A radiotracer targeting pan-KRAS mutant protein, its preparation method and application

Abstract

This invention discloses a radiotracer targeting pan-KRAS mutant proteins, its preparation method, and its applications. The tracer uses small molecule ligands that specifically recognize multiple KRAS mutant proteins as targeting modules, and is coupled to diagnostic radionuclides through an optimized linkage system. This tracer is independent of specific mutation sites and can broadly and specifically bind to multiple high-frequency KRAS mutant subtypes, including G12C, G12D, and G12V. Based on PET / SPECT imaging, this tracer enables rapid, non-invasive, systemic, visualized, and quantitative assessment of KRAS mutant protein expression load in living tumors, overcoming the invasiveness, spatiotemporal limitations, and tumor heterogeneity challenges of traditional biopsies. It provides a novel molecular imaging tool for accurate companion diagnosis of KRAS-mutant tumors, screening of patients benefiting from pan-KRAS inhibitors, and monitoring efficacy, possessing significant clinical translational value.

Description

Technical Field

[0001] This invention relates to the fields of radiopharmaceutical chemistry and nuclear medicine molecular imaging technology, and in particular to a radiotracer targeting pan-KRAS (Pan-KRAS) mutant protein, its preparation method, and its application. Background Technology

[0002] The Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently mutated oncogenes in human malignant tumors and a key factor driving the occurrence and progression of various malignant tumors. Clinical data show that KRAS mutations are widely present in highly lethal malignant tumors such as pancreatic cancer (approximately 90%), colorectal cancer (approximately 40%), and non-small cell lung cancer (approximately 30%). Therefore, KRAS mutations have become a crucial target in the field of tumor diagnosis and treatment.

[0003] Currently, the gold standard for clinical detection of KRAS mutations mainly relies on invasive tissue biopsies combined with gene sequencing and PCR techniques. This genotype-based detection model has inherent limitations: First, biopsies carry the risk of trauma and infection, and it is difficult to monitor KRAS mutation status in real time; second, due to the high spatiotemporal heterogeneity of tumors, single-point biopsies cannot comprehensively reflect the overall mutation status of the lesion tissue, easily leading to missed diagnoses or misdiagnoses; more importantly, this detection method mainly focuses on qualitative analysis at the gene level and cannot quantify the abundance of KRAS mutant protein expression (i.e., mutant protein load) in the tumor in vivo. Therefore, there is an urgent clinical need for a real-time, accurate, comprehensive, and non-invasive method for assessing KRAS mutations.

[0004] Nuclear medicine molecular imaging technology (PET / SPECT) can provide whole-body, non-invasive, real-time visualization of biological targets, making it an ideal means to solve the aforementioned dilemmas. However, due to the smooth surface of the KRAS protein and the lack of deep binding pockets for traditional small molecule drugs, it has long been considered an "undrugable" target. Because of the lack of suitable ligands that can specifically bind to the KRAS protein as radionuclide targeted delivery carriers, the development of nuclear medicine molecular imaging probes targeting KRAS has stagnated, making it difficult to achieve in vivo dynamic monitoring of this target.

[0005] In recent years, significant breakthroughs have been achieved in this field with in-depth understanding of the structure and functional mechanisms of KRAS. Covalent inhibitors targeting specific KRAS-G12C mutations (such as Sotorasib and Adagrasib) have been approved for marketing; meanwhile, pan-KRAS inhibitors capable of broadly binding to multiple KRAS mutations (such as BI-2493 and RMC-6236) have become a focus of research and development and have shown potential in clinical trials. The discovery of these high-affinity ligands has provided a solid foundation for the development of imaging agents. Currently, specific imaging probes based on KRAS-G12C inhibitors have been reported, but due to the covalent binding mechanism, these probes can only recognize KRAS-G12C mutations and cannot recognize other high-frequency mutation subtypes, including G12D, G12V, G13D, and Q61H. Clinically, there is still a lack of a broad-spectrum nuclear medicine molecular imaging probe that can overcome mutation site limitations, target the conserved pocket of pan-KRAS, and simultaneously detect multiple mutation subtypes.

[0006] Therefore, the development of radiotracers using novel pan-KRAS ligands that can broadly identify multiple KRAS mutant proteins and non-invasively quantitatively assess their expression load in vivo is of urgent clinical need and significant scientific value for achieving accurate subtyping of KRAS mutant tumors, screening patients who may benefit from KRAS inhibitors, and real-time monitoring of the efficacy of KRAS inhibitor targeted therapy. Summary of the Invention

[0007] This invention addresses two main challenges of existing clinical gold standards for detection (tissue biopsy combined with gene sequencing): invasiveness, inability to overcome the spatiotemporal heterogeneity of tumors, and the inability to qualitatively detect genotypes while failing to reflect the expression load of mutant proteins. It also overcomes the limitation of existing KRAS imaging probes that can only identify the single G12C mutant subtype. This invention provides a radiotracer that utilizes positron emission tomography (PET) or single-photon emission computed tomography (SPECT) for rapid, non-invasive, dynamic, and quantitative imaging of the expression load of KRAS mutant proteins in living tumors. Furthermore, it describes its application in precise companion diagnosis of KRAS-related tumors, screening patients who benefit from pan-KRAS inhibitors, and monitoring treatment efficacy. The tracer of this invention does not rely on covalent binding to cysteine ​​residues, thus possessing the ability to broadly identify multiple high-frequency KRAS mutant subtypes (such as G12C, G12D, G12V, G12A, G13D, or Q61H) and quantitatively visualize their in vivo expression load.

