the

99mTc-labeled

complexes were over 70%, and the radiochemical purities were more than 95%

after radio-HPLC purification. The Re complexes were obtained by reacting the ligands with the [Re(CO)3(H2O)3]+ intermediate and characterized by 1H NMR, 13C NMR, HRMS and IR. Identification of the

99mTc-labeled

complexes was

achieved by comparing their HPLC retention time with those of the corresponding rhenium analogs (Figure 1). The matched HPLC retention time of the 99mTc-labeled complex and the corresponding Re complex provided evidence for the certification of the structure.

Scheme 1. Structure and synthetic route of 99mTc/Re-Ln (n=2-5). Reagents and conditions: (a) formic acid, acetic anhydrate, 95 oC, 3 h; (b) TFP, DCC, DMF, r.t., overnight; (c) Burgess reagent, DCM, r.t., overnight; (d) TEA, DCM, r.t., 4 h; (e) [Re(CO)3(H2O)3]+, 90 oC, 3 h; (f) [99mTc(CO)3(H2O)3]+, pH = 5~6, 100 oC, 30 min.

ACS Paragon Plus Environment

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Figure 1. Comparative HPLC analysis of Re-L2 and 99mTc-L2 (a), Re-L3 and 99mTc-L3 (b), Re-L4 and 99mTc-L4 (c), and Re-L5 and 99mTc-L5 (d).

CDK kinase inhibition assay The IC50 values of rhenium analogs and positive control compound to CDK4 and CDK2 are shown in Table 1. The result showed that the complexes Re-Ln (n=2-5) had high affinity to CDK4, and the affinity decreased with the length of carbon chains. In addition, all of the four complexes showed higher CDK4 selectivity over CDK2. The IC50 values of palbociclib are consistent with the data reported previously. Table 1. The IC50 values of rhenium analogs and palbociclib

IC50 (nM)

Compound Re-L2 Re-L3 Re-L4 Re-L5 palbociclib

CDK4

CDK2

89.7 142.1 187.5 197.2 2.5

> 2000 > 2000 > 2000 > 2000 1110

Stability assessment and partition coefficient After maintaining at room temperature for 4 h or incubating in mouse serum at 37 °C for 4 h, the radiochemical purities of ACS Paragon Plus Environment


the

99mTc-labeled

Page 12 of 21

complexes were still over 95% (Figure 2), which showed that the complexes exhibited good stability in

vitro. As estimated by measuring the partition coefficient between 1-octanol and PBS (pH 7.4), the log P values of the 99mTclabeled tracers were between 1.476 and 1.681 (Table 2), which demonstrated that these 99mTc-labeled tracers were lipophilic. Additionally, the HPLC retention time of the 99mTc-labeled complexes was positively related to the log P value and the length of the carbon chain between the isocyanide group and the target vector. Compounds with a shorter carbon chain tended to exhibit a lower log P value and a shorter HPLC retention time. Table 2. The partition coefficient values of 99mTc-labeled tracers and their corresponding radio-HPLC retention times

Compound

n

Log P

Retention time (min)

99mTc-L2

2 3 4 5

1.476 ± 0.022 1.520 ± 0.034 1.616 ± 0.066 1.681 ± 0.083

18.817 19.117 19.200 19.433

99mTc-L3 99mTc-L4 99mTc-L5

Figure 2. Stability results for 99mTc-L2 (black), 99mTc-L3 (red), 99mTc-L4 (green), and 99mTc-L5 (blue) after being maintained at room temperature (a) or incubated in mouse serum at 37 °C (b) for 4 h.

Metabolic study in vivo The result of metabolic study in vivo of 99mTc-L4 was shown in Figure 3. 99mTc-L4 kept intact in mouse blood and urine. In mouse liver, there was one metabolite shown in the HPLC profile, indicating the hepatic metabolism of the complex.


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Figure 3. HPLC profiles of metabolite of 99mTc-L4 in blood (a), urine (b), and liver (c) 1 h post-injection.

Cell studies The cell studies were carried out on CDK4/6 overexpressed MCF-7 cell line.25, 26 In vitro cell uptake studies showed that all four 99mTc-labeled tracers exhibited substantial uptake in MCF-7 tumor cells, and the uptake increased over time from 15 to 120 min (Figure 4). Among the four 99mTc-labeled tracers, 99mTc-L3 showed the highest cellular uptake (98.97 ± 1.14% ID/mg protein at 15 min and 200.29 ± 15.90% ID/mg protein at 120 min). Inhibited by palbociclib and the corresponding nonradiolabeled ligand, the cellular uptake of all the CDK4/6-specific uptake mechanism. When

99mTc-labeled

tracers decreased significantly (Figure 5), indicating a

99mTc-L2, 99mTc-L3, 99mTc-L4

and

99mTc-L5

were pretreated with palbociclib,

their cell uptake decreased by 55.0%, 58.8%, 23.0% and 27.8%, respectively. When 99mTc-L2, 99mTc-L3, 99mTc-L4 and 99mTcL5 were pretreated with the corresponding unlabeled ligand, their cell uptake decreased by 49.7%, 64.0%, 66.3% and 67.1%, respectively.

