Radiosynthesis and characterization of a carbon-11 PET tracer for receptor-interacting protein kinase 1

Abstract

Introduction:

Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a Ki of 0.91 nM that can suppress necroptosis in mouse and human cells. To develop a radiotracer for investigating the RIPK1 in vivo, we radiosynthesized [¹¹C]TZ7774 and performed preliminary in vitro and in vivo evaluations in rodents and macaque.

Methods:

Synthesis of the demethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [¹¹C]TZ7774 was achieved through TZ7790 reacting with [¹¹C]methyl iodide via N-methylation. Ex vivo biodistribution of [¹¹C]TZ7774 was performed in normal Sprague-Dawley rats. Characterization of [¹¹C]TZ7774 in response to inflammation was performed using ex vivo biodistribution study in normal and LPS treated (10mg/kg) C57BL/6 mice, and in vitro autoradiography and immunohistochemistry in the spleen. MicroPET brain study of [¹¹C]TZ7774 in the macaque was also performed.

Results and Conclusions:

The radiosynthesis of [¹¹C]TZ7774 was achieved with good radiochemical yield (30–40%, decay corrected to the end of bombardment (EOB)), high chemical purity (>90%), high radiochemical purity (>99%), and high molar activity (>207 GBq/μmol, decay corrected to EOB). Distribution studies in Sprague-Dawley rats showed [¹¹C]TZ7774 has a high brain uptake of 0.53 (%ID/gram) at 5 min post injection; pancreas, spleen, kidney, and liver also showed a relatively high initial uptake of 0.49, 0.41, 0.62, and 0.95 at 5 min respectively. Uptake of [¹¹C]TZ7774 increased in LPS treated C57BL/6 mice by 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. In vitro autoradiography study also revealed increased uptake of [¹¹C]TZ7774 in the spleen of LPS treated mice. Further characterization with immunohistochemistry confirmed increased expression of RIPK1 in red and white pulp in the spleen of mice pre-treated with LPS. MicroPET demonstrated that [¹¹C]TZ7774 had good initial brain uptake in macaque with an (SUV) of ~3.7 at 6–10 min, and quickly washed out from brain. These data confirm successful radiosynthesis of a RIPK1 specific radiotracer [¹¹C]TZ7774. Our preliminary studies showed good response to LPS-induced inflammation in rodents and good uptake in macaque brain. [¹¹C]TZ7774 has a potential to image RIPK1 related necroptosis and inflammatory processes.

Graphical abstract

1. Introduction

Necroptosis is a regulated form of necrosis that mediates inflammatory or lytic cell death and can trigger an innate immune response. It is a regulated cell death controlled by the upstream kinase activity of receptor-interacting protein kinase 1 (RIPK1) and its downstream mediators can be activated in apoptosis-deficient conditions [1]. RIPK1 is a crucial mediator of cell survival, cell death programming, and inflammatory signaling. Emerging evidence suggests that RIPK1 is involved in both apoptosis and necroptosis signal regulation of inflammatory cytokines. It plays an essential role in a variety of pathological and physiological processes [2]. In RIPK1 dependent necroptosis and inflammatory signaling, the carboxyl-terminus of RIPK1 contains the death domain that interacts with tumor necrosis factor receptor 1 (TNFR1) and triggers the assembly of complex Ι, promoting the expression of a wide range of prosurvival and proinflammatory cytokines through nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling [3–5]. On the other hand, internalization and recruitment of Fas-associated death domain (FADD), caspase-8, and RIPK3 to the RIPK1-containing Complex Ι can trigger the subsequent formation of cytoplasmic complex ΙΙ, driving cell death signaling. In Complex II, when caspase-8 is inhibited, RIPK1 can activate RIPK3 and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) to disrupt the integrity of plasma membrane and thus induce necroptosis. In contrast, when caspase-8 is activated, RIPK1 in complex I can be converted into a highly insoluble form and activate RIPK1-dependent apoptosis [6–8]. Due to its essential role in both apoptosis and necroptosis, as well as the regulation of inflammatory cytokines, RIPK1 dysfunction can contribute to a wide range of human diseases including immune and inflammatory diseases, neurodegenerative diseases, and cancers [7, 9–11]. Inhibition of the RIPK1 has emerged as a promising therapeutic strategy for treating these diseases.

To date, several lead RIPK1 inhibitors have been reported as shown in Figure 1. Nec-1 (1) and its improved analog Nec-1s (2) were the first reported small molecule RIPK1 inhibitors and were widely used to investigate the RIPK1 functions in different disease models [7, 12, 13]. However, their micromolar potency (EC₅₀ = 0.49 μM vs. 0.21 μM), poor metabolic stability (t_1/2 < 5 min) and off-target binding activities narrow their therapeutic potential [14]. Investigators focused on optimizing lead structures Nec-1 (1) or Nec-1s (2), explorations of diversified structural compounds, and pharmacological characterizations of these new analogues [15–19]. The pharmaceutical company, GlaxoSmithKline plc (GSK), first reported a potent lead compound 4 that contained 5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one pharmacophore with an IC₅₀ value of 1.0 nM for binding toward RIPK1, low lipophilicity (log D = 3.8), and good solubility (~450 μM at pH 7.4) [18]. Currently, compound 4 is in phase II clinical trials for patients with chronic immune inflammatory disorders, including active ulcerative colitis (NCT02903966) and psoriasis (NCT02776033). Further optimization of compound 4 structure by replacing the 3-(S)-N-1H-(5-benzyl-1,2,4-triazole-3-carboxamide group using 3-((S)-2-benzyl-3-chloro-7-oxo-4,5,6,7-tetrahydro-2H-indazol-6-yl) group and introducing a carbonitrile group at 8 position of 5-methyl-4-oxo-2,3,4,5- tetrahydrobenzo[b][1,4]oxazepine group, led to compound 5. Pharmacological characterizations reveal that compound 5 is highly potent for RIPK1 (Ki = 0.91 nM) and can penetrate the blood brain barrier to inhibit RIPK1 kinase after oral administration. It significantly suppressed necroptotic cell death in both mouse and human cells [19]. In addition, compound 5 showed promising specificity for RIPK1 kinase over 406 kinases tested using a reaction biology corporation kinase panel [19]. Future clinical trial studies will determine if compound 5 has therapeutic potential. Positron emission tomography has been used widely in clinical practice and preclinical/clinical research to quantitatively measuring protein expression changes in tissues in living animals and humans. Therefore, a suitable RIPK1 PET radiotracer may provide a unique tool to investigate pathophysiological functions of RIPK1 in diseases and help to evaluate the therapeutic efficacy of different strategies to inhibit RIPK1.

