Carbon-11 labeled larotrectinib compounds and methods of making the same

Abstract

The application discloses a kind of carbon-11 labeled Larotrectinib compounds, and the chemical structural formula of the compound is as follows: The compound can be obtained by mixing reaction with carbon-11 labeled fluorine gas, or carbon-11 labeled carbon dioxide can be introduced into the solution containing BEMP and then POCl3 is added, and the reaction is generated again with the reaction. Compared with the prior art, the synthesis of short half-life carbon-11 labeled Larotrectinib is successfully realized, the radiochemical purity of the label is high, and it is suitable for the demand of short half-life carbon-11 labeled Larotrectinib in clinic.

Description

Technical Field

[0001] This invention relates to the field of chemical drug synthesis, particularly to in vivo imaging agents for the Trk receptor subtype in refractory solid tumors, specifically to radiocarbon compounds based on the novel tyrosine receptor kinase (TRK) inhibitor Larotrectinib. 11 C]-Larotrectinib and its preparation method. Background Technology

[0002] Developed by Loxo Oncology, larotrectinib is a broad-spectrum oncology drug used for all patients with tumors expressing tropomyosin receptor kinase (TRK), rather than targeting tumors in a specific anatomical location. This small-molecule TRK inhibitor exhibits strong selectivity for TRK; by inhibiting the TRK signaling pathway, larotrectinib can suppress tumor growth. Larotrectinib is a potent, orally administered TRK inhibitor with consistent and durable antitumor activity in TRK fusion tumors. It is suitable for a wide range of patients of various ages and tumor types, with indications across 13 different tumor types. It is well-tolerated and effective against various solid tumors in adults and children, including salivary gland cancer, infantile fibrosarcoma, lung cancer, thyroid cancer, colon cancer, melanoma, cholangiocarcinoma, gastrointestinal stromal cancer (GIST), breast cancer, and various sarcoma types. On July 13, 2016, the US FDA (http: / / www.chemdrug.com / article / 11 / ) granted larotrectinib Orphan Drug and Breakthrough Therapy designations for the treatment of unresectable or metastatic solid tumors in adults and children with TRK fusion gene mutations. Larotrectinib is expected to become the first treatment to be developed and approved simultaneously in adults and children, and the first molecularly targeted cancer therapy that transcends all traditionally defined tumor types. The structure of larotrectinib is shown below:

[0003]

[0004] However, current methods for evaluating the distribution of drug molecules in the body and their efficacy typically employ positron-emitting radiopharmaceuticals. 18 F]-deoxyglucose ( 18F-FDG), indirectly evaluating the efficacy of drugs against tumors and related diseases, and, moreover, radiopharmaceuticals that emit positrons [ 18 F]-deoxyglucose ( 18 FDG uptake is also high in non-tumor tissues and inflammatory cellular components, which may lead to false-positive results in tumor imaging due to the presence of inflammation. Therefore, how to track the distribution of larotrectinib in humans or animals and its status in solid tumors in vivo, to directly assess the physiological function of larotrectinib in tumors, and to evaluate its efficacy and prognosis, is a challenging problem.

[0005] PET (Positron emission tomography) is a non-invasive imaging technique that allows for the visualization and quantitative assessment of the physiological and biochemical functions and pharmacological processes of drug molecules at the in vivo molecular level. Larotrectinib, a small molecule inhibitor of tropomyosin receptor kinase (TRK), exhibits strong selectivity for TRK and can be used as a PET imaging molecule. This allows for precise localization of Larotrectinib molecules in vivo to evaluate tumor status, enabling online tracking of its distribution in animals or humans and its status in solid tumors. It provides a direct assessment of the physiological functions and pharmacological processes of Larotrectinib in animals, humans, and tumors, evaluating its efficacy and prognosis. This is currently the most advanced technique using positron-emitting radiopharmaceuticals. 18 F]-deoxyglucose ( 18 There is currently no more effective technical means that can compare with F-FDG.

[0006] Carbon-11 has the characteristics of emitting positrons, high radioactivity, and suitability for in vivo experiments. However, carbon-11 has a short half-life (20.38 minutes), and how to complete the labeling of carbon-11 in a short time is a thorny problem to obtain and realize the clinical application of carbon-11 labeled compounds.

[0007] WO2016 / 077841A1 discloses a method for preparing Larotrectinib sulfate, the reaction route of which is as follows:

[0008]

[0009] However, the step of preparing compound VI from compound V takes 30 to 90 minutes, and the step of preparing compound I-HS from compound VI takes as long as 9 to 19 hours at a reaction temperature of 50°C. This is unacceptable and infeasible for obtaining carbon-11 labels with short half-lives.

[0010] CN109705124A discloses a method for preparing a radioactive fluorine-labeled Larotrectinib compound, the reaction of which includes:

[0011]

[0012] The specific operation process is as follows: (1) Dissolve triphosgene (9.91g, 33.38mmol) in 50ml tetrahydrofuran, add 5-chloropyrazolo[1,5-a]pyrimidin-3-amine (16.86g, 0.1mol) and triethylamine (0.47g, 4.64mmol), and stir the reaction system at 25 degrees for 1 hour. A suspension of 5-chloro-3-isocyanatopyrazolo[1,5-a]pyrimidine is obtained. (2) In a 250 mL reactor, sodium bicarbonate solution (80 mL, 0.5 M, 40 mmol) was added, followed by (S)-pyrrolo-3-ol (79 g, 55 mmol). The above suspension (62 mL, 100 mmol, calculated as 5-chloropyrazolo[1,5-a]pyrimidin-3-amine) was added dropwise, maintaining the temperature at approximately 0°C. After the addition was complete, the mixture was stirred at 0°C for 3 hours, then heated to 50°C and reacted for another 5 hours. The mixture was cooled to room temperature and extracted three times with 100 mL of ethyl acetate each time. The organic phases were combined, washed with 2N hydrochloric acid, then with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was then subjected to silica gel column chromatography, eluted with ethyl acetate / petroleum ether (5:5, v / v). The reaction time for the two steps described above is still as long as 6 hours, which is unacceptable and infeasible for obtaining carbon-11 labeled compounds with short half-lives. Summary of the Invention

[0013] The purpose of this invention is to overcome the defects of the prior art by providing a carbon-11 labeled Larotrectinib compound and its preparation method.