[0008] In a first aspect, the present invention provides a radiotracer targeting pan-KRAS mutant proteins, which is achieved through the following technical solution.

[0009] A radiotracer targeting pan-KRAS mutant proteins, comprising compounds of general formula (I):

[0010]

[0011] (I)

[0012] in:

[0013] Ring A is selected from substituted phenyl, 5- to 10-membered heteroaryl, or 5- to 10-membered heterocyclic groups; and ring A is optionally substituted by 1 to 3 or fewer substituents: hydroxyl, oxo (=O), halogen, cyano, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 haloalkyl; ring A and its substituents together constitute a small molecule targeting ligand backbone capable of specifically and non-covalently binding to pan-KRAS mutant proteins;

[0014] L represents a functionalized linker group, selected from single bonds or C1-C bonds with or without substitution. 10 Alkylene, polyethylene glycol chain (PEG2-PEG) 12 A linear or branched chain structure composed of one or more groups from the following groups: amide bond, thiourea bond, succinimide group, 1,2,3-triazolyl group, aminocarboxylic acid ester chain, and polypeptide chain; the linking group L is intended to regulate the pharmacokinetic properties of the molecule and provide a suitable site for radiolabeling.

[0015] R is the signal output module, which can be independently selected from any of the following:

[0016] a. A bifunctional chelating group that is not coordinated with a radionuclide;

[0017] b. Complexes formed by the coordination of bifunctional chelating groups with radioactive or non-radioactive metal nuclides;

[0018] c. Non-metallic radioactive isotopes or non-radioactive isotopes that are directly or covalently linked to a linker group L via a prosthetic group.

[0019] As one embodiment of the present invention, a radiotracer targeting pan-KRAS mutant protein may also be selected from pharmaceutically acceptable salts, stereoisomers or prodrugs of compounds represented by general formula (I).

[0020] In a preferred embodiment of the present invention, ring A is selected from substituted phenyl, 5- to 6-membered heteroaryl, or 5- to 6-membered saturated heterocyclic group containing 1-2 nitrogen atoms.

[0021] In a more preferred embodiment of the present invention, ring A is a piperazine ring substituted with a methyl group, wherein one nitrogen atom of the piperazine ring is linked to the pyrimidine ring of the main skeleton of the compound shown in general formula (I) (containing a thiophene-pyrimidine core), and the other nitrogen or carbon atom is linked to a linking group L. This modification site points to the external solvent-exposed region of the pan-KRAS protein, thereby minimizing interference with the non-covalent binding affinity to the core.

[0022] As a specific embodiment of the present invention, the present invention also provides a specific core structure derived from the above general formula, as shown in formula (II):

[0023]

[0024] (II)

[0025] Or its pharmaceutically acceptable salt, stereoisomer, or prodrug;

[0026] Where L and R are defined as above.

[0027] As one embodiment of the present invention, the bifunctional chelating group in the signal output module R includes DOTA, NOTA, HBED-CC, CB-TE2A, TETA, DTPA, DFO, RESCA, RESCA-TFP or Macropa, as well as the above-mentioned bifunctional chelating group skeleton derivatives, the derivatives having branch structures for covalent coupling attached to the skeleton, the branch structures being selected from glutaric acid arms, substituted or unsubstituted benzyl groups, and C1-C6 alkyl groups.

[0028] As one embodiment of the present invention, the nuclides used for positron emission tomography (PET) include 68 Ga、 18 F, 64 Cu、 124 I, 89 Zr; nuclides used in single-photon emission computed tomography (SPECT) include 99 mTc, 123 I, 111 In.

[0029] As a specific embodiment of the present invention, the present invention also provides a radioactive tracer derived from formula (II), the structure of which is as shown in formula (II). As shown in the image:

[0030]

[0031] ( );

[0032] The structure of the radiolabeled precursor is shown in formula (III):

[0033]

[0034] ( ).

[0035] The present invention can also replace the signal output module R in the above-mentioned specific compound with other adapter modules, wherein the adapter module can be any of the following:

[0036] a. A bifunctional chelating agent that matches other radioactive metal nuclides, or a complex formed by coordination of the bifunctional chelating agent with other radioactive nuclides selected from... 64 Cu、 89 Zr、 99m Tc, 111 In;

[0037] b. A nonmetallic radioisotope module directly or covalently linked via a cofactor, wherein the nonmetallic radioisotope is selected from... 18 F, 123 I, 124 I or 131 I.

[0038] Secondly, the present invention provides a method for preparing a radiotracer targeting pan-KRAS mutant protein, which is achieved through the following technical solution.

[0039] The method includes key steps such as radiolabeled precursor synthesis and radiolabeling. For different types of nuclides, this invention provides the following specific labeling strategies:

[0040] 1. For 68 Ga、 64 Cu、 111 Radioactive metal ions such as In:

[0041] A chelating agent coordination method was employed. The targeting group and linker arm were coupled with a bifunctional chelating agent (such as a DOTA derivative) to prepare a labeled precursor. In a specific pH buffer system, the precursor was reacted with a solution of radioactive nuclide produced by elution or cyclotron accelerator, such as […]. 68 Ga]GaCl3、[ 64 Cu]CuCl2 or [ 111 The target product is obtained by mixing In, InCl3, etc., carrying out a coordination reaction at a specific temperature, followed by purification by solid phase extraction or high performance liquid chromatography.