Fig. 1.

Structures of selected RIPK1 inhibitors.

However, PET imaging quantification of RIPK1 has been limited by a lack of suitable RIPK1 PET radiotracers. Recently, Dr. Wang and his colleagues reported radiosynthesis of [¹⁸F]CNY-07 (3) through replacement of the chlorine with fluorine-18 in the aromatic moiety of Nec-1s (2). They also performed a preliminary evaluation of [¹⁸F]CNY-07 (3) in rodents, but have not yet demonstrated that [¹⁸F]CNY-07 could be a suitable radiotracer for imaging RIPK1 in vivo for human [20]. Compound 5 is highly potent for RIPK1 (Ki = 0.91 nM), and its structure contains a methylamine group. Therefore, our group used N–methylation of its demethyl precursor reacting with [¹¹C]methyl iodide to radiosynthesize [¹¹C]5, renamed as [¹¹C]TZ7774. We also performed preliminary evaluations of [¹¹C]TZ7774 in normal and LPS-treated mice and performed microPET brain imaging of [¹¹C]TZ7774 in a macaque. Our results indicated that [¹¹C]TZ7774 has excellent potential as a PET radiotracer for imaging RIPK1 in vivo.

2. Materials and methods

2.1. General

All reagents and solvents (ACS or HPLC grade) were purchased from Sigma-Aldrich (St Louis, MO, USA) and used as received unless otherwise stated. ¹H (400 MHz) and ¹³C (100 MHz) NMR spectra were recorded at room temperature on a 400 MHz Varian spectrometer. Chemical shifts are reported in δ units (ppm) downfield relative to the chemical shift of tetramethylsilane (TMS). Signals are quoted as s (singlet), d (doublet), dd (double doublet), dt (double triplet), t (triplet), q (quartet) or m (multiplet). High-resolution mass spectra (HRMS, m/z) were acquired with Bruker MaXis 4G Q-TOF mass spectrometer with an electrospray ionization source.

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals in Washington University and approved by the Institutional Animal Care and Use Committee (IACUC) of Washington University in St. Louis, Missouri, USA. All animal studies were conducted in the Preclinical Imaging Facility and the nonhuman primate microPET facility at the Washington University School of Medicine in St. Louis.

2.2. Chemistry

2.2.1. O-(5-bromo-2-nitrophenyl)-N-(tert-butoxycarbonyl)-L-serine (6).

To the suspension of NaH (60%, 1.818 g, 120 mmol) in dimethylformamide (DMF) (70 mL) was added a solution of (S)-2-((tertbutoxycarbonyl)amino)-3-hydroxypropanoic acid (commercially available, 4.66 g, 22.7 mmol) in DMF (20 mL) at 0 °C over 20 minutes. After stirring at 0 °C for 1 h, 4-bromo-3-nitrobenzonitrile (commercially available, 5.0 g, 22.7 mmol) was added to the reaction mixture at 0 °C. The mixture was stirred at 0 °C for 2 h and then stirred at room temperature over two days. The mixture was neutralized using ice-water and 1 M HCl (50 mL) at 0 °C and then extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and dried over MgSO₄. After filtration, the organic solution was concentrated under reduced pressure to give the crude compound 6 as white solid (11.3 g, 81% yield), which was used for the next step reaction without further purification. HRMS (ESI) calcd for C₁₄H₁₆BrN₂O₇ [M - H]⁺ 403.0146. Found [M - H]⁺ 403.0149.

2.2.2. O-(2-amino-5-bromophenyl)-N-(tert-butoxycarbonyl)-L-serine (7).

A mixture of 6 (6.1 g, 15.0 mmol), zinc (5 g) in Acetic acid (20 mL) was stirred at 0 °C for 3 h. The reactant mixture was filtered through Celite, and then concentrated under reduced pressure to give the crude 7 as a black solid (6.9 g, >99% yield), which was used for the next reaction without further purification. LC-MS calcd for C₁₄H₁₈BrN₂O₅ [M - H]⁺ 373.1. Found [M - H]⁺ 372.9.

2.2.3. Tert-butyl (S)-(8-bromo-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-carbamate (8).

Triethylamine (TEA) (1.12 mL, 11.0 mmol) was added to a mixture of 7 (3.75 g, 10.0 mmol) and Hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (4.19 g, 11.0 mmol) in Dimethyl sulfoxide (DMSO) (40 mL) at room temperature. The mixture was stirred at room temperature for 1 h. The reactant mixture was diluted with water, and the precipitate was collected by filtration and washed with water (3 × 10 mL). The solid was dissolved in EtOAc. The solution was dried using MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, Hexane/AcOEt: 10/1 to 3/1) to give 8 (1.49 g, 39%) as a light-brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 1.32 (9H, s), 4.21−4.35 (3H, m), 6,99 (1H, d, J= 9.1 Hz), 7.11 (1H, d, J= 7.6 Hz), 7.27−7.32 (2H, m), 9.98 (1H, s). HRMS (ESI) calcd for C₁₄H₁₇BrN₂O₄Na [M + Na]⁺ 379.0264, found: 379.0261.

2.2.4. (S)-3-amino-8-bromo-5-methyl-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (9).

Cesium carbonate (1.35 g, 4.15 mmol) was added to a solution of 8 (1.06 g, 2.97 mmol) in DMF (10 mL). Then iodomethane (0.223 ml, 3.56 mmol) was added and the reaction was stirred for 3 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, and concentrated under reduced pressure. The solid was crystallized from EtOA to give 9 (489 mg, 45%) as a colorless solid. ¹H NMR (400 MHz, DMSO-d6) δ 1.37 (9H, s), 3.35 (3H, s), 4.28−4.57 (3H, m), 7.40−7.42 (2H, m), 7.46−7.49 (1H, m). HRMS (ESI) calcd for C₁₅H₁₉BrN₂O₄Na [M + Na]⁺ 393.0420, found: 393.0422.