[0014] This invention proposes that Larotrectinib can be prepared by radiolabeling with carbon-11 radionuclides to form [ 11[C]-Larotrectinib, a novel TRK positron emission tomography (PET) tracer, is used for online in vivo PET imaging. It exhibits TRK specificity and utilizes PET to detect changes in TRK kinase distribution, enabling the detection of TRK fusion protein expression in tumor tissues. This provides reliable diagnostic and molecular phenotypic data for clinical examination and can also be used for in vitro cell receptor and drug molecule screening and evaluation. Preliminary results from this invention show... 11 C]-Larotrectini molecule as a TRK kinase [ 11 The potential imaging potential of C] positron emission tomography tracers.

[0015] To achieve the objectives of this invention, the following technical solutions can be used:

[0016] A carbon-11 labeled Larotrectinib compound has the following chemical structural formula:

[0017]

[0018] This invention provides a method for preparing the above-mentioned carbon-11 labeled Larotrectinib compound, the method comprising the following steps:

[0019] (1) The compound shown in Formula 1, the compound shown in Formula 2, and carbon-11 labeled fluorine phosgene were mixed and reacted to obtain the compound shown in Formula 3.

[0020] Formula 1:

[0021] Formula 2:

[0022] Formula 3:

[0023] Wherein, R is a hydrogen or hydroxyl protecting group;

[0024] Optionally, (2) when R is a hydroxyl protecting group, the hydroxyl protecting group of the compound shown in Formula 3 is removed.

[0025] Preferably, the hydroxyl protecting group is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, triphenylmethyl, p-methoxytriphenylmethyl, dimethoxytriphenylmethyl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyloxymethyl, 3,4-dimethoxybenzyl, tetrahydropyran, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, etc. 9-fluorenylmethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, pivaloyl, methanesulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16 alkylyl, C3-C16 enoyl, C3-C6 alkynyl, C4-C10 cycloalkyl, C7-C16 aromatic acyl, C4-C10 heterocyclic alkylyl.

[0026] Preferably, the mixing reaction process in step (1) includes: introducing carbon-11 labeled phosgene into a solution containing the compound shown in Formula 1 and the compound shown in Formula 2.

[0027] Preferably, the concentration of the compound represented by Formula 1 in the solution is 0.01 to 1000 mmol / L, more preferably, the concentration is 0.1 to 200 mmol / L, and most preferably, the concentration is 1 to 20 mmol / L.

[0028] Preferably, the molar ratio of the compound shown in Formula 1 to the compound shown in Formula 2 is 1:0.1 to 10; more preferably, the molar ratio is 1:0.5 to 5; and most preferably, the molar ratio is 1:1 to 3.

[0029] Preferably, the time for introducing carbon-11 labeled fluorine phosgene is 1 to 10 minutes, more preferably, the time is 2 to 7 minutes, and most preferably, the time is 4 to 6 minutes.

[0030] Preferably, the reaction solvent for the mixed reaction is selected from acetonitrile.

[0031] Preferably, when R is a hydroxyl protecting group, the compound shown in Formula 3 is reacted at 60–180 °C under the action of acid, potassium carbonate or magnesium bromide to remove the hydroxyl protecting group, thereby obtaining the carbon-11 labeled Larotrectinib compound.

[0032] Preferably, when R is a hydroxyl protecting group, steps (1) and (2) are completed in a one-pot process.

[0033] This invention provides another method for preparing the above-mentioned carbon-11 labeled Larotrectinib compound, the method comprising the following steps:

[0034] (1) Carbon-11 labeled carbon dioxide was bubbled into a solution containing BEMP and the compound shown in Formula 1. After the reaction was complete, POCl3 was added, and the reaction was continued to obtain the compound shown in Formula 4.

[0035] Formula 1:

[0036] Formula 4:

[0037] (2) The compound shown in Formula 4 reacts with the compound shown in Formula 2 to obtain the compound shown in Formula 3.

[0038] Formula 2:

[0039] Formula 3:

[0040] Wherein, R is a hydrogen or hydroxyl protecting group;

[0041] Optionally, (3) when R is a hydroxyl protecting group, the hydroxyl protecting group of the compound shown in Formula 3 is removed.

[0042] Preferably, the hydroxyl protecting group is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, triphenylmethyl, p-methoxytriphenylmethyl, dimethoxytriphenylmethyl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyloxymethyl, 3,4-dimethoxybenzyl, tetrahydropyran, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, etc. 9-fluorenylmethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, pivaloyl, methanesulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16 alkylyl, C3-C16 enoyl, C3-C6 alkynyl, C4-C10 cycloalkyl, C7-C16 aromatic acyl, C4-C10 heterocyclic alkylyl.

[0043] Preferably, the concentration of the compound represented by Formula 1 in the solution is 0.01 to 1000 mmol / L, more preferably, the concentration is 0.1 to 200 mmol / L, and most preferably, the concentration is 1 to 20 mmol / L.

[0044] Preferably, the volume percentage concentration of BEMP in the solution is 1-30%, more preferably, the volume percentage concentration is 1-10%, and even more preferably, the volume percentage concentration is 3-6%.

[0045] Preferably, in step (1), the introduction of carbon-11 labeled carbon dioxide is stopped after the radioactivity captured by the solution reaches its peak. More preferably, the reaction is allowed to proceed for 0.5 to 5 minutes after the introduction of carbon-11 labeled carbon dioxide is stopped. Most preferably, the reaction is allowed to proceed for 1 to 2 minutes.

[0046] Preferably, the molar ratio of POCl3 to the compound shown in Formula 1 is 1 to 50:1, more preferably, the molar ratio is 2 to 20:1, and most preferably, the molar ratio is 3 to 10:1.

[0047] Preferably, in step (1), after adding POCl3, the reaction time is 0.1 to 3 minutes, more preferably, 0.2 to 2 minutes, and even more preferably, 0.5 to 1 minute.

[0048] Preferably, the molar ratio of the compound shown in Formula 4 to the compound shown in Formula 2 is 1:0.1 to 100, and more preferably, the molar ratio is 1:1 to 100.

[0049] Preferably, the reaction solvent in step (1) is selected from acetonitrile.

[0050] Preferably, when R is a hydroxyl protecting group, the compound shown in Formula 3 is reacted at 60–180 °C under the action of acid, potassium carbonate or magnesium bromide to remove the hydroxyl protecting group, thereby obtaining the carbon-11 labeled Larotrectinib compound.

[0051] Preferably, steps (1), (2) and / or (3) are completed in a single pot.