[0042] As a specific embodiment of the present invention, a method for preparing the above-mentioned radiotracer targeting pan-KRAS mutant protein includes the following steps:

[0043] S1. Synthesis of radiolabeled precursors

[0044] a. Mix the pan-KRAS inhibitor BI-2493, BocHN-PEG4-bromide, and N,N-diisopropylethylamine in a molar ratio of 1:(1.0-10):(1.5-20) thoroughly and stir at 50-55 °C for 12-48 hours. After the reaction, separate and purify the product by HPLC, and then obtain the intermediate product BI2493-PEG4-NHBoc by rotary evaporation and lyophilization.

[0045] b. Add 95% trifluoroacetic acid to the prepared BI2493-PEG4-NHBoc, wherein the mass-volume ratio of BI2493-PEG4-NHBoc to trifluoroacetic acid is 1 mg : (10-1000) μL, react for 0.5-2 hours, and after the reaction is completed, separate and purify by HPLC, and obtain the intermediate product BI2493-PEG4-NH2 by rotary evaporation and lyophilization.

[0046] c. Mix the prepared BI2493-PEG4-NH2 with p-SCN-Bn-DOTA at a molar ratio of 1:(1.1-50.0), adjust the pH to 8.5-9.0, and react at 37 ℃ for 1-24 hours; after the reaction is completed, separate and purify by HPLC, and obtain the radiolabeled precursor BI2493-PEG4-DOTA by rotary evaporation and lyophilization.

[0047] S2. Preparation of radioactive tracers

[0048] a. To [Activity of 37-1850 MBq] 68 Sodium acetate solution was added to the Ga]GaCl3 solution to adjust the pH of the reaction system to 4.0-5.0;

[0049] b. Add 10-200 nmol of the radiolabeled precursor BI2493-PEG4-DOTA prepared in step S1 to the solution obtained in step S2a, and react at 90-105 °C for 5-20 minutes; after the reaction, perform solid-phase extraction purification, dilution, and filtration to obtain the target product, wherein the molar activity of the target product is not less than 1 GBq / μmol.

[0050] Preferably, the molar activity of the target product is 13.5-100 GBq / μmol.

[0051] 2. For 18 F

[0052] The primary method employed is nucleophilic fluorination. Precursor molecules with easily leaving groups (such as nitro, trifluoromethanesulfonate, etc.) are designed and synthesized. In the automated synthesis module, activated […] 18[F]fluoride ions undergo nucleophilic substitution reactions with precursors, or via […]. 18 The F prosthetic group is coupled.

[0053] 3. For 99m Tc

[0054] The method employs ligand exchange or direct labeling. In the presence of a reducing agent (such as stannous chloride), the labeled precursor containing suitable coordinating atoms undergoes ligand exchange or direct coordination with pertechnetate to form a stable complex.

[0055] 4. For 123 I, 124 Iodine-1 radionuclides: Electrophilic or nucleophilic substitution strategies are employed. Precursors containing organotin or organoboron groups are designed and synthesized, and then subjected to electrophilic substitution reactions with radioactive iodine in the presence of oxidants (such as NBS or chloramine-T); or radioactive iodine is introduced through isotope exchange.

[0056] Thirdly, the present invention provides the use of a radiotracer targeting pan-KRAS mutant proteins, which is achieved through the following technical solution.

[0057] The use of the above-mentioned radiotracer targeting the pan-KRAS mutant protein in the preparation of products for positron emission tomography or single-photon emission computed tomography, said products comprising any one of the following:

[0058] a. Products for quantitative assessment of KRAS mutant subtypes;

[0059] b. Products for quantitative visualization of the expression load of KRAS mutant proteins in in vivo tumor tissues: By obtaining the radioactive uptake value of the tracer by the living tumor, the expression abundance of KRAS mutant proteins in tumor tissues can be quantitatively visualized.

[0060] c. Products that non-invasively screen patients who may benefit from pan-KRAS inhibitor therapy, especially tumor patients with high expression of KRAS mutant protein;

[0061] d. Products for dynamically monitoring the efficacy of pan-KRAS inhibitor anti-tumor therapy;

[0062] e. Non-invasive imaging products for pan-KRAS-mutant tumors: used for non-invasive whole-body detection of the presence of KRAS-mutant lesions.

[0063] As one embodiment of the present invention, the KRAS mutation subtypes include one or more of G12C, G12D, and G12V.

[0064] Fourthly, the present invention provides a kit for targeting pan-KRAS mutant proteins, which is achieved through the following technical solutions.

[0065] A kit for targeting pan-KRAS mutant protein, comprising the aforementioned radiotracer targeting pan-KRAS mutant protein.

[0066] As a specific embodiment of the present invention, a kit for targeting pan-KRAS mutant protein includes:

[0067] a. Radiolabeled precursors not coordinated with radionuclides, specifically compounds as shown in formula (III);

[0068] b. One or more pharmaceutically acceptable reagents selected from labeled buffers, antioxidants, pH adjusters, or radiochemical stabilizers;

[0069] This kit allows hospital radiology pharmacies to perform rapid, standardized, and immediate labeling operations.

[0070] This application has the following beneficial effects:

[0071] 1. Originality: This invention successfully constructed a radioactive molecular imaging probe targeting pan-KRAS mutant protein, achieving non-invasive visualization of this target for the first time, filling a technological gap in the field, and realizing a shift from invasive genotyping to non-invasive protein functional imaging diagnostic mode.