2.2.5. (S)-3-amino-8-bromo-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (10).

To a 50 mL round-bottom-flask was added 9 (530 mg, 2.0 mmol), Trifluoroacetic acid (5 mL) and dichloromethane (10 mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 24 hours. After the reaction was completed, the solvent was removed by high vacuum to give 10 as a light-brown solid (187 mg, 37% yield). This product was directly used for the next step reaction without further purification. HRMS (ESI) calcd for C₁₀H₁₂BrN₂O₂ [M + H]⁺ 271.0077, found: 271.0079.

2.2.6. (S)-3-amino-8-bromo-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (11).

11 was obtained (121 mg, 33%) as a red solid using similar procedure of making compound 10. ¹H NMR (400 MHz, DMSO-d6) δ 4.37 (m, 2H). 4.53 (m, 1H), 7.06 (d, J = 8.6 Hz, 1H), 7.32 (m, 1H), 7.36 (m, 1H), 8.40 (s, 2H), 10.55 (s, 1H); ¹³C NMR (100 MHz, DMSO-d6) δ 51.05, 71.60, 116.93, 124.39, 125.08, 127.85, 129.16, 149.28, 168.27. HRMS (ESI) calcd for C₉H₁₀BrN₂O₂ [M + H]⁺ 256.9921, found: 256.9920.

2.2.7. Ethyl 1-benzyl-5-hydroxy-1H-pyrazole-3-carboxylate (12).

Added diethyl oxaloacetate sodium salt (commercially available, 21.0 g, 100 mmol) to a mixture of benzylhydrazine dihydrochloride (commercially available, 15.9 g, 100 mmol) and potassium carbonate (20.7 g, 150.0 mmol) in ethanol (EtOH) (600 mL). The reaction mixture was stirred at 90 °C overnight. After stirring at room temperature for 2 days, the mixture was acidified using 6 M HCl (70 mL) at 0 °C and extracted with EtOAc (300 mL×2). The combined organic layer was washed using brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residual solid was triturated with MeCN (50 mL), and the collected solid was washed using acetonitrile (MeCN, 20 mL) to give 12 (8.12 g, 33%) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 1.21 (3H, t, J = 7.1 Hz), 4.17 (2H, q, J = 7.1 Hz), 5.13 (2H, s), 5.80 (1H,s), 7.14−7.29 (5H, m), 11.59 (1H, s). HRMS (ESI) calcd for C₁₃H₁₅N₂O₃ [M + H]⁺ 247.1078, found: 247.1075.

2.2.8. Ethyl 1-benzyl-5-chloro-4-formyl-1H-pyrazole-3-carboxylate (13).

To a solution of 12 (6.16 g, 25.0 mmol) in DMF (15 mL) was drop wisely added phosphoryl trichloride (30.6 mL, 200 mmol) at room temperature. The reaction mixture was stirred at 90 °C for 7 h. The solvent was removed by rotary evaporation under reduced pressure. The residue was added to saturated NaHCO₃ aqueous solution at 0 °C and extracted with EtOAc. The organic layer was separated, washed using brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column with eluting mobile phase of 20−35% EtOAc in hexane to give 13 (2.27 g, 31%) as a pale-yellow solid. ¹ H NMR (400 MHz, CDCl₃) δ 1.40 (3H, t, J = 7.1 Hz), 4.45 (2H, q, J = 7.1 Hz), 5.42 (2H, s), 7.27 (5H, m), 10.39 (1H, s). HRMS (ESI) calcd for C₁₄H₁₄ClN₂O₃ [M + H]⁺ 293.0688, found: 293.0686.

2.2.9. Ethyl 1-benzyl-5-chloro-4-(2-methoxyvinyl)-1H-pyrazole-3-carboxylate (14).

To a mixture of (methoxymethyl)triphenylphosphonium chloride (commercially available, 8.57 g, 25.0 mmol) in tetrahydrofuran (THF) (25 mL) was added t-BuOK (2.80 g, 25.0 mmol) over 1 min at 0 °C. The mixture was stirred at 0 °C for 10 min, and then a solution of 13 (1.80 g, 6.0 mmol) in THF (10 mL) was added into the reaction mixture over 20 mins. The reaction mixture was stirred at 0 °C for 30 minutes, and then warmed to 20−25 °C for 12 h. The reaction mixture was diluted using EtOAc (200 mL) and washed with water (1 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column with mobile phase (Hexane/EtOAc, 10/1 to 4/1) to give 14 (0.72 g, 37%) as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆) δ 1.23 (3H, t, J = 7.1 Hz), 3.58 (3H, s), 4.21 (2H, q, J = 7.1 Hz), 5.21 (1H, d, J = 6.6 Hz), 5.41 (2H, s), 6.26 (1H, d, J = 6.6 Hz), 7.14−7.16 (2H, m), 7.27−7.34 (3H, m). HRMS (ESI) calcd for C₁₆H₁₈ClN₂O₃ [M + H]⁺ 321.1001, found: 321.1000.

2.2.10. Ethyl 1-benzyl-5-chloro-4-(2-oxoethyl)-1H-pyrazole-3-carboxylate (15).

To a solution of 14 (613.5 mg, 2.0 mmol) in THF (5 mL) was added 6 M HCl aqueous (2 mL, 12.00 mmol) at room temperature. The mixture was stirred for 2 hours at room temperature. Then the reaction mixture was heated to 60 °C and stirred for an additional 30 minutes. The reaction mixture was neutralized with saturated NaHCO₃ aqueous solution and brine, and then extracted using EtOAc. The organic layer was dried over Na₂SO₄, and concentrated under reduced pressure to give 15 (533 mg, 87%). This product was directly used for the next step reaction without further purification. 1H NMR (400 MHz, CDCl₃) δ 1.36 (t, J = 7.2 Hz, 3H), 3.78 (s, 2H), 4.38 (dd, J = 14.0, 7.0 Hz, 2H), 5.43 (s, 2H), 7.22 (s, 2H), 7.30 (t, J = 7.2 Hz, 3H), 9.66 (s, 1H). HRMS (ESI) calcd for C₁₅H₁₆ClN₂O₃ [M + H]⁺ 307.0844, found: 307.0841.