[0052] This invention also provides an intermediate for synthesizing carbon-11 labeled Larotrectinib compounds, the chemical structural formula of which is as follows:

[0053]

[0054] This invention also provides an intermediate for synthesizing carbon-11 labeled Larotrectinib compounds, the chemical structural formula of which is as follows:

[0055]

[0056] Wherein, R is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, triphenylmethyl, p-methoxytriphenylmethyl, dimethoxytriphenylmethyl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, tetrahydropyran group, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenemethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, tert-pentanoyl, methanesulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16 alkyl, C3-C16 enoyl, C3-C6 ynyl, C4-C10 cycloalkyl. C7-C16 aryl acyl, C4-C10 heterocyclic acyl.

[0057] Beneficial effects

[0058] Compared with the prior art, the present invention has the following beneficial technical effects:

[0059] (1) The present invention successfully prepared a carbon-11 labeled Larotrectinib compound that has been desired but not yet obtained in the field.

[0060] (2) The present invention also provides a method for preparing carbon-11 labeled Larotrectinib compounds. Compared with the prior art, the preparation method of the present invention can significantly shorten the carbon-11 labeling reaction time, making it possible to rapidly synthesize the corresponding radioactive tracer with short carbon-11 half-life in a short time, thereby successfully realizing the synthesis of short half-life carbon-11 labeled Larotrectinib.

[0061] (3) The preparation process provided by this invention requires fewer types of reaction reagents and less amount of reaction precursors. The operation steps are simple and easy to implement, and the post-processing is simple. It can prepare carrier-free radiolabeled compounds with high radiochemical purity. It is suitable for the clinical demand for short half-life carbon-11 labeled Larotrectinib.

[0062] (4) The present invention provides [ 11 C]-Larotrectinib has the property of emitting positrons. With the help of PET-CT positron emission tomography, the distribution of Larotrectinib compounds in vivo and in tumors can be directly displayed, providing a new imaging agent for early tumor diagnosis. Attached Figure Description

[0063] Figure 1 Micro PET / micro CT imaging of tumor-bearing (H2228) mice. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

[0065] Unless otherwise stated, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.

[0066] In the following embodiments, the various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following examples are commercially available.

[0067] A carbon-11 labeled Larotrectinib compound has the following chemical structural formula:

[0068]

[0069] In some embodiments of the present invention, the above-mentioned carbon-11 labeled Larotrectinib compound is prepared by the following method, which includes the following steps:

[0070] (1) The compound shown in Formula 1, the compound shown in Formula 2, and carbon-11 labeled fluorophosgene ([ 11 The mixture of C]-COF2) reacts to give the compound shown in Formula 3.

[0071] Formula 1:

[0072] Formula 2:

[0073] Formula 3:

[0074] Wherein, R is a hydrogen or hydroxyl protecting group;

[0075] Optionally, (2) when R is a hydroxyl protecting group, the hydroxyl protecting group of the compound shown in Formula 3 is removed.

[0076] In some embodiments of the present invention, the hydroxyl protecting group is selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), methyl (Me), benzyl (Bn), triphenylmethyl (Tr), p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), tert-butyl (tBu), methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), benzyloxymethyl (BOM), p-methoxybenzyl (PMB), p-methoxybenzyloxy Methyl methyl (PMBOM), 3,4-dimethoxybenzyl (DMB), tetrahydropyran (THP), methoxycarbonyl (Moc), ethoxycarbonyl (Eoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenemethoxycarbonyl (Fmoc), trifluoroacetyl (TfAc), chloroacetyl (CAc), dichloroacetyl (DAc), phenylacetyl (Bz), pivaloyl (Pv), methanesulfonyl (Ms), benzylsulfonyl (Bs), allylsulfonyl (Bs), allyloxycarbonyl (Als), C1-C16 alkyl acyl, C3-C16 enoyl acyl, C3-C6 alkynyl acyl, C4-C10 cycloalkyl, C7-C16 aromatic acyl, C4-C10 heterocyclic alkyl acyl.

[0077] In some embodiments of the present invention, the C1-C16 alkyl acyl group is selected from formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, and hexadecanoyl.

[0078] In some embodiments of the present invention, the C3-C16 enoyl group is selected from acryloyl, 2-butenoyl, 4-pentenoyl, 5-hexenoyl, 2-ethylacryloyl, 3,3-dimethyl-2-methylenebutyryloyl, 6-heptenoyl, 7-octenyl, 3-methyl-hept-6-enoyl, 5-methyl-hept-6-enoyl, and 8-nonenoyl.

[0079] In some embodiments of the present invention, the C3-C6 acetylacetyl group is selected from propynoyl, 2-butynoyl, 4-pentynoyl, 5-hexynoyl, 6-heptynoyl, 7-octynoyl, and 8-nonynoyl.

[0080] In some embodiments of the present invention, the C4-C10 cycloalkyl group is selected from cyclopropylformyl, cyclobutylformyl, cyclopentylformyl, cyclohexylformyl, 2-methylcyclohexylformyl, and 2,6-dimethylcyclohexylformyl.

[0081] In some embodiments of the present invention, the C7-C16 aryl group is selected from benzoyl, p-methylbenzoyl, m-methylbenzoyl, o-methylbenzoyl, 4-bibenzoyl, 1-naphthoyl, 2-naphthoyl, 1-methyl-2-naphthoyl, 6-methyl-2-naphthoyl, 3-quinolinecarboxyl, 8-quinolinecarboxyl, 9-anthracarboxyl, and 9-acridinylcarboxyl.

[0082] In some embodiments of the present invention, the mixing reaction process in step (1) includes: passing carbon-11 labeled phosgene into a solution containing the compound shown in Formula 1 and the compound shown in Formula 2.

[0083] Those skilled in the art can determine the concentration of the compound represented by Formula 1 in the solution during synthesis based on the required amount for online in vivo imaging. In some embodiments of the present invention, the concentration of the compound represented by Formula 1 in the solution is 0.01 to 1000 mmol / L, more preferably 0.1 to 200 mmol / L, and most preferably 1 to 20 mmol / L.

[0084] In some embodiments of the present invention, the molar ratio of the compound represented by Formula 1 to the compound represented by Formula 2 is 1:0.1 to 10. Specifically, the molar ratios are 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.5, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. More preferably, the molar ratio is 1:0.5 to 5, and most preferably, the molar ratio is 1:1 to 3.

[0085] Carbon-11 labeled fluorinated phosgene is bubbled into a solution containing compounds shown in Formula 1 and Formula 2. The compound shown in Formula 1 or Formula 2 can be tracked using thin-layer chromatography (TLC). The fluorinated phosgene is stopped once the reactants have substantially disappeared. The exhaust gas is absorbed with sodium hydroxide solution, and the pipeline is purged with an inert gas such as helium.