[0072] 2. Broad-spectrum recognition capability: The tracer of this invention can simultaneously and specifically recognize multiple high-frequency mutant subtypes (G12C, G12D, G12V, G12A, G13D or Q61H, etc.), breaking through the detection limitations of existing KRAS-G12C specific probes and realizing broad-spectrum imaging with one agent for multiple detections.

[0073] 3. Non-invasive whole-body imaging: Through a single PET / SPECT whole-body scan, the expression level of KRAS mutant protein in lesions throughout the patient's body can be non-invasively and panoramically assessed, effectively avoiding the invasive risks, sampling errors, and diagnostic biases caused by the spatiotemporal heterogeneity of tumors in traditional puncture biopsy.

[0074] 4. Dynamic Quantitative Monitoring: The tracer of this invention can dynamically and repeatedly perform non-invasive quantification of KRAS protein load in tumors before and after treatment. By accurately monitoring the molecular response during treatment, it provides an objective basis for dynamically adjusting individualized treatment plans.

[0075] 5. Strong clinical translational potential: This invention provides diversified labeling schemes adapted to PET / SPECT, using core radionuclides (such as...) 68 Ga、 18 F, 99m With a stable supply of Tc and mature labeling technology, it is easy to integrate into the existing clinical radiopharmaceutical production and quality control system, providing a solid foundation for rapid clinical application. Attached Figure Description

[0076] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0077] Figure 1 This is a schematic diagram of the chemical synthesis route of DOTA-pKRASi, the precursor molecule of the targeted pan-KRAS radiotracer in Example 1 of this invention.

[0078] Figure 2 This is the high-resolution mass spectrum of the precursor molecule DOTA-pKRASi in Example 1 of this invention;

[0079] Figure 3 This is a high-performance liquid chromatogram of the precursor molecule DOTA-pKRASi from Example 1 of this invention;

[0080] Figure 4 This is the radioactive tracer of the present invention. 68 The preparation process and quality control results of Ga]Ga-DOTA-pKRASi are shown in the figure. A. Schematic diagram of the radiolabeling process; B. Radio-TLC spectrum, showing that the labeling rate and radiochemical purity are both >99%.

[0081] Figure 5 This is the radioactive tracer of the present invention. 68 Figures showing the in vitro stability and in vivo pharmacokinetic properties of Ga]Ga-DOTA-pKRASi; A. Changes in radiochemical purity at different time points after incubation in 0.01M phosphate-buffered saline (PBS) and 5% human serum albumin (HSA); B. Blood clearance curve and pharmacokinetic parameter fitting plot after intravenous injection in healthy mice.

[0082] Figure 6 This invention [ 68 Ga-DOTA-pKRASi uptake diagrams in cell lines with different KRAS mutation states; where A. probe uptake in KRAS-G12C mutant cell lines (MIA PaCa-2 and BXPC-3). KRAS-G12C B. In vitro uptake curves of the probe over time in KRAS-G12D mutant cell lines (ASPC-1 and BXPC-3) and wild-type control cell lines; KRAS-G12DC. In vitro uptake curves of the probe over time in KRAS-G12V mutant cell lines (SW620 and BXPC-3) and wild-type control cell lines; KRAS-G12V D. In vitro uptake curves over time in the wild-type control cell line (BXPC-3); E. Comparison of 2-hour uptake of the probe in each endogenous KRAS mutant cell line and its corresponding exogenous overexpression / transfected cell line; KRAS-G12C BXPC-3 KRAS-G12D BXPC-3 KRAS-G12V F. Results of 2-hour specific blocking assays of the probe in three endogenous KRAS mutant cell lines (MIA PaCa-2, ASPC-1, SW620);

[0083] Figure 7 This invention [ 68 Micro-PET / CT imaging of Ga]Ga-DOTA-pKRASi in healthy Kunming mice at different time points; where, A. representative Micro-PET / CT images of the probe at different time points after injection (30 minutes, 60 minutes, 120 minutes and 240 minutes); B. bar chart of radioactive uptake values ​​(%ID / mL) of major organs (heart, liver, lung, kidney and muscle) at corresponding time points obtained from quantitative analysis of imaging results;

[0084] Figure 8 This invention [ 68 Biodistribution data of Ga]Ga-DOTA-pKRASi in healthy mice in Kunming;

[0085] Figure 9 This invention [ 68 Micro-PET / CT images of Ga]Ga-DOTA-pKRASi in nude mouse models carrying KRAS-G12C mutant tumors (conventional and overexpression);

[0086] Figure 10 This invention [ 68 Micro-PET / CT images of Ga]Ga-DOTA-pKRASi in nude mouse models carrying KRAS-G12D mutant tumors (conventional and overexpression);

[0087] Figure 11 This invention [ 68 Micro-PET / CT images of Ga]Ga-DOTA-pKRASi in nude mouse models carrying KRAS-G12V mutant tumors (conventional and overexpression);