2.2.11. Ethyl (S)-1-benzyl-4-(2-((8-bromo-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)amino)ethyl)-5-chloro-1H-pyrazole-3-carboxylate (17).

2-picoline boran (367 mg, 3.4 mmol) was added into a solution of 15 (504 mg, 2.0 mmol) and 11 (612 mg, 2.0 mmol) in acetic acid (5.0 mL, 2.6 mmol) and methanol (MeOH) (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 h. The mixture was quenched using saturated NaHCO₃ aq. at room temperature and extracted using EtOAc. The organic layer was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column with mobile phase of 50−70% EtOAc in hexane to give 17 as a colorless oil (471 mg, 43%). ¹H NMR (400 MHz, CDCl3) δ 1.36 (3H, t, J = 7.1 Hz), 2.59−2.70 (1H, m), 2.80−2.85 (2H, m), 2.89−2.93 (1H, m), 3.59 (1H, dd, J = 10.4, 5.9 Hz), 4.11 (1H, m), 4.36 (3H, m), 5.39 (2H, s), 6.85 (1H, d, J = 8.4 Hz), 7.18−7.19 (3H, m), 7.23−7.34 (4H, m), 8.12 (1H, s). HRMS (ESI) calcd for C₂₄H₂₅BrClN₄O₄ [M + H]⁺ 547.0743, found: 547.0737.

2.2.12. (S)-3-(2-benzyl-3-chloro-7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepine-8-carbonitrile (TZ7790).

To a solution of 17 (273 mg, 0.50 mmol) in toluene (30 mL) was added trimethylaluminum in toluene (0.75 mL, 1.50 mmol) at room temperature. After stirring at 100 °C for 4 h under argon gas protection, the reaction mixture was heated to 120 °C and stirred overnight at 120 °C. The reaction mixture was cooled to room temperature and quenched in water, and then diluted with EtOAc. A saturated Rochelle salt aqueous solution was added to the mixture. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column with a mobile phase of 50−100% EtOAc in hexane to give the product (49 mg, 48%) as an off-white solid. Continually, to a solution of the off-white solid (49 mg, 0.25 mmol) and dicyanozinc (Zn(CN)₂) (37 mg, 0.31 mmol) in DMF (5 mL) was added Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)_4, 121 mg, 0.04 mmol) at room temperature. The mixture was stirred at 100 °C under argon gas protection for 2 h. The mixture was quenched in water at room temperature and extracted by EtOAc. The organic layer was separated, washed with water and brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by a silica gel column with a mobile phase of 75% EtOAc in hexane to give the product a yellow solid (19 mg, 21%). ¹H NMR (400 MHz, CDCl3) δ 2.72 (1H, dt, J = 15.5, 5.3 Hz), 2.92 (1H, ddd, J = 15.2, 9.7, 5.3 Hz), 3.61–3.75 (1H, m), 3.79–3.91 (1H, m), 4.44 (1H, dd, J = 11.5, 5.0 Hz), 4.60 (1H, m), 5.25–5.56 (3H, m), 6.81 (1H, d, J = 8.5 Hz), 7.15 (1H, dd, J = 8.5, 2.0 Hz), 7.27−7.34 (3H, m), 7.43−7.49 (1H, m), 7.52−7.54 (1H, m), 7.64−7.68 (1H, m), 8.02 (1H, s); ¹³C NMR (100 MHz, CDCl3) δ 19.6, 29.7, 47.3, 53.9, 57.3, 71.1, 117.3, 117.7, 122.6, 123.3, 124.9, 127.3, 127.5, 127.8, 128.3, 128.4, 128.5, 128.7, 131.9, 132.0, 132.1, 134.9, 141.9, 149.0, 161.0, 170.0. HRMS (ESI) calcd for C₂₃H₁₉ClN₅O₃ [M + H]⁺ 448.1171, found: 448.1171. Analytical HPLC > 95% purity

2.2.13. (3S)-3-(2-Benzyl-3-chloro-7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)-5-methyl-4-oxo-2,3,4,5-tetrahydro-1,5-benzoxazepine-8-carbonitrile (TZ7774).

Compound TZ7774 was obtained (5 mg, 29%) as a white solid using similar procedure of making compound TZ7790 ¹H NMR (400 MHz, CDCl3) δ 2.69 (1H, dt, J = 15.7, 4.8 Hz), 3.05 (1H, ddd, J = 15.6, 10.3, 5.1 Hz), 3.39 (3H, s), 3.51−3.61 (1H, m), 4.15−4.27 (1H, m), 4.45 (1H, dd, J = 10.2, 7.9 Hz), 4.69 (1H, dd, J= 11.9, 10.0 Hz), 5.40 (2H, s), 5.89 (1H, dd, J= 11.7, 7.9 Hz), 7.20−7.38 (6H, m), 7.48 (1H, d, J= 1.9 Hz), 7.57 (1H, dd, J= 8.3, 1.9 Hz); ¹³C NMR (100 MHz, CDCl3) δ 19.7, 35.6, 45.3, 51.2, 53.9, 74.9, 110.2, 117.6, 118.0, 123.4, 123.8, 126.7, 127.7, 128.2, 128.7, 129.8, 135.0, 141.5, 141.9, 149.9, 160.7, 169.1. HRMS (ESI) calcd for C₂₄H₂₁ClN₅O₃ [M + H]⁺ 461.1328, found: 461.1317. Analytical HPLC > 95% purity

2.3. Radiochemistry

The radiosynthesis of [¹¹C]TZ7774 was achieved through the demethyl precursor TZ7790 reacting with [¹¹C]methyl iodide via N-methylation.