[0086] In some embodiments of the present invention, the time for introducing carbon-11 labeled fluorine phosgene is 1 to 10 minutes, more preferably 2 to 7 minutes, and most preferably 4 to 6 minutes.

[0087] In some embodiments of the present invention, the reaction solvent for the mixed reaction is selected from acetonitrile.

[0088] In some embodiments of the present invention, when R is a hydroxyl protecting group, the compound of formula 3 generated in step (1) does not need to be separated and purified, and can be directly used for the removal of the hydroxyl protecting group in step (2), that is, steps (1) and (2) are completed in one pot.

[0089] The one-pot method described in this invention refers to the direct use of the intermediate product obtained in the previous step in the reaction of the next step without separation and purification.

[0090] Step (2) above can be performed using conventional methods in the art to remove the hydroxyl protecting group (R) from the compound shown in Formula 3.

[0091] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0092] The compound shown in Formula 3 was heated to 60–100 °C in an acidic solution and reacted for 1–5 minutes. After cooling and neutralization to neutrality, it was separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0093] In some embodiments of the present invention, the acid solution may be selected from hydrochloric acid with a concentration of 6 mol / L.

[0094] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0095] The compound shown in Formula 3 was mixed with magnesium bromide, heated to 120–180 °C, reacted for 2–7 minutes, cooled, neutralized to neutral, and then separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0096] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0097] The compound shown in Formula 3 was mixed with potassium carbonate, heated to 80–120 °C, reacted for 5–15 minutes, cooled, neutralized to neutral, and then separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0098] In some embodiments of the present invention, the method of concentration to dryness may be vacuum concentration to dryness, nitrogen blowing to dryness, or evaporation to dryness.

[0099] In some embodiments of the present invention, Waters C-18 Sep-Pak is used as the eluent for HPLC separation and purification.

[0100] In some embodiments of the present invention, when the one-pot method is used, the reaction in step (1) can be quenched with HPLC eluent / water (1 / 1, v / v) before the removal of the hydroxyl protecting group in step (2).

[0101] In some embodiments of the present invention, the above-mentioned carbon-11 labeled Larotrectinib compound is prepared by the following method, which includes the following steps:

[0102] (1) Carbon-11 labeled carbon dioxide was bubbled into a solution containing BEMP and the compound shown in Formula 1. After the reaction was complete, POCl3 was added, and the reaction was continued to obtain the compound shown in Formula 4.

[0103] Formula 1:

[0104] Formula 4:

[0105] (2) The compound shown in Formula 4 reacts with the compound shown in Formula 2 to obtain the compound shown in Formula 3.

[0106] Formula 2:

[0107] Formula 3:

[0108] Wherein, R is a hydrogen or hydroxyl protecting group;

[0109] Optionally, (3) when R is a hydroxyl protecting group, the hydroxyl protecting group of the compound shown in Formula 3 is removed.

[0110] In some preferred embodiments of the present invention, the hydroxyl protecting group is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, triphenylmethyl, p-methoxytriphenylmethyl, dimethoxytriphenylmethyl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, tetrahydropyran, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenmethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, tert-pentanoyl, methanesulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16 alkylyl, C3-C16 Enyl, C3-C6 alkynyl, C4-C10 cycloalkyl, C7-C16 aromatic acyl, C4-C10 heterocyclic alkanoyl.

[0111] In some embodiments of the present invention, the C1-C16 alkyl acyl group is selected from formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, and hexadecanoyl.

[0112] In some embodiments of the present invention, the C3-C16 enoyl group is selected from acryloyl, 2-butenoyl, 4-pentenoyl, 5-hexenoyl, 2-ethylacryloyl, 3,3-dimethyl-2-methylenebutyryloyl, 6-heptenoyl, 7-octenyl, 3-methyl-hept-6-enoyl, 5-methyl-hept-6-enoyl, and 8-nonenoyl.

[0113] In some embodiments of the present invention, the C3-C6 acetylacetyl group is selected from propynoyl, 2-butynoyl, 4-pentynoyl, 5-hexynoyl, 6-heptynoyl, 7-octynoyl, and 8-nonynoyl.

[0114] In some embodiments of the present invention, the C4-C10 cycloalkyl group is selected from cyclopropylformyl, cyclobutylformyl, cyclopentylformyl, cyclohexylformyl, 2-methylcyclohexylformyl, and 2,6-dimethylcyclohexylformyl.

[0115] In some embodiments of the present invention, the C7-C16 aryl group is selected from benzoyl, p-methylbenzoyl, m-methylbenzoyl, o-methylbenzoyl, 4-bibenzoyl, 1-naphthoyl, 2-naphthoyl, 1-methyl-2-naphthoyl, 6-methyl-2-naphthoyl, 3-quinolinecarboxyl, 8-quinolinecarboxyl, 9-anthracarboxyl, and 9-acridinylcarboxyl.

[0116] Those skilled in the art can determine the concentration of the compound represented by Formula 1 in the solution during synthesis based on the required amount for online in vivo imaging. In some embodiments of the present invention, the concentration of the compound represented by Formula 1 in the solution is 0.01 to 1000 mmol / L, more preferably 0.1 to 200 mmol / L, and most preferably 1 to 20 mmol / L.

[0117] In some embodiments of the present invention, the volume percentage concentration of BEMP in the solution is 1% to 30%, specifically, the volume percentage concentrations are 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 14%, 16%, 18%, 20%, 24%, 28%, and 30%. Preferably, the volume percentage concentration is 1% to 10%, and more preferably, the volume percentage concentration is 3% to 6%.

[0118] In some embodiments of the present invention, in step (1), after the radioactivity captured by the solution reaches its peak, the introduction of carbon-11 labeled carbon dioxide is stopped. More preferably, after stopping the introduction of carbon-11 labeled carbon dioxide, the reaction is allowed to proceed for 0.5 to 5 minutes, specifically 0.5 minutes, 0.6 minutes, 0.8 minutes, 1 minute, 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, 1.6 minutes, 1.7 minutes, 1.8 minutes, 1.9 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, or 5 minutes. Most preferably, the reaction is allowed to proceed for 1 to 2 minutes.

[0119] In some embodiments of the present invention, the molar ratio of POCl3 to the compound represented by Formula 1 is 1 to 50:1. Specifically, the molar ratio of POCl3 to the compound represented by Formula 1 is 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 12:1, 14:1, 16:1, 18:1, or 20:1. More preferably, the molar ratio is 2 to 20:1, and most preferably, the molar ratio is 3 to 10:1.