[0088] Figure 12 It is in the tumor tissue of this invention 68 The correlation analysis results of Ga-DOTA-pKRASi PET uptake and KRAS mutant protein expression levels are shown in the figure. Among them, A.KRAS-G12C mutant tumor tissues (MIA PaCa-2 and BX PC-3) KRAS-G12C A. Immunohistochemical staining image of tumor tissue; B. Linear correlation analysis of probe PET uptake (%ID / mL) and relative expression level of KRAS-G12C in tumor tissue; C. KRAS-G12D mutant tumor tissues (ASPC-1 and BX PC-3) KRAS-G12D Immunohistochemical staining images of tumor tissues; D. Linear correlation analysis of probe PET uptake (%ID / mL) and relative expression level of KRAS-G12D in tumor tissues; E. KRAS-G12V mutant tumor tissues (SW620 and BX PC-3) KRAS-G12V Immunohistochemical staining image of ) ; F. Linear correlation analysis of probe PET uptake (%ID / mL) and relative expression level of KRAS-G12V in tumor tissue;

[0089] Figure 13 This invention [ 68 Safety evaluation results of Ga]Ga-DOTA-pKRASi (blood routine results);

[0090] Figure 14 This invention [ 68 Safety evaluation results of Ga]Ga-DOTA-pKRASi (blood biochemistry test results). Detailed Implementation

[0091] Preferred embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Before description, it should be understood that the terminology used in the specification and appended claims should not be construed as limited to its general and dictionary meaning, but rather should be interpreted based on the principle of allowing the inventors to appropriately define the terminology for the best interpretation, and based on its meaning and concept corresponding to the technical level of the invention. Therefore, the description herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention; thus, it should be understood that other equivalent implementations and modifications can be made without departing from the spirit and scope of the invention.

[0092] The invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can all be purchased from relevant material sales companies.

[0093] Example 1: Synthesis of the radiolabeled precursor DOTA-pKRASi

[0094] This embodiment describes in detail the synthesis method of the compound represented by general formula (II). The specific synthesis method is as follows:

[0095] Step 1: As Figure 1 As shown, the pan-KRAS inhibitor (S)-2-amino-3'-(2-((S)-2-methylpiperazin-1-yl)pyrimidin-4-yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carboxynitrile (BI-2493) (CAS No.: 2937344-16-4) was used as the starting material. First, 50 mg of BI2493 was weighed and dissolved in 5 mL of ultra-dry acetonitrile. Then, 2.0 eq of (14-bromo-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl ester (BocHN-PEG4-bromide) (CAS No.: 1392499-32-9) and 3.0 eq of N,N-diisopropylethylamine (DIPEA) were added, and the mixture was thoroughly mixed. The mixture was stirred at 50 °C for 16 hours. After the reaction was completed, the product was separated and purified by HPLC, and obtained by rotary evaporation and lyophilization to yield the intermediate product BI2493-PEG4-NHBoc.

[0096] Step 2: Take 50 mg of BI2493-PEG4-NHBoc obtained in Step 1, add 1 mL of 95% trifluoroacetic acid (TFA), react for 1 hour to remove the tert-butyloxycarbonyl (Boc), and after the reaction is completed, use HPLC to separate and purify, rotary evaporate and freeze dry to obtain the intermediate product BI2493-PEG4-NH2;

[0097] Step 3: Weigh 50 mg of the intermediate product BI2493-PEG4-NH2 obtained in Step 2 into a 2 mL vial, add 0.5 mL of ultra-dry N,N-dimethylformamide (DMF) to dissolve it completely, then add 202.3 mg of chelating agent 2,2',2'',2'''-(2-(4-isothiocyanobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (p-SCN-Bn-DOTA) (CAS No.: 127985-74-4), adjust the pH to 8.5 using DIPEA, and react at 37 ℃ for 2 hours. After the reaction is complete, separate and purify the product using HPLC, then rotary evaporate and lyophilize to obtain the radiolabeled precursor BI2493-PEG4-DOTA, named DOTA-pKRASi.

[0098] The structure of the obtained compound DOTA-pKRASi was determined by high-resolution mass spectrometry (HMS). Figure 2 ) and analytical high performance liquid chromatography (HPLC) Figure 3 It was confirmed that the purity met the requirements for subsequent radiolabeling.

[0099] Example 2: Radioactive tracer [ 68 Preparation and Quality Control of Ga-DOTA-pKRASi

[0100] This embodiment provides [ 68 The standard operating procedure for radioactive labeling and quality control of Ga-DOTA-pKRASi is as follows:

[0101] 1. Labeling reaction:

[0102] (1) Use 3 mL of 0.05 M hydrochloric acid from 68 Ge / 68 The Ga generator was rinsed out of [ 68 Ga]GaCl3 solution;

[0103] (2) Add 180 µL of 1 mol / L sodium acetate solution to the reaction flask to adjust the pH of the reaction system to 4.5;

[0104] (3) Add the labeled precursor DOTA-pKRASi (100 µg) prepared in Example 1 and mix well;

[0105] (4) React at 95 °C for 15 minutes.

[0106] 2. Solid-phase extraction purification:

[0107] (1) After the reaction is complete, add 5 mL of pure water to cool the reaction solution;

[0108] (2) Load the reaction solution onto a Sep-C18 Light solid-phase extraction column pretreated with ethanol and water;

[0109] (3) Pass the column through 5 mL of pure water to remove free radicals. 68 Ga;

[0110] (4) Elute the target product with 0.5 mL of 80% ethanol aqueous solution.

[0111] 3. Formulation preparation:

[0112] The eluent was diluted with 5 mL of physiological saline to bring the final ethanol concentration to below 10% (v / v), and then filtered through a 0.22 µm sterile filter membrane to obtain a clear, sterile, pyrogen-free solution. 68 Ga]Ga-DOTA-pKRASi injection.