Production of [¹¹C]CH₃I followed the reported method [21]. Briefly, [¹¹C]CH₃I was produced on-site from [¹¹C]CO₂ using a GE PETtrace MeI Microlab. Up to 1.4 Ci of [¹¹C]carbon dioxide was produced from the JSW BC-16/8 cyclotron by irradiating a gas target of 0.5% O₂ in N₂ for 15 – 30 min with a 40 μA beam of 16 MeV protons in the Barnard Cyclotron Facility of Washington University School of Medicine. After the [¹¹C]CO₂ was converted to [¹¹C]CH₄ using a nickel catalyst [Shimalite-Ni (reduced), Shimadzu, Japan P.N.221–27719] in the presence of hydrogen gas at 360 °C; the [¹¹C]CH₄ was further converted to [¹¹C]CH₃I by reaction with iodine in the gas phase at 690 °C. Approximately 12 min following the end-of-bombardment (EOB), several hundred millicuries of [¹¹C]CH₃I were delivered in the gas phase to the hot cell where the radiosynthesis was accomplished.

Precursor TZ7790 (0.6 mg, 1.5 μmol) was dissolved in 200 μL anhydrous DMF, then powder NaOH (2 mg) was added to the solution. The reaction mixture was transferred into a 1.5 mL V-shape reaction vessel with a septum cap. Radioactive [¹¹C]CH₃I gas was bubbled into the solution within 2 – 4 minutes. Then the reaction vial was sealed and heated at 80 °C for 5 minutes using an oil bath, agitating the reaction vessel twice during the heating period. The reaction vial was raised above the oil bath; the reaction was quenched in 2.0 mL of HPLC mobile phase (acetonitrile/Ammonium Formate solution 48/52, pH = ~ 4.5); and then loaded onto a semi-preparative HPLC system equipped with a C18 column (250 × 9.6 mm, 5 μM) with a mobile phase flow rate of 4.0 mL/min. A UV detector set for a wavelength of 254 nm permitted purification. A radioactive product was collected from 11 to 14 minutes using a glass vial prefilled with milli-Q water (50 mL), then the solution was passed through a C-18 Sep-Pak cartridge (Part No. WAT020515, Waters Corporation, Milford, MA). After using 20 mL of Milli-Q water to rinse the C-18 cartridge, the radioactive product was eluted into a dose vial using 0.6 mL absolute ethanol, followed by 5.4 mL of saline (v/v) to formulate the injection dose solution for administration to an animal.

An aliquot of the dose sample (20 μL) was injected onto the analytic HPLC system for authentication by co-injection with the cold standard compound TZ7774, the chemical and radiochemical purity, and molar activity of the final dose were determined. The analytic HPLC system for quality control consists of a reversed-phase HPLC analytical column (Agilent Zorbax SB-C18, 4.6 mm × 250), UV wavelength of 254 nm, mobile phase of acetonitrile/0.1 M ammonium formate buffer (v/v, 70/30, pH = 4.5) with the flow rate of 1.0 mL/min. Under this condition, [¹¹C]TZ7774 has a retention time of T_R = 4.8 min, 30–40% radiochemical yield (decay corrected to EOB), >207 GBq/μmol molar activity (decay corrected to EOB), >90% chemical purity and >99% radiochemical purity.

2.4. Ex vivo biodistribution study in rodents

To investigate the tissue distribution and kinetics of [¹¹C]TZ7774 in living animals, the ex vivo biodistribution study was performed using male adult Sprague Dawley (SD) rats by following our previously reported procedure [22]. In brief, approximate 7.4 MBq/100 μL of [¹¹C]TZ7774 was administrated to the animal intravenously under 2–3% isoflurane/oxygen anesthesia. Rats were euthanized at 5, 30, and 60 minutes post-injection (n = 4 for each group). Tissues including blood, heart, lung, muscle, fat, pancreas, spleen, kidney, liver, thymus, small intestine, and brain were collected, weighed, and counted on a Beckman 8000 automated gamma counter (Beckman, Brea, CA). To check the distribution of [¹¹C]TZ7774 in different brain regions, including the cerebellum, brain stem, cortex, thalamus, hippocampus, striatum, and the rest of the brain was collected, weighed, and counted. The tissue uptake of radiotracer was calculated as weight, background, and decay-corrected percent injected dose per gram (%ID/g).

To check the uptake of [¹¹C]TZ7774 in response to lipopolysaccharides (LPS) induced inflammation, the ex vivo biodistribution study of [¹¹C]TZ7774 was performed in adult male C57BL/6 mice under normal and LPS pre-treated conditions. After 24 hours of intraperitoneal administration of LPS (10 mg/kg), approximate 3.7 MBq/100 μL of radiotracer was administrated to the animal via tail vein injection. Tissues including blood, lung, liver, spleen, kidney, muscle, fat, heart, thyroid, pancreas, thymus, small intestine, and brain were collected, weighed, and counted as described above. The tracer uptake (%ID/g) was calculated as described above.

2.5. In vitro autoradiography study of the mice spleen

In vitro autoradiography study was carried out using frozen sections from spleens of normal and LPS treated mice. In brief, 12-micron sections were preincubated with buffer at room temperature for 10 minutes and then incubated with 0.74 MBq/slide of [¹¹C]TZ7774 for 30 minutes at room temperature. After that, all slides were washed for 2 minutes in each of the following buffers sequentially: 1x TBST, 20% EtOH in 1x TBST, 40% EtOH in 1X TBST, 1X TBS. Sections were then exposed immediately to BAS-IP MS 2025 Storage Phosphor Screen (Cytiva, Amersham, UK) overnight, and visualized by a Fuji BioImaging Analyzer FLA-7000 (Fuji Photo Film, Tokyo, Japan). Photostimulated luminescence was quantified with Multi Gauge (Fuji Photo Film, Tokyo, Japan). Data were background corrected and calculated as photo-stimulated luminescence signals per square millimeter (PSL/mm²).