[0120] In some embodiments of the present invention, in step (1), after adding POCl3, the reaction is carried out for 0.1 to 3 minutes, specifically 0.1 minutes, 0.2 minutes, 0.3 minutes, 0.4 minutes, 0.5 minutes, 0.6 minutes, 0.7 minutes, 0.8 minutes, 0.9 minutes, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, and 3 minutes. Preferably, the reaction is carried out for 0.2 to 2 minutes, and more preferably, for 0.5 to 1 minute.

[0121] In some embodiments of the present invention, the molar ratio of the compound shown in Formula 4 to the compound shown in Formula 2 is 1:0.1 to 100. More preferably, in order to make full use of the compound shown in Formula 4, an excess of the compound shown in Formula 2 may be added, such as making the molar ratio of the compound shown in Formula 4 to the compound shown in Formula 2 1:1 to 100, for example 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or a range thereof.

[0122] In some embodiments of the present invention, the reaction solvent in step (1) is selected from acetonitrile.

[0123] In some embodiments of the present invention, the intermediates generated in each step of the reaction do not need to be separated and purified, and can be directly used for the next step of processing, that is, steps (1), (2) and / or steps (3) are completed in one pot.

[0124] The one-pot method described in this invention refers to the direct use of the intermediate product obtained in the previous step in the reaction of the next step without separation and purification.

[0125] Step (3) above can be performed using conventional methods in the art to remove the hydroxyl protecting group (R) on the compound shown in Formula 3.

[0126] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0127] The compound shown in Formula 3 was heated to 60–100 °C in an acidic solution and reacted for 1–5 minutes. After cooling and neutralization to neutrality, it was separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0128] In some embodiments of the present invention, the acid solution may be selected from hydrochloric acid with a concentration of 6 mol / L.

[0129] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0130] The compound shown in Formula 3 was mixed with magnesium bromide, heated to 120–180 °C, reacted for 2–7 minutes, cooled, neutralized to neutral, and then separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0131] In some embodiments of the present invention, the hydroxyl protecting group on the compound represented by Formula 3 is removed by the following method:

[0132] The compound shown in Formula 3 was mixed with potassium carbonate, heated to 80–120 °C, reacted for 5–15 minutes, cooled, neutralized to neutral, and then separated and purified by preparative HPLC. The collected eluent was concentrated to dryness to obtain the carbon-11 labeled Larotrectinib compound.

[0133] In some embodiments of the present invention, the method of concentration to dryness may be vacuum concentration to dryness, nitrogen blowing to dryness, or evaporation to dryness.

[0134] In some embodiments of the present invention, Waters C-18 Sep-Pak is used as the eluent for HPLC separation and purification.

[0135] In some embodiments of the present invention, when using a one-pot method, the reaction in step (2) can be quenched with HPLC eluent / water (1 / 1, v / v) before the dehydroxylation protecting group treatment in step (3).

[0136] The chemical structural formula of BEMP described in this invention is as follows: CAS: 98015-45-3.

[0137] This invention also provides an intermediate for synthesizing carbon-11 labeled Larotrectinib compounds, the chemical structural formula of which is as follows:

[0138]

[0139] This invention also provides an intermediate for synthesizing carbon-11 labeled Larotrectinib compounds, the chemical structural formula of which is as follows:

[0140]

[0141] Wherein, R is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, triphenylmethyl, p-methoxytriphenylmethyl, dimethoxytriphenylmethyl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, tetrahydropyran group, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenemethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, tert-pentanoyl, methanesulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16 alkyl, C3-C16 enoyl, C3-C6 ynyl, C4-C10 cycloalkyl. C7-C16 aryl acyl, C4-C10 heterocyclic acyl.

[0142] In some embodiments of the present invention, the C1-C16 alkyl acyl group is selected from formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, and hexadecanoyl.

[0143] In some embodiments of the present invention, the C3-C16 enoyl group is selected from acryloyl, 2-butenoyl, 4-pentenoyl, 5-hexenoyl, 2-ethylacryloyl, 3,3-dimethyl-2-methylenebutyryloyl, 6-heptenoyl, 7-octenyl, 3-methyl-hept-6-enoyl, 5-methyl-hept-6-enoyl, and 8-nonenoyl.

[0144] In some embodiments of the present invention, the C3-C6 acetylacetyl group is selected from propynoyl, 2-butynoyl, 4-pentynoyl, 5-hexynoyl, 6-heptynoyl, 7-octynoyl, and 8-nonynoyl.

[0145] In some embodiments of the present invention, the C4-C10 cycloalkyl group is selected from cyclopropylformyl, cyclobutylformyl, cyclopentylformyl, cyclohexylformyl, 2-methylcyclohexylformyl, and 2,6-dimethylcyclohexylformyl.

[0146] In some embodiments of the present invention, the C7-C16 aryl group is selected from benzoyl, p-methylbenzoyl, m-methylbenzoyl, o-methylbenzoyl, 4-bibenzoyl, 1-naphthoyl, 2-naphthoyl, 1-methyl-2-naphthoyl, 6-methyl-2-naphthoyl, 3-quinolinecarboxyl, 8-quinolinecarboxyl, 9-anthracarboxyl, and 9-acridinylcarboxyl.

[0147] Examples 1-5:

[0148]

[0149] Step 1: [ 11 Synthesis of the intermediate (compound 4) of [C]-3-(isocyanato)pyrazolo[1,5-a]pyrimidine analogue

[0150]

[0151] A 1 mL V-shaped reaction flask was purged with nitrogen (10 mL / min) for 5 minutes, then 100 μL of acetonitrile, 5 μL of BEMP, and 100 μg of compound 1 were added. The mixture prepared by the cyclotron was then added. 11 CO2 was purged into the above acetonitrile solution under a nitrogen flow (10 mL / min), and the radioactivity captured by the solution was measured until it reached its peak using a near-end small radiation detector. After reacting for 1 minute, 100 μl of freshly prepared POCl3 (0.2% v / v) acetonitrile solution was added, and after reacting for 30 seconds, a sample was taken for analysis to obtain compound 4, which was used directly in the next reaction without purification.

[0152] Step Two, [ 11 Preparation of C]-3-R-Larotrectinib compound

[0153]

[0154] In the reaction mixture of step one, 100 μL of acetonitrile solution of compound 2 (0.2 mg) was added. After reacting for 1 minute, a sample was taken for analysis. TLC tracking showed that the labeled precursor compound 4 had essentially disappeared. Radioactive TLC tracking of a 1-2 μL sample of the reaction mixture revealed […]. 11 The product labeled with C]-3-R-Larotrectinib, namely compound 3, was used directly in the next reaction without purification.