Figure 4. MCF-7 in vitro cell uptake assay performed by incubating MCF-7 cells (2 × 105 cells/well in 24-well plates) with radio-HPLC purified 99mTc-L2

(black squares),

99mTc-L3

(red spheres),

99mTc-L4

(blue triangles) or

99mTc-L5

(green triangles) at 37 °C over 15, 30, 60, 90 and 120



Page 14 of 21

min. The protein content in each well was measured by the BCA assay. The results are expressed as the percent uptake of the injected dose per milligram protein ± the standard deviation of triplicate wells.

Figure 5. In vitro uptake by MCF-7 cells incubated with 99mTc-labeled tracers alone (black column) at 37 °C over 120 min, with 20 μM palbociclib (light gray column) or with 20 μM corresponding ligand (deep gray column) to block CDK4/6 binding. Statistical analysis was carried out using a bilateral t test of equal variance (* P < 0.05).

Biodistribution experiments The biodistribution results of the 99mTc-labeled tracers in Balb/c nude mice bearing MCF-7 tumors at 2 h post-injection are shown in Table 3. All four 99mTc-labeled complexes had high tumor accumulation and 99mTc-L2 exhibited the highest tumor uptake (2.69 ± 0.26% ID/g). Regarding the target/nontarget ratio, 99mTc-L2 showed the highest tumor/blood ratio, and 99mTcL4 showed the highest tumor/muscle ratio. In the case of nontarget tissues, all four complexes showed high uptake in the liver, indicating that the metabolism of the complexes was through a hepatobiliary pathway. In addition, from 99mTc-L5,

99mTc-L2

to

the liver uptake increased (49.07 ± 3.20% ID/g and 92.46 ± 11.58% ID/g for 99mTc-L2 and 99mTc-L5, respectively),

and the kidney uptake decreased (12.69 ± 0.92% ID/g and 6.41 ± 1.60% ID/g, respectively), which was in accordance with ACS Paragon Plus Environment

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the lipophilicity of the radiotracers. Considering that high liver uptake may have a negative effect on tumor imaging, the development of radiotracers with lower lipophilicity might be the main focus of a subsequent study. The possible approaches might be introducing an oligoethyleneoxy linker between the target group and the chelator or changing the bifunctional chelator to others that can be labeled by 99mTc with some hydrophilic co-ligands. The uptake in the thyroid and stomach of the 99mTc-labeled complexes was low, suggesting that no 99mTcO4- was present in the mice after injection of the 99mTc-labeled complexes. Table 3. Biodistribution of 99mTc-labeled tracers in Balb/c nude mice bearing MCF-7 tumors at 2 h p.i. (% ID/g ± SDa, n = 3)

a

99mTc-L2

99mTc-L3

99mTc-L4

99mTc-L5

Heart

2.29 ± 0.34

1.80 ± 0.26

1.79 ± 0.39

6.08 ± 4.50

Liver

49.07 ± 3.20

51.05 ± 4.63

62.1 ± 6.71

92.46 ± 11.58

Lung

6.01 ± 0.65

5.21 ± 0.83

4.84 ± 0.70

5.10 ± 0.96

Kidneys

12.69 ± 0.92

11.76 ± 2.37

10.97 ± 1.48

6.41 ± 1.60

Spleen

7.05 ± 0.76

4.30 ± 0.84

9.88 ± 1.39

24.52 ± 4.43

Stomach

1.59 ± 0.61

1.51 ± 0.51

1.95 ± 1.79

2.31 ± 1.10

Bone

2.04 ± 0.43

0.92 ± 0.28

1.13 ± 0.43

1.85 ± 0.64

Muscle

0.88 ± 0.13

0.45 ± 0.09

0.38 ± 0.10

0.38 ± 0.13

Intestine

3.93 ± 0.86

3.18 ± 0.90

1.92 ± 0.76

2.90 ± 0.90

Tumor

2.69 ± 0.26

1.02 ± 0.12

1.43 ± 0.10

1.17 ± 0.06

Blood

6.38 ± 1.15

3.51 ± 0.86

5.52 ± 0.92

5.80 ± 0.70

Thyroidb

0.05 ± 0.01

0.02 ± 0.01

0.03 ± 0.01

0.03 ± 0.02

T/Bc

0.42

0.29

0.26

0.20

T/Md

3.06

2.27

3.76

3.08

SD is standard deviation; b The uptake in the thyroid is expressed as % ID; c T/B = tumor-to-blood ratio; d T/M = tumor to muscle ratio.