2.6. Immunohistochemical study of the mice spleen

To understand the role of RIPK1 in response to LPS-induced inflammation, we next did immunohistochemistry of spleen from normal control and LPS-treated mice. Immunohistochemical staining of RIPK1 was carried out in adjacent frozen sections from the autoradiograph study as previously published [23]. In brief, 12-micron sections were pre-warmed to room temperature and fixed with 4% paraformaldehyde for 15 minutes. Sections were then washed and blocked with 5% horse serum in PBS for 1 hour followed by incubation with BLOXALL (Vector Laboratories, Burlingame, CA) to block endogenous peroxidase. After that, slides were incubated with an anti-RIPK1 antibody (ThermoFisher, Waltham, MA) at 4 ºC overnight. Slides were then washed in PBS followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature, and then developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

2.7. MicroPET brain imaging study in cynomolgus macaque

To check if [¹¹C]TZ7774 can image RIPK1 in the brain, microPET imaging of [¹¹C]TZ7774 was performed on non-human primate brain as previously published [24]. A microPET Focus-220 scanner (Concorde/CTI/Siemens Microsystems, Knoxville, TN) was used for data collection as described [25]. Male macaque, weighing ~10 kg, was scanned under anesthesia and kept at 37 ºC with a heat water blanket. Dynamic PET scans were collected from 0–120 min after intravenous administration of ~0.23 GBq of radiotracer. PET time frames were: 3 × 1 min, 4 × 2 min, 3 × 3 min, and 20 × 5 min. A filtered back projection method was used to reconstruct PET images with dead time, scatter, randomes, and attenuation correction (volume size: 12 × 128 × 95, voxel size: 1.898 × 1.898 × 0.796 mm³ in the x, y, and z direction). The final reconstructed resolution was 2.0 mm full width at half-maximum for all three demotions at center of the field of view. For quantitative analyses, dynamic PET images were co-registered to a standardized monkey MRI template using an automated image registration program ‘Fuse It’ in PMOD software 4.02 (PMOD Technologies, Zürich, Switzerland) [26]. Predefined brain ROIs from the template were transformed to the co-registered PET images to obtain time-activity curves (TACs). The uptake of radioactivity was standardized to body weight and the dose of radioactivity injected to yield standardized uptake value (SUV).

2.8. Statistical analysis

All data were analyzed with Prism 7.0 (GraphPad Software, San Diego, CA). Two-way ANOVA followed by Šídák multiple comparisons test was used to compare the percent injected dose per gram of tissues in the biodistribution study. Student t-test compared autoradiographic data from normal and LPS treated mice. A P-value ≤ 0.05 was considered to be statistically significant difference.

3. Results

3.1. Chemistry and radiochemistry

Compound TZ7774 was reported as a potent ligand for RIPK1 with a Ki value of 0.91 nM. It also has a methylamine group in its structure, allowing incorporating a [¹¹C]methyl group through N-methylation of its demethyl amino precursor. To investigate RIPK1, we first synthesized compound TZ7774 according to the reported procedure with necessary modifications [19]. We then synthesized the demethyl amino precursor TZ7790, which reacted with [¹¹C]methyl iodide via N-methylation to afford [¹¹C]TZ7774.

The synthesis of compound TZ7774 and demethyl amino precursor TZ7790 was achieved by following Scheme 1. We first synthesized key intermediates 10, 11, and 15, followed by reductive amination to generate intermediate 16 and 17, then cyclization and cyanation by bromide reacting with Zn(CN)₂ in the presence of Pd(PPh₃)₄ to afford TZ7774 and TZ7790. Briefly, 4-bromo-substituted fluoronitrobenzenes reacting with (S)-2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoic acid in the presence of sodium hydride afforded intermediate 6 in 81% yield. Followed by reducing the nitro group using zinc, intramolecular amination gave Boc-protected 8 with a 36% yield. After N-methylation, using iodomethane and cesium carbonate gave compound 9 in 69% yield. Using trifluoroacetic acid to remove the Boc group, compounds 10 and 11 were generated. The compound 15 started with benzylhydrazine hydrochloride smoothly to treat commercially available diethyl oxaloacetate sodium salt to afford intermediates 12. Following the hydroxy group conversion in 12 to chloride and oxidation using phosphoryl chloride, aldehyde 13 was obtained with a 31% yield. Then, compound 14 was obtained through the Wittig reaction between aldehyde 13 and (methoxymethyl)-triphenylphosphonium chloride in the presence of potassium tert-butoxide (t-BuOK). The key intermediate 15 was synthesized in 87% yield, followed by hydrolysis using hydrochloric acid. Compounds 16 and 17 were generated by a reductive amination reaction to connect two parts of the product. Cyclization and cyanation using dicyanozinc afforded the standard compound TZ7774 and the precursor TZ7790.

Scheme 1.

Synthesis of standard compound TZ7774 and precursor TZ7790. Agents and reaction condtions: (a) sodium hydride (60%), DMF, 0 °C-r.t.; (b) zinc powder, acetic acid, 0 °C; (c) HATU, TEA, DMSO, r.t.; (d) MeI, Cs₂CO₃, DMF, r.t.; (e) trifluoromethylacetic acid, r.t.; (f) acetic acid, benzene, 100 °C; (g) phosphoryl chloride, DMF, 90 °C; (h) (methoxymethyl)triphenylphosphonium, ^tBuOK, THF, 0 °C-r.t.; (i) 6 M HCl aq/THF, r.t.; (j) 5, 2-picoline boran, acetic acid, intermediate 10, MeOH, r.t.; (k) 5, 2-picoline boran, acetic acid, intermediate 11, MeOH, r.t.; (l) AlMe₃, toluene, 100 °C; (m) dicyanozinc, Pd(PPh₃)₄, DMF, 100 °C, under argon gas.

The radiosynthesis of [¹¹C]TZ7774 was accomplished by N-[¹¹C]methylation of its N-demethyl precursor TZ7790 with [¹¹C]CH₃I under basic condition (2.0 mg of powder NaOH) in 200 μL DMF, heated at 80 °C for 5 minutes as shown in Scheme 2. The radioactive product was purified on a semi-preparative reversed-phase HPLC column combined with solid-phase extraction. Under this condition, the radiosynthesis of [¹¹C]TZ7774 was successfully achieved with good radiochemical yield (30–40%, 1.85 GBq, decay corrected to EOB), >90% chemical purity and >99% radiochemical purity, and high molar activity ( >207 GBq/μmol, decay corrected to EOB).