[0155] Step 3, [ 11 C]-3-R-Larotrectinib was deprotected to obtain [ 11 C]-Larotrectinib

[0156]

[0157] The reaction in step two was quenched with 750 μL of HPLC eluent / water (1 / 1, v / v). Then, 0.25 mL of 6 N hydrochloric acid was added to the reaction mixture, and the mixture was heated to 80 °C for 3 minutes. The reaction system was cooled in an ice bath at 0 °C and then neutralized to neutral by adding 10% NaHCO3 aqueous solution. The target product was purified by preparative HPLC: the neutralized mixture was diluted with buffer (60:40 CH3CN:H2O + 0.1 N ammonium formate, 2 mL) and passed through Waters C-18 Sep-Pak, which had been activated by washing sequentially with ethanol (1 mL) and water (5 mL). Sep-Pak was washed with water (2 mL), and the desired product was eluted with ethanol (1 mL). After preparative HPLC purification, the eluent of the target product was collected and concentrated to dryness under vacuum at 70 °C. The radiotracer was redissolved in 10 mL of physiological saline containing 0.25% Tween 80 and 5% ethanol, and then filtered through a sterile filter membrane and collected in a sterile, pyrogen-free V-shaped bottle to obtain a sterile, pyrogen-free radiotracer preparation for small animal PET imaging studies.

[0158] Product identification, radiochemical purity, and specific activity were assessed using analytical HPLC with an internal standard under different elution conditions. 810 mCi (29 GBq) was generated using a cyclotron. 11 In CO2, 48-98 mCi (2.1-3.5 GBq) participates in the tracer, with a preparation time of 25-30 min. The radiochemical purity of the product is greater than 97.2%, and the chemical purity is greater than 98.4%. 11 The uncorrected radiochemical yield of C-Larotrectinib was 12.5% ​​relative to that in a V-type reaction flask, and specific activities of 2200-5800 mCi / umol (83.3-208 GBq / umol) were obtained in the final formulation.

[0159] Example 6:

[0160] Step 1: [ 11 Synthesis of the intermediate (compound 4) of [C]-3-(isocyanato)pyrazolo[1,5-a]pyrimidine analogue

[0161] Same as step one in Example 1.

[0162] Step Two, [ 11 Preparation of C]-Larotrectinib compounds

[0163]

[0164] In the reaction mixture of step one, 100 μl of (S)-3-pyrrolidone (0.15 mg) in acetonitrile solution was added. After reacting for 1 minute, a sample was taken for analysis. TLC tracking showed that the labeled precursor compound 4 had essentially disappeared. Radioactive TLC tracking of a 1-2 μl sample of the reaction mixture revealed […]. 11 The product was labeled with [C]-Larotrectinib. The reaction was quenched with 750 μl of ethanol / water (1 / 1, v / v). The mixture was purified by preparative HPLC (the specific procedure is the same as in Example 1). The eluent of the target product was collected and concentrated to dryness under vacuum at 70 °C to obtain [C]. 11 [C]-Larotrectinib compound.

[0165] Examples 7-12:

[0166]

[0167] Step 1, [ 11 Synthesis of C]COF2;

[0168]

[0169] Cyclone accelerator nuclear reaction 14 N(p,α) 11 C generated [ 11 [C]CO2 (3–4 GBq) is passed sequentially through a hot molybdenum target (875°C), a sodium hydroxide absorption bottle, and a liquid nitrogen-cooled stainless steel conical flask containing 0.4 g AgF2. The entire process takes 11–12 minutes. 11 The radiochemical yield (RCY) of C]COF2 was 71 ± 2%. Under a helium flow rate of 5 mL / min, 0.4 g of AgF2 was sufficient to almost quantitatively […]. 11 C]CO is converted to [ 11 [C]COF2. No [C]COF2 was detected in the NaOH system. 11C]COF2 or [ 11 C]CO2 and AgF2 can also be recycled.

[0170] References, preparation [ 11 C]Fluorophosgene.Jimmy E.Jakobsson et al.,(2020).[11C]Carbonyldifluoride-a new and highly efficient[11C]carbonyl group transferagent.Angew.Chem.Int.Ed.,DOI: 10.1002 / anie.201915414

[0171] Step Two, [ 11 Preparation of C]-3-R-Larotrectinib compound

[0172]

[0173] The [prepared in step one] 11 [C]COF2 was bubbled into a 0.5 mL acetonitrile solution containing 1.0 μmol of compound 1 and 1.2 μmol of compound 2 for approximately 5.5 min. The tail gas was then passed into a sodium hydroxide solution. The pipeline was purged with helium gas. Sampling and analysis showed that the TLC-tagged precursor compound 1 had largely disappeared. Radioactive TLC analysis of the reaction mixture (1-2 μL) revealed […]. 11 The C]-3-R-Larotrectinib-labeled product was used directly in the next reaction without purification.

[0174] Step 3, [ 11 Preparation of deprotected [C]-3-R-Larotrectinib [ 11 C]-Larotrectinib

[0175]

[0176] The reaction mixture from step two was quenched with 750 μL of HPLC eluent / water (1 / 1, v / v). Magnesium bromide (0.6 mg) was added to the reaction mixture, and the mixture was heated to 155°C for 5 minutes. The reaction was then cooled in an ice bath at 0°C, and neutralized to neutral by adding 10% NaHCO3 aqueous solution. After preparative HPLC separation and purification (the specific procedure is the same as in Example 1), the eluent of the target product was collected and concentrated to dryness under vacuum at 70°C. The product was redissolved in 10 mL of physiological saline containing 0.25% Tween 80 and 5% ethanol, and the solution was collected in a sterile, pyrogen-free V-shaped flask after filtration through a sterile filter membrane to obtain a sterile, pyrogen-free radiotracer formulation.

[0177] Product identification, radiochemical purity, and specific activity can be evaluated using analytical HPLC with internal standard under different elution conditions. 85 mCi (3-4 GBq) generated by a cyclotron […]. 11 In CO2, 5.5-7.5 mCi (0.2-0.3 GBq) participates in the tracer, with a preparation time of 18-19 min. The product has a radiochemical purity greater than 98.2% and a chemical purity greater than 98.4%. 11 The uncorrected radiochemical yield of C]-Larotrectinib was 10% relative to that in the V-type reaction flask, and a specific activity of 22.2 Ci / μmol was obtained in the final formulation.