The result of in vivo inhibition study was shown in Figure 6. When pretreated with palbociclib, the tumor uptake of complex 99mTc-L4

decreased significantly, indicating the specificity of 99mTc-L4 to CDK4/6.



Page 16 of 21

Figure 6. Biodistribution of 99mTc-L4 without and with palbociclib in Balb/c nude mice bearing MCF-7 tumors at 1 h p.i. (* P < 0.05).

Micro-SPECT/CT imaging Since 99mTc-L4 showed the highest tumor/muscle ratio among the four radiotracers, it was selected for the Micro-SPECT/CT imaging study. Micro-SPECT/CT images were obtained on a Balb/c nude mouse bearing an MCF-7 tumor at 30 min (Figure 7A), 60 min (Figure 7B) and 120 min (Figure 7C) after intravenous injection of

99mTc-L4.

The images were expressed as

sagittal plane (a), coronal plane (b) and transverse plane (c). From these images, we can see that there was significant accumulation in the tumor at 30 min post-injection. Up to 120 min post-injection, tumor accumulation was still evident, suggesting good tumor retention of 99mTc-L4. The high abdomen uptake was in consistent with the result of biodistribution study, from which we could see the high uptake of complex

99mTc-L4

in liver as well as in kidneys and spleen. When

coinjected with palbociclib, radioactivity accumulation in the tumor was clearly absent (Figure 8B), and the ROI value decreased to 0.33 ± 0.05 from 3.91 ± 0.85 (without palbociclib), providing more evidence for the specificity for CDK4/6. To our knowledge, few studies have investigated radiolabeled CDK4/6 inhibitors as tumor imaging agents. In 2010, Lena Koehler et al reported two compounds ([124I]CKIA and [124I]CKIB) and evaluated their potential for tumor imaging.15 However, the results showed that [124I]CKIA and [124I]CKIB had low tumor uptake from small animal PET and autoradiography studies. The increasing thyroid uptake from 5 min to 60 min of [124I]CKIA and [124I]CKIB indicated ACS Paragon Plus Environment

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radioiodination in vivo, which showed the limitation of these compounds as tumor imaging agents. In contrast, the

99mTc-

labeled complexes in this work showed tumor uptake from the biodistribution and Micro-SPECT/CT studies, and no 99mTcO4appeared in the mice after injection of the complexes.

Figure 7. Micro-SPECT/CT images of 99mTc-L4 in a Balb/c nude mouse bearing an MCF-7 tumor at 30 min (A), 60 min (B) and 120 min (C) post-injection. The images are shown as sagittal plane (a), coronal plane (b) and transverse plane (c).



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Figure 8. SPECT/CT images of 99mTc-L4 without (A) or with (B) palbociclib (100 μg) in a Balb/c nude mouse bearing an MCF-7 tumor at 60 min post-injection. The images are shown as sagittal plane (a), coronal plane (b) and transverse plane (c).

Conclusion In this study, four isocyanide-containing palbociclib derivatives were successfully synthesized and radiolabeled with [99mTc(CO)3(H2O)3]+ intermediate with high radiochemical purity. These 99mTc-labeled complexes exhibited lipophilicity and good stability in vitro. The results of the kinase inhibition assay demonstrated that the corresponding Re complexes had high affinity for CDK4. A cell study using MCF-7 tumor cells showed that the uptake of these 99mTc-labeled complexes could be blocked by palbociclib and the corresponding non-radiolabeled ligand, indicating that the uptake was CDK4/6 specific. Biodistribution studies in nude mice bearing MCF-7 tumors showed that the

99mTc-labeled

accumulation at 2 h post-injection. The micro-SPECT/CT study showed that complex

complexes had obvious tumor

99mTc-L4

had visible uptake in the

tumor site and that the accumulation could be blocked significantly by palbociclib. These results demonstrated that the 99mTclabeled complexes in this work might have the potential for the tumor image targeting of CDK4/6.


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Supporting information Details of chemical synthesis routes, characterization of isocyanide-containing active esters and Ex vivo autoradiography

Author Information Corresponding Author *[email protected]

ORCID: Junbo Zhang: 0000-0003-3549-6483 Present Addresses No. 19 Xinjiekou Wai Boulevard, Haidian District, Beijing. College of Chemistry, Beijing Normal University, 100875, P. R. (China).

Author Contributions † Xiaoqing Song and Qianqian Gan have contributed equally to this work. Notes The author(s) confirm that this article content has no conflicts of interest.

Acknowledgments The work was financially supported by the National Natural Science Foundation of China (21771023) and the project of the Beijing Municipal Science and Technology Commission (Z181100002218033).

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Table of Content graphic

99mTc-L4

had obvious accumulation in the tumor site on the micro-SPECT image and the uptake could be blocked by palbociclib, suggesting it would be a promising tumor imaging agent to target CDK4/6.


Synthesis and Biological Evaluation of Novel 99mTc-Labeled

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