Scheme 2.

Radiosynthesis of [¹¹C]TZ7774.

3.2. Ex vivo biodistribution study of [¹¹C]TZ7774 in rodents

The tissue distribution of [¹¹C]TZ7774 in adult male SD rats was summarized in Figure 2. At 5 min post-injection, relatively high uptake of [¹¹C]TZ7774 were observed in the brain, pancreas, spleen, kidney, and liver at 0.53, 0.49, 0.41, 0.62, 0.95 (%ID/g) respectively (Figure 2A). The high brain uptake of [¹¹C]TZ7774 indicated [¹¹C]TZ7774 has a good blood-brain barrier permeability. Within the brain, a radiotracer uptake of 0.49, 0.68, 0.44, 0.54, 0.49, 0.59 was observed in cerebellum, brain stem, cortex, thalamus, hippocampus, and striatum respectively (Figure 2B). Notably, [¹¹C]TZ7774 washed out relatively fast from the brain, the brain uptake was 0.53 at 5 min, 0.14 at 30 min, 0.07 at 60 min. The brain uptake ratio was ~7.60 for 5 min versus 60 min. The radioactivity was gradually accumulated in the small intestine for the peripheral tissues, suggesting a possible hepatobiliary clearance into small intestine.

Fig. 2.

Tissue distribution of [¹¹C]TZ7774 in normal SD rats. (A) The uptake of [¹¹C]TZ7774 in normal rats. At 5 min, the pancreas, kidney, liver, brain, and small intestine had high initial uptake; liver displayed the highest tracer uptake (%ID/g) at 5 min with ~1.0. (B) The high initial uptake was observed in the brain at 5 min, the order of [¹¹C]TZ7774 uptake observed in the brain from highest to lowest is brain stem > striatum > thalamus > hippocampus > cerebellum > cortex.

To test if the uptake of [¹¹C]TZ7774 can be impacted by the LPS-induced acute inflammation, biodistribution studies of [¹¹C]TZ7774 were performed in normal and LPS treated C57BL/6 mice. Our biodistribution data showed that LPS pre-treated mice have an increased uptake of [¹¹C]TZ7774 in several tissues, including liver, spleen, and kidney (Two-way ANOVA: F(1,65) = 21.26, p < 0.0001). LPS pre-treatment caused 40.9%, 90.4%, and 54.9% increase of [¹¹C]TZ7774 in liver, spleen, and kidney respectively (Figure 3).

Fig. 3.

The tissue distribution of [¹¹C]TZ7774 in normal control and LPS (10 mg/kg, 24 hours prior to the injection of the radiotracer) pre-treated mice. The tracer uptake (%ID/g) in LPS treated mice was significantly higher in liver, spleen, and kidney than that in normal control mice (Two-way ANOVA: F(1,65) = 21.26, p < 0.0001, n = 4; Šídák multiple comparison test: * p ≤ 0.05).

3.3. In vitro autoradiograph and immunohistochemical studies in the normal and the LPS treated mouse spleen

In vitro autoradiograph study of [¹¹C]TZ7774 was carried out on tissues from normal and LPS pre-treated C57BL/6 mice. Our autoradiograph study data showed a significant increase of [¹¹C]TZ7774 in the spleen of LPS treated mice compared to healthy normal control mice (Figure 4). The uptake of [¹¹C]TZ7774 increased >1.4-fold in the spleen of LPS pre-treated mice with a p-value of 0.034 respectively (Figure 4). To confirm if the increased uptake of [¹¹C]TZ7774 in the LPS treated mice spleen was caused by increased expression of RIPK1 in the spleen, Immunohistochemical studies of RIPK1 in the spleen mice were performed in the control and LPS-treated mice. Our studies showed an increased level of RIPK1 in the spleen of LPS-treated mice compared to normal control mice, particularly, the expression of RIPK1 was elevated in the red pulp of the spleen in LPS-treated mice. In addition, a group of RIPK1 positive cells was identified in the white pulp of the LPS-treated spleen (Figure 5). Whereas in the normal control mice, only a relatively low level of RIPK1 was observed in the red pulp of the spleen, and a negligible amount of RIPK1 positive cells in the white pulp of the spleen was observed (Figure 5). Our immunohistochemistry study indicated that the increased spleen uptake of [¹¹C]TZ7774 in the LPS pre-treated mice may reflected the increased expression of RIPK1 caused by the LPS.

Fig. 4.

In vitro autoradiograph studies of [¹¹C]TZ7774 in the spleen of normal control and LPS treated mice. Autoradiograph analysis indicated that LPS treated mice have a significant increase of [¹¹C]TZ7774 uptake in the spleen (t-test: p < 0.05).

Fig. 5.

Immunohistochemistry staining of RIPK1 in the spleen of normal control and LPS treated mice. Immunohistochemistry staining using RIPK1 specific antibody indicated an overall increase of RIPK1 expression in the spleen of LPS treated mice compared to that in the normal mice. RIPK1 expression was elevated in the red pulp of the spleen after LPS treatment (red arrow); a group of RIPK1 positive cells was also identified in the white pulp of the spleen in LPS treated mice compared to the normal control mice (blue arrow).

3.4. MicroPET macaque brain imaging study

MicroPET in macaque showed that [¹¹C]TZ7774 entered the brain very well; the tissue time-activity curve showed that the brain uptake (standardized uptake value, SUV) reached a maximum value of ~3.75 at 5 min, and decreased to 1.53 at 60 min as shown in Figure 6A&B. Within the brain, the cerebellum, thalamus, putamen, and hippocampus displayed a high tracer uptake, and the basal frontal cortex region displayed the lowest uptake as shown in Figure 6B–C.

Fig. 6.

The microPET brain imaging study of [¹¹C]TZ7774 in the non-human primate. (A) A high uptake (standard uptake value) of [¹¹C]TZ7774 was observed in the macaque brain; (B) The brain regional time tissue activity curves of [¹¹C]TZ7774; (C-D) Representative PET images of [¹¹C]TZ7774 and co-registered with MRI images.