[0178] Example 13:

[0179] [ 11 Preparation of deprotected [C]-3-R-Larotrectinib [ 11 C]-Larotrectinib

[0180] The reaction mixture from step 2 of Example 7 was quenched with 750 μL of HPLC eluent / water (1 / 1, v / v). Anhydrous K₂CO₃ (0.86 mg) was added to the reaction mixture, and the mixture was heated to 100°C for 10 min. The reaction was then cooled in an ice bath at 0°C and neutralized to neutral by adding 15 μL of 10% HCl aqueous solution. The reactants were further diluted with buffer (60:40 CH₃CN:H₂O + 0.1N ammonium formate, 2 mL) and passed through Waters C-18 Sep-Pak, which had been activated by washing sequentially with ethanol (1 mL) and water (5 mL). Wash Sep-Pak with water (2 mL), elute the desired product with ethanol (1 mL), rinse into a sterile vacuum bottle, dry under nitrogen at 60°C for 20 minutes, and reconstitute with saline containing 100 μL of a 25% vitamin C aqueous solution and 100 μL of a 20% Tween 80 ethanol solution to obtain […]. 11 C]-Larotrectinib marker injection.

[0181] The sample was analyzed by radiochemical TLC (silica gel plate, 100% ethyl acetate developing layer) to confirm radiochemical transformation (RCC). Product identification and purity were determined by radiochemical HPLC (60:40CH3CN:H2O+0.1N ammonium formate, Phenomenex Luna C-18 column) and radiochemical TLC (silica gel plate, 100% ethyl acetate developing layer). The radiochemical purity of the product was >93%, and the chemical purity was >95%. The product was identified using a co-injection method with radioactive and non-radioactive reference standards, with peak positions confirmed by dual detection using a radioactive detector and a non-radioactive UV detector. The radiochemical yield was determined by adding the amino precursor (compound 1) to acetonitrile diluted […]. 11 C]CO2 or [ 11 The percentage of radioactivity separated as the final product from the activity measure of the C]COF2 solution in the V-tube, without decay correction. 11 The uncorrected radiochemical yield of C]-Larotrectinib relative to that in a V-type reaction flask ([ 11 C]CO2 or [ 11 The C]COF2 content is typically 1.85 mCi, which is 28.56%, and a specific activity of 19.28 Ci / μmol is obtained in the final formulation. The removal rate of the hydroxyl protecting group R is 97%.

[0182] Example 14

[0183] Step 1, [ 11 Synthesis of C]COF2

[0184] Same as step one in Example 3.

[0185] Step Two, [ 11 Preparation of C]-Larotrectinib compounds

[0186]

[0187] The [prepared in step one] 11 [C]COF2 was bubbled through a 0.5 mL acetonitrile solution containing compound 1 (2.5 mg) and (S)-3-pyrrolidone (0.9 mg) for approximately 5.5 min. The tail gas was then passed through a sodium hydroxide solution. The pipeline was purged with helium. Sampling and analysis showed that the TLC-tagged precursor 2 had essentially disappeared. Radioactive TLC-tagged a sample (1-2 μL) of the reaction mixture was detected to contain […]. 11 The target product was labeled with C]-Larotrectinib. After the target product was prepared and purified by HPLC (the specific process is the same as in Example 1), the eluent of the target product was collected and concentrated to dryness under vacuum at 70°C.

[0188] This invention develops a novel TRK PET probe with TRK specificity, which is used to detect the expression of TRK fusion proteins in tumor tissues, thereby providing reliable diagnostic and molecular phenotypic data for clinical examination. It can also be used for in vitro cell receptor and drug molecule screening and evaluation.

[0189] Trk plays a crucial role in the physiology, development, and function of the central and peripheral nervous systems. Normal Trk receptors bind to extracellular ligands, forming dimers and activating the intracellular kinase domain through phosphorylation and activation. This triggers the activation of downstream signaling pathways, promoting cell proliferation and differentiation. NTRK gene rearrangements and fusions are common in various cancers (such as colorectal cancer, non-small cell lung cancer, breast cancer, glioma, astrocytoma, infantile fibrosarcoma, thyroid cancer, and gastrointestinal stromal tumors), encoding various Trk fusion proteins. These fusion proteins can activate downstream pathways and promote tumor formation and cell proliferation even without ligands. Therefore, Trk fusion proteins have become broad-spectrum drug targets and diagnostic biomarkers in the field of cancer.

[0190] Currently, the main clinical techniques for detecting NTRK fusions include NGS sequencing, FISH, and IHC. This invention modifies existing small molecule compounds targeting Trk fusion proteins to create molecular probes that specifically diagnose Trk fusion protein expression. This provides reliable molecular typing data for the molecular diagnosis of tumor characteristics and guides the clinical use of related Trk fusion proteins.

[0191] Example 15 [ 11 Radioactive distribution of C-Larotrectinib in normal mice:

[0192] Experimental protocol

[0193] Male mice were randomly divided into 5 groups of 5 mice each. 37 MBq of [11C]-Larotrectinib imaging agent was injected via the tail vein. Mice were euthanized by decapitation at 5, 15, 30, 45, and 60 minutes. Brain, lung, heart, liver, spleen, stomach, intestine, kidney, and muscle tissues and organs were removed, washed with physiological saline, weighed, and their radioactivity counts (cpm) were measured using a gamma counter. Blood and urine were also collected, weighed, and their radioactivity counts (cpm) were measured using a gamma counter. After time decay correction, the percentage of injected dose per gram of tissue (%ID / g wet tissue) was calculated.

[0194] Experimental results

[0195] Table 1 shows the radiobiological distribution of [11C]-Larotrectinib in normal mice.

[0196] [11C]-Larotrectinib is rapidly absorbed in the body, with the highest distribution in the lungs, heart, liver, kidneys, and brain. The radioactivity distribution of [11C]-Larotrectinib reaches its peak 5 minutes after intravenous injection, and rapidly decreases in all tissues after 15 minutes; 30 minutes after administration, the radioactivity levels in all organs show a significant decrease.