[0113] 4. Quality Control:

[0114] The product was analyzed using radio-TLC, and the results showed that ( Figure 4(B) The product's labeling rate and radiochemical purity (RCP) are greater than 99%, meeting the standards for in vivo application.

[0115] Performance testing

[0116] I. Evaluation of in vitro stability and pharmacokinetics

[0117] In vitro stability: 3.7 MBq [ 68 Ga-DOTA-pKRASi was incubated with 0.01 M phosphate-buffered saline (PBS, pH 7.4) and 5% human serum albumin (HSA) at 37 °C. 2 μL samples were taken at 0, 30, 60, and 120 minutes, and the radiochemical purity was determined by Radio-TLC (stationary phase: silica gel impregnated glass fiber strip; developing solvent: saturated EDTA and physiological saline 1:1). The results showed ( Figure 5 After incubation for 2 hours under the above conditions, the radiochemical purity of the tracer remained above 95%, indicating that the tracer has excellent in vitro stability.

[0118] Blood pharmacokinetics: Healthy Kunming rats (n=5, 4-5 weeks old, 18-20 g) were injected intravenously via the tail vein with 100 μL of […]. 68 Ga]Ga-DOTA-pKRASi probe (3.7 MBq) was used to collect blood samples from the orbital cavity at 1, 5, 10, 15, 30, 45, 60, 90, 120, and 240 minutes after drug administration. The radioactivity of each tube was detected using a gamma counter, and the percentage injection dose rate per unit mass (%ID / g) was calculated at each time point. The pharmacokinetic parameters were analyzed using GraphPad Prism 9.0 software.

[0119] The results show that ( Figure 5 The probe (t1 / 2β) conforms to the characteristics of a two-compartment model, with a distribution half-life (t1 / 2α) of 1.68 minutes and an elimination half-life (t1 / 2β) of 89.77 minutes. These results indicate that the probe can be rapidly cleared from the blood, which is beneficial for increasing the target-to-background ratio and makes it suitable for PET imaging.

[0120] II. In vitro cell uptake and specificity verification

[0121] This experiment aims to verify the probe's ability to recognize general KRAS.

[0122] Cell models: BXPC-3 (WT), MIAPaCa-2 (G12C), ASPC-1 (G12D), SW620 (G12V), and constructed BXPC-3 KRAS-G12C / D / VOverexpression cell lines. The specific construction process of the above-mentioned overexpression cell lines was as follows: Wild-type BXPC-3 cells in logarithmic growth phase were seeded in culture dishes and transfected using recombinant lentiviruses customized by Heyuan Biotechnology Co., Ltd., carrying KRAS-G12C, KRAS-G12D, or KRAS-G12V mutant genes, respectively; during transfection, 1 mg / mL of lentiviral infection adjuvant (Polybrene) was added to improve infection efficiency; after transfection, the medium was replaced with complete medium containing 1 μg / mL puromycin for pressure selection, and the culture was continuously changed to remove uninfected cells, finally amplifying to obtain cell lines stably overexpressing the corresponding KRAS mutant protein. Polybrene and puromycin used in the transfection process were both purchased from Heyuan Biotechnology Co., Ltd.

[0123] Experimental method: After culturing the above cells to the logarithmic growth phase, they were seeded into 24-well plates, 2 × 10⁶ cells per well. 5 Cells were cultured overnight, and the culture medium in the 24-well plate was discarded. The cells were washed twice with pre-cooled PBS (0.01 M, pH 7.4) buffer. [The experimental group was then treated with...] 68 Ga-DOTA-pKRASi probe (approximately 74 kBq / well, i.e., 2 µCi / well), with an additional 100 μg of the radiolabeled precursor (DOTA-pKRASi) prepared in Example 1 added to the blocking group; after incubation in an incubator for 10, 30, 60, and 240 minutes, the supernatant was removed, and the cells were washed twice with cold PBS. 200 μL of NaOH (1.0 M) was added to the corresponding wells for cell lysis, and the cells were collected into γ-counter tubes. The radioactivity of each well was detected using a γ-counter. Uptake rate is expressed as %AD / 10. 6 cells.

[0124] like Figure 6 As shown, the probe was taken up in cells with three common mutant types: G12C, G12D, and G12V, confirming its pan-KRAS binding characteristics. Furthermore, the uptake in the overexpressing strain was significantly higher than in the common mutant and WT strains. In the 2-hour blocking experiment, excessive cryotherapy significantly inhibited probe uptake, demonstrating that the probe specifically binds to the KRAS mutant protein.

[0125] III. Micro-PET / CT Experiment in Healthy Mice

[0126] Injecting [the substance] into the tail vein of healthy Kunming rats 68The Ga-DOTA-pKRASi (4.5 MBq) probe was used, and Micro-PET / CT images were acquired at 30, 60, 120, and 240 minutes post-injection. The image acquisition was performed using a PET / SPECT / CT trimodal imaging system from Medtronic, with each scan lasting 10 minutes. 2% isoflurane anesthesia was used throughout the acquisition process. After acquisition, attenuation correction and reconstruction (CT-AC reconstruction) were performed based on the CT data using the instrument's built-in software. Regions of interest (ROIs) were delineated using P-MOD data processing software, and uptake data for major organs (such as the heart, liver, and lungs) were plotted. Statistical analysis was performed using GraphPad Prism 9.0 software.