4. Discussion

RIPK1 is a crucial mediator in multiple signaling pathways regulating inflammatory responses and cell death programming. The pathology of many CNS disorders is characterized by RIPK1-mediated necroptosis and neuroinflammation [27]. Inhibiting RIPK1 using its antagonists may provide an innovative approach to the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis [28], Alzheimer’s diseases [29], multiple sclerosis [30], ischemic stroke [31], and traumatic brain injury [32]. To date, investigators have made tremendous efforts to develop and evaluate RIPK1 specific inhibitors for therapies. We focused on radiosynthesis of an RIPK1 specific radioligand [¹¹C]TZ7774 and tested whether [¹¹C]TZ7774 could be a potential RIPK1 PET radioligand to quantify RIPK1 expression changes in vivo.

We first designed and synthesized the demethyl precursor TZ7790 used to make [¹¹C]TZ7774 through the N-methylation of demethyl precursor TZ7790 reacting with [¹¹C]methyl iodide under the conventional procedure. The injection dose of [¹¹C]TZ7774 for in vitro and in vivo biological studies was formulated using 10% of absolute alcohol in saline. An aliquot sample of injection dose was authenticated using an analytical HPLC system for quality control as described above. The synthesis of TZ7774 and [¹¹C]TZ7774 are smooth without any challenges.

The biodistribution studies in normal SD rats indicated that the uptake of [¹¹C]TZ7774 was high in the majority of tested tissues including lung, liver, spleen, kidney, and small intestine. In particular, [¹¹C]TZ7774 penetrates the blood-brain barrier with good initial brain uptake. In addition, microPET in a macaque revealed good brain uptake (SUV) of [¹¹C]TZ7774 with a maximum value of 3.7 at 6–10 min post tracer injection. Within the brain, the uptake of [¹¹C]TZ7774 was high in the striatum, thalamus, hippocampus, and cerebellum. Good brain uptake in both SD rat and macaque suggested that [¹¹C]TZ7774 has potential to measure brain RIPK1 expression for neurological diseases and psychiatric abnormalities.

RIPK1 is a key regulator of inflammation and can directly regulate inflammatory cytokine expression. Activation of RIPK1 promotes inflammation in response to infectious diseases; for example, RIPK1 induced antiviral gene transcription in Zika virus infected neurons [33]; mice expressing a kinase-dead form of RIPK1 displayed increased susceptibility to West Nile virus infection [33]. RIPK1 activity is required to promote inflammatory gene expression in myeloid cells after administrating a sub-lethal dose of LPS in the mouse [34]. In fact, administration of LPS causes a rapid and transient rise in TNFα levels and induces an acute inflammation in mice because LPS binds to TLR4 and leads to the formation of Myd88 and TRIF. Myd88 further activates the early phase of NF-κB, whereas TRIF recruits RIPK1 to activate the late phase of NF-κB, promoting NF-κB pathway activation to mediate the expression of both proinflammatory and prosurvival genes [10]. In our study, after intraperitoneal administration of LPS into C57BL/6 mice, compared to the normal control mice, mice pre-treated with LPS led the uptake of [¹¹C]TZ7774 increased approximately 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. Our data did not detect significant difference of the brain uptake of LPS-pre-treated mice compared to the control mice, which might resulted from that LPS effecting on the changes of RIPK1 expression in the brain was much fast (3–6 hrs) and then gradually recovered normal at 24 hrs. Future study needs to focus on check the LPS effect the RIPK1 expression in early time (3–6 hrs) for acute inflammatory response caused by LPS, not 24 hrs.

Interestingly, the spleen showed the highest increase of [¹¹C]TZ7774 uptake in response to LPS induced acute inflammation. Further characterization using in vitro autoradiography and immunohistochemistry studies in the control and LPS treated mouse spleen confirmed that the increased uptake of [¹¹C]TZ7774 was due to the upregulation of RIPK1 expression in the spleen of LPS treated mice. As the largest lymphoid organ in the body, the spleen plays a pivotal role in host immune function during inflammation. In the LPS induced inflammation, LPS activated macrophages produce a variety of inflammatory cytokines via activation of TLRs signaling which can further regulate RIPK1 related pro-death kinase activity in response to the inflammation [35]. While the most abundant type of macrophage in the spleen is the red pulp macrophage, in our case, an overall increase of RIPK1 expression in the red pulp of the spleen was observed in LPS-treated mice, possibly attributed to the LPS-induced macrophage necroptosis. In addition, immunohistochemistry also identified a group of RIPK1 positive cells in the white pulp of the LPS-treated mice but not in the spleen from normal mice. RIPK1 is expressed in lymphoid tissues such as lymph nodes, thymus, and spleen, and it regulates lymphocyte survival and death [36]. It is critical for mature T-cell survival and proliferation [37], as well as B cell development [36]. Mutations of RIPK1 can cause immunodeficiencies and autoinflammation [10]. In particular, several phosphoacceptor sites have been recognized that can regulate RIPK1 activity upon pathogen infection and RIPK1 plays an important role in inflammation response [2]. Our study indicated an increase of RIPK1 in both red and white pulp of LPS treated spleen, possibly due to LPS-induced macrophage necroptosis. Detailed characterization of the exact role of RIPK1 upregulation is needed. In addition, further characterizations of [¹¹C]TZ7774 in different animal models of diseases and its pharmacological properties are warranted to determine if [¹¹C]TZ7774 is worth transferring into clinical evaluation for human use.

5. Conclusion

We successfully synthesized the ¹¹C-labelled RIPK1 specific radioligand [¹¹C]TZ7774 with good radiochemical yield, high chemical and radiochemical purity, and high molar activity. Animal studies in rodent and macaque revealed that [¹¹C]TZ7774 has good blood-brain barrier penetration with high initial brain uptake, and has a good response to the LPS-induced acute inflammation in the spleen. Our study demonstrated the feasibility of imaging RIPK1 expression changes in vivo and [¹¹C]TZ7774 has potential to be a PET radiotracer for imaging RIPK1 expression in living animals.