[0197] The radioactivity was moderately distributed in the brain, with 0.988 ± 0.056 ID / g at 5 min, 0.268 ± 0.043% ID / g at 15 min, and 0.123 ± 0.088% ID / g at 30 min, indicating rapid clearance of [11C]-Larotrectinib from the brain. Higher radioactive uptake and distribution were observed in organs such as the lungs, heart, liver, kidneys, and blood. Lower radioactive uptake and distribution were observed in organs such as the spleen, stomach, intestines, muscles, and bones. The radioactivity distribution in liver and kidney tissues at 5 min was 2.654 ± 0.420% ID / g and 1.373 ± 0.312% ID / g, respectively, and at 60 min were 0.231 ± 0.039% ID / g and 0.153 ± 0.165% ID / g, respectively. Clearance from the blood was rapid, with only 0.010 ± 0.001% of the injected dose retained 30 min after injection. 88% of the dose was cleared from the urinary tract within 45 minutes after injection. The tracer showed high hepatic uptake, peaking within 5 minutes at 2.654 ± 0.420% ID / g (0.345 ± 0.003% ID / g within 45 minutes), and rapid hepatic clearance also indicated low absorption in liver tissue. The kidneys are the primary excretory organ for [11C]-Larotrectinib, indicating that [11C]-Larotrectinib is metabolized by the hepatobiliary system and excreted via the renal-urinary system.

[0198] Table 1. Radioactivity distribution of the tracer [11C]-Larotrectinib in normal mice 1 hour after injection (%ID / g±SD, n=5).

[0199]

[0200] Example 16 [11C]-Larotrectinib PET / CT imaging in tumor-bearing mice

[0201] Experimental protocol

[0202] Male tumor-bearing mice (gene-fused human lung adenocarcinoma cell line H2228) were randomly divided into 5 groups of 5 mice each. After being injected with 37 MBq[11C]-Larotrectinib via the tail vein, PET / CT imaging was performed at 5 min, 15 min, 30 min, 45 min and 60 min after injection.

[0203] Experimental results

[0204] Table 2 summarizes the radiobiodistribution of the tracer [11C]-Larotrectinib in tumor-bearing mice. The radioactive material was effectively cleared from the blood, with only 0.010 ± 0.001% of the dose remaining in whole blood. Its primary excretion pathway was very similar to that of normal mice. At 5, 15, 30, and 60 min post-injection, the renal uptake of the tracer was 1.899 ± 0.655, 0.689 ± 0.004, 0.352 ± 0.142, and 0.118 ± 0.112% ID / g, respectively. Compared with the biodistribution data from normal mice, higher radioactive uptake was observed in the liver, with results of 3.864 ± 0.564% ID / g at 5 min post-injection and 0.167 ± 0.012% ID / g at 60 min post-injection. The uptake concentration of the H2228-transplanted tumor reached its maximum (4.545 ± 1.688% ID / g) 15 minutes after injection. At 30 and 45 minutes post-injection, the tracer concentration in the tumor remained high, exceeding that of all normal tissues, retaining 2.888 ± 0.456 and 2.166 ± 1.234% ID / g, respectively, and tumor washing was visible. This indicates that the radioactive clearance of tumor tissue is much slower than that of other positively expressed organs (such as the heart, liver, and kidneys).

[0205] Table 2. Radiobiodistribution of tracer [11C]-Larotrectinib in vivo within 1 hour after injection into tumor-bearing mice (gene fusion human lung adenocarcinoma cell line H2228) (%ID / g±SD, n=5).

[0206]

[0207] The data above show that [11C]-Larotrectinib is rapidly absorbed in the body. At 5 minutes, it is more distributed in the lungs, liver, spleen, kidneys, heart, and brain, and radioactive accumulation is visible in the bladder. At 15 minutes, the radioactivity is mainly distributed in the lungs, heart, liver, and kidneys, with more radioactivity appearing in the bladder. After 30 minutes, the liver and kidney shadows become significantly lighter, and the radioactive accumulation in the bladder also decreases. After 45 minutes, the liver and kidneys are basically not visible, and the radioactive accumulation in the bladder disappears.

[0208] Finally, due to the rapid clearance of radioactive materials from the body, the tumor-to-background ratio was high. For example, the ratios of tumor to blood and tumor to muscle remained at 36.1 and 28.3 times, respectively, from 33.6 and 32.0 times at 15 minutes after injection.

[0209] Micro PET / micro CT imaging of tumor-bearing (H2228) mice using [11C]-Larotrectinib is as follows: Figure 1 As shown, the H2228 xenograft tumor remained clearly visible after 45 minutes, producing high-quality, high-contrast microPET / microCT images with a good tumor-to-background ratio.

[0210] The results indicate that the combination of [11C]-Larotrectinib imaging agent and PET / CT imaging is feasible for tumor assessment. It not only allows for intuitive and dynamic observation of the distribution and metabolic characteristics of [11C]-Larotrectinib imaging agent, but also enables quantitative analysis.

[0211] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a carbon-11 labeled Larotrectinib compound, comprising the following steps: (1) mixing a compound shown in formula 1, a compound shown in formula 2 and carbon-11 labeled fluorine gas to obtain a compound shown in formula 3, Formula 1: , Formula 2: , Formula 3: , wherein, R is hydrogen or a hydroxyl protecting group; the concentration of the compound shown in formula 1 in the solution is 2 mmol / L; the molar ratio of the compound shown in formula 1 to the compound shown in formula 2 is 1:1.2; the time for passing in the carbon-11 labeled fluorine gas is 5.5 min; optionally, when R is a hydroxyl protecting group, removing the hydroxyl protecting group of the compound shown in formula 3; the mixing reaction process comprises: passing in the carbon-11 labeled fluorine gas into the solution containing the compound shown in formula 1 and the compound shown in formula 2.

2. The method of claim 1, wherein: the hydroxyl protecting group is selected from trimethylsilyl, tert-butyldimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, methyl, benzyl, trityl, p-methoxytrityl, dimethoxytrityl, tert-butyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, p-methoxybenzyl, p-methoxybenzyloxymethyl, 3,4-dimethoxybenzyl, tetrahydropyranyl, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, phenylacetyl, tert-butylacetyl, methylsulfonyl, benzylsulfonyl, allylsulfonyl, allyloxycarbonyl, C1-C16alkanoyl, C3-C16alkenoyl, C3-C6alkynoyl, C4-C10cycloalkyl, C7-C16aroyl, C4-C10heterocycloalkanoyl.

3. The method of claim 1, wherein: the reaction solvent of the mixing reaction is selected from acetonitrile.

4. The method of claim 1, wherein: when R is a hydroxyl protecting group, the compound shown in formula 3 is reacted under the action of an acid, potassium carbonate or magnesium bromide at 60-180℃ to remove the hydroxyl protecting group, to obtain the carbon-11 labeled Larotrectinib compound.

5. The method of claim 1, wherein: when R is a hydroxyl protecting group, step (1) and step (2) are completed by one-pot method.

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