Douglas D. Dischino

Synthesis and evaluation of candidate PET radioligands for corticotropin-releasing factor type-1 receptors

INTRODUCTION A radioligand for measuring the density of corticotropin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)-pyrazin-2(1H)-one scaffold. METHODS CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF1 receptors (displacement of [(125)I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF1 receptor ligand, [(3)H]BMS-728300. RESULTS The three ligands selected for development showed high binding affinity (IC50 values, 0.3-8nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8-4.4). [(3)H]BMS-728300 and the two (18)F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50-120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [(18)F]BMS-721313, [(18)F]BMS-732098 and [(11)C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro. CONCLUSIONS Candidate CRF1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF1 receptors.

Synthesis of gadolinium (±)-10-(1-hydroxypropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyltriacetate via tribenzyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate

The synthesis of monogadolinium 10-(1-hydroxypropan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyltriacetate 12 has been achieved through a multistep sequence. The key step in the synthesis was the reaction of the previously unknown tribenzyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate 6 with ethyl 2-(trifluoromethylsulphonyloxy)propionate 4 to give 7. Reduction of 7 with LiBH4/9-OMe-BBN, followed by hydrogenolysis of the protecting groups (Z groups) yielded 2(1,4, 7,10-tetraazacyclododec-1-yl)propanol 10. The monosubstituted macrocycle 10 was alkylated with bromoacetic acid and then complexed with gadolinium oxide to yield the title compound.

Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti–PD-L1 adnectin after 18F-fluorine labeling. Methods: An anti–PD-L1 adnectin was labeled with 18F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generate 18F-BMS-986192. 18F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1–negative (PD-L1(–)) and PD-L1–positive (PD-L1(+)) subcutaneous tumors. 18F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. Results: 18F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled with 18F to yield a PET radioligand for assessing PD-L1 expression in vivo. 18F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, P < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(–)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti–PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1–specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1–rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E–01 mSv/MBq. Conclusion: 18F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies with 18F-BMS-986192 are under way to measure PD-L1 expression in human tumors.

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [(18)F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.

Synthesis of the monosodium salt of carbon-14 labeled paclitaxel (Taxol®) 2'-ethyl carbonate 7-phosphonooxymethyl ether, a potential prodrug of paclitaxel

The monosodium salt of Carbon-14 labeled paclitaxel (Taxol®) [N3′-14COPh] 2′-ethyl carbonate 7-phosphonooxymethyl ether, was prepared from C-14 labeled paclitaxel [N3′-14COPh] in 5 steps. The radiochemical purity of the final product was greater than 99% and the specific activity was 25 μCi/mg.

Synthesis of 3H-labeled 2-hydroxy-N-[(1,3,3-trimethyl-[4,5,6-3H]cyclohexyl)methyl]-5-azidobenzamide, a photoaffinity analog of an influenza fusion inhibitor

Synthesis of 3 H-labeled 2-hydroxy-N-[(1,3,3-trimethyl-[4,5,6- 3 H]cyclohexyl)methyl]-5-azidobenzamide, a photoaffinity analog of an influenza fusion inhibitor, is reported. Tritiation of a mixture of N-(t-butoxycarbonyl)-1,3,3-trimethylcyclohex-4 (or 5)-enylmethylamine via T 2 and Pd/C, followed by coupling of the deprotected tritiated amine with acetyl 5-azidosalicylic acid chloride yielded the penultimate product. Subsequent deprotection and normal phase HPLC purification yielded the target compound with a radiochemical purity > 99% and a specific activity of 63 Ci/mmol.

Lead pig for use with screw cap vials.

A lead pig specifically designed for use with solid screw cap vials is described. Methods: The salient features of this design include a compressible polyethylene sleeve, which is housed in the lower section of the pig, and a partially recessed O-ring, which is housed in the upper section of the pig. These features permit both the vial and the cap to be secured independently without the risk of overtightening. Fingertip radiation exposure associated with repetitively opening and closing a screw cap vial containing a sample of 18F was monitored by use of a fingertip dosimeter. Results: The cumulative fingertip radiation exposure resulting from opening and closing a screw cap vial containing 200 MBq of 18F-FDG 3 times was 307 μSv without the aid of this lead pig, as compared with 6 μSv when using this device. Conclusion: Use of this lead pig with screw cap vials can significantly reduce fingertip radiation exposure and decrease the likelihood of accidental radioactive contamination of research personnel.

Stereoselective reduction via lithium borotritide: synthesis of3H-labeled 2-hydroxy-N-[(5-hydroxy-[5-3H]-1,3,3-trimethylcyclohexyl)methyl]-5-methylbenzamide

3H-labeled 2-hydroxy-N-[(5-hydroxy-[5-3H]-1,3,3-trimethyl-cyclohexyl)methyl]-5-methylbenzamide, 1, was prepared in one step by the reduction of the corresponding ketone with high specific activity LiBT4. The reduction was stereoselective yielding the desired cis alcohol in a ratio of >20 : 1. Purification of the compound was achieved on normal-phase HPLC. The radiochemical purity of the final product was greater than 99.5% and the specific activity was 29 Ci/mmol. Copyright