[0127] The results show that ( Figure 7 The probe is primarily metabolized through the hepatobiliary system, excreted via the intestines, and partially cleared through the kidneys and urinary system. It exhibits extremely low uptake in non-target tissues such as muscle and brain, resulting in a clean background and facilitating the highlighting of tumor lesions.

[0128] IV. Biodistribution of healthy mice

[0129] 3.7 MBq was injected intravenously into the tail vein of healthy female Kunming rats. 68 The mice were injected with Ga]Ga-DOTA-pKRASi and sacrificed at 30, 60, 120 and 240 minutes after injection. The main organs and tissues were removed, weighed and the radioactivity count was measured using a gamma counter.

[0130] In vitro biological distribution experiment ( Figure 8 The results of the Micro-PET / CT experiments were further quantified. The probe was distributed in the liver, intestines, and kidneys, while its radioactivity in the blood, muscles, and bones decreased rapidly over time. This confirms that the probe has good in vivo clearance kinetics.

[0131] V. Micro-PET / CT Experiment in Tumor-Bearing Rats

[0132] This experiment demonstrates the probe's ability to broadly identify different mutations and quantitatively assess protein load at the in vivo level.

[0133] Five-week-old female BALB / c nude mice (weighing 15-17 g) were selected to construct a system carrying MIAPaCa-2 (G12C), ASPC-1 (G12D), SW620 (G12V), and BXPC-3. KRAS-G12C BXPC-3 KRAS-G12D BXPC-3 KRAS-G12V Subcutaneous xenograft model in nude mice. 5 × 10⁵ tumor cells were injected subcutaneously into the right axilla of mice. 6The above-mentioned tumor cells in logarithmic growth phase (suspended in 100 μL PBS) were used. Mice were observed daily after inoculation, and tumor growth was monitored. When the tumor diameter reached 0.8-1.0 cm, 3.7 MBq of [a specific drug / method] was injected via the tail vein. 68 Ga]Ga-DOTA-pKRASi was used to acquire Micro-PET / CT images at different time points (30, 60, 120, and 240 minutes) after injection.

[0134] like Figures 9-11 As shown, the tracer exhibits significant radioactive accumulation at the tumor site, and the uptake in the overexpression model is significantly higher than that in the conventional mutation model. In vivo imaging results are consistent with in vitro cell experiments, directly confirming that the tracer provided by this invention can non-invasively and specifically visualize different KRAS mutation subtypes and can differentiate the KRAS protein expression load of tumors based on uptake intensity.

[0135] VI. In tumor tissue 68 Correlation analysis between Ga-DOTA-pKRASi PET uptake and KRAS mutant protein expression level

[0136] This experiment aims to confirm, through the "gold standard" of in vitro pathology, that the uptake signal of the radioactive tracer provided by this invention at the in vivo level can accurately and quantitatively reflect the actual expression load of KRAS mutant proteins in tumor cells.

[0137] Experimental methods:

[0138] (1) Tissue sampling and processing: After the above-mentioned micro-PET / CT in vivo imaging of tumor-bearing mice, the tumor-bearing mice in each group (conventional mutation group and G12C / D / V overexpression group) were euthanized immediately. The tumor tissue was completely removed, weighed and fixed in 10% neutral formalin solution for 24 hours, followed by routine dehydration, clearing, paraffin embedding and paraffin embedding.

[0139] (2) Immunohistochemical (IHC) staining: Paraffin-embedded tumor tissue was serially sectioned (4 µm thick). After routine off-target treatment, hydration, and antigen retrieval, a specific anti-KRAS primary antibody (Anti-pan-KRAS antibody, Abcam, ab275875) was added and incubated overnight at 4 °C. After washing, HRP-labeled secondary antibody was added and incubated at room temperature. Finally, DAB staining kit was used for staining, hematoxylin counterstaining of cell nuclei, and dehydration and mounting were performed.

[0140] (3) Data collection and quantitative analysis:

[0141] In vitro protein quantification: IHC images were acquired and analyzed using ImageJ software to calculate the percentage of cells positive for KRAS protein, which served as an indicator of the true expression load of mutant proteins in histological studies.

[0142] Quantitative analysis of in vivo images: From the Micro-PET / CT data of tumor-bearing mice mentioned above, the region of interest (ROI) of the tumor site of each mouse was delineated, and its quantitative analysis data was obtained.

[0143] (4) Statistical correlation analysis: The PET in vivo quantitative data of each mouse were matched one-to-one with the corresponding IHC in vitro quantitative data, and linear correlation analysis was performed using GraphPad Prism software.

[0144] Experimental results are as follows Figure 12 As shown, the immunohistochemical (IHC) pathological staining results are consistent with the signal intensity of in vivo Micro-PET images. In tumor sections of the conventional mutation group, the brownish-yellow positive staining of the KRAS mutant protein was weak and relatively scarce, reflecting its basal endogenous expression level; while diffuse brownish-yellow strong positive staining was observed in tumor tissue sections of each overexpression group, confirming the high abundance of the target protein in the model from the pathological "gold standard" level. Further statistical correlation analysis revealed a significant linear positive correlation between the in vivo radioactive uptake of this tracer at the tumor site and the proportion of KRAS-positive cells in ex vivo tumor tissue (R0). 2 > 0.9, P < 0.001).

[0145] The above results indicate that the targeted pan-KRAS radiotracer provided by this invention can accurately and realistically reflect the expression load of KRAS mutant proteins in tumor cells by providing specific uptake signals in living tumors.

[0146] seven,[ 68 Security evaluation of Ga-DOTA-pKRASi

[0147] This experiment aims to evaluate the potential acute toxicity of the tracer described in this invention to experimental animals under high-dose administration conditions, with a focus on its effects on the blood system and liver and kidney function.

[0148] Animal grouping: Twenty healthy Kunming rats (half male and half female, 6-8 weeks old) were randomly and evenly divided into two groups:

[0149] Control group: 100 µL of phosphate-buffered saline (PBS) was injected via the tail vein.

[0150] Experimental group: 100 µL containing 2.46 mCi (approximately 91 MBq) of [ ] was injected via the tail vein. 68Ga]Ga-DOTA-pKRASi solution (dosage setting: based on the proposed human clinical imaging dose of 0.1 mCi / kg, the interspecies dose conversion was performed according to the body surface area method recommended by the FDA. The chemical mass dose received by the experimental group mice was equivalent to 100 times the standard human dose).

[0151] On days 1 and 7 after drug administration, five mice from each group were collected blood via enucleation. A portion of the whole blood was anticoagulated with EDTA and used for routine blood tests, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), and platelet count (PLT). The other portion of blood was centrifuged to separate serum for blood biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA-S), and uric acid (UA) to assess liver and kidney function.

[0152] Blood routine test data such as Figure 13 As shown, on days 1 and 7 after injection of a 100-fold overdose probe, the WBC, RBC, HGB, and PLT levels in the experimental group mice were all within the normal reference range, and there was no statistically significant difference compared with the PBS control group (P > 0.05). This indicates that the tracer has no significant inhibitory or destructive effect on bone marrow hematopoietic function.

[0153] Blood biochemistry results as follows Figure 14 As shown, serum ALT, AST, UA, and CREA-S levels in the experimental group mice did not show abnormal increases at either time point, and there was no statistically significant difference compared with the PBS control group (P > 0.05), indicating that high-dose administration did not cause acute liver and kidney toxicity or liver and kidney dysfunction.

[0154] The above results indicate that even at extremely high doses, equivalent to 100 times the human imaging dose, [ 68 Ga]Ga-DOTA-pKRASi continued to demonstrate excellent biocompatibility, without causing significant systemic toxicity, hematological toxicity, or liver and kidney damage. This result provides a reliable safety basis for subsequent clinical trials.

[0155] In summary, this invention comprehensively verifies the feasibility, broad-spectrum specificity, safety, and clinical translational potential of the radiotracer targeting pan-KRAS mutant proteins provided by this invention from multiple dimensions, including chemical synthesis, radiolabeling, in vitro stability, cellular uptake experiments, in vivo pharmacokinetics and imaging of various tumor-bearing models, and drug safety.

[0156] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A radiotracer targeting pan-KRAS mutant protein, characterized in that: The radiolabeled precursor of the radioactive tracer is shown in formula (III): ； The structure of the radioactive tracer is shown in formula (IV): 。 2. A method for preparing the radiotracer targeting the pan-KRAS mutant protein as described in claim 1, characterized in that: Includes the following steps: S1. Synthesis of radiolabeled precursors a. Mix the pan-KRAS inhibitor BI-2493, BocHN-PEG4-bromide, and N,N-diisopropylethylamine thoroughly in a molar ratio of 1:(1.0-10.0):(1.5-20.0), and stir at 50-55 °C for 12-48 hours. After the reaction, separate and purify the product by HPLC, and then obtain the intermediate product BI2493-PEG4-NHBoc by rotary evaporation and lyophilization. b. Add 95% trifluoroacetic acid to the prepared BI2493-PEG4-NHBoc, wherein the mass-volume ratio of BI2493-PEG4-NHBoc to trifluoroacetic acid is 1 mg : (10-1000) μL, react for 0.5-2 hours, and after the reaction is completed, separate and purify by HPLC, and obtain the intermediate product BI2493-PEG4-NH2 by rotary evaporation and lyophilization. c. Mix the prepared BI2493-PEG4-NH2 with p-NCS-Bz-DOTA at a molar ratio of 1:(1.1-50.0), adjust the pH to 8.5-9.0, and react at 37 ℃ for 1-24 hours; after the reaction is completed, separate and purify by HPLC, and obtain the radiolabeled precursor BI2493-PEG4-DOTA by rotary evaporation and lyophilization. S2. Preparation of radioactive tracers a. To [Activities of 37-1850 MBq] 68 Sodium acetate solution was added to the Ga]GaCl3 solution to adjust the pH of the reaction system to 4.0-5.0; b. Add 10-200 nmol of the radiolabeled precursor BI2493-PEG4-DOTA prepared in step S1 to the solution obtained in step S2a, and react at 90-105℃ for 5-20 minutes; after the reaction, perform solid-phase extraction purification, dilution, and filtration to obtain the target product, wherein the molar activity of the target product is not less than 1 GBq / μmol.

3. The use of the radiotracer targeting the pan-KRAS mutant protein as described in claim 1 in the preparation of products for positron emission tomography or single-photon emission computed tomography, characterized in that: The product is selected from any one of the following: a. Products for quantitative assessment of KRAS mutant subtypes; b. Products that quantitatively visualize the expression load of KRAS mutant proteins in in vivo tumor tissues; c. Non-invasive imaging products for pan-KRAS-mutant tumors.

4. The application according to claim 3, characterized in that: KRAS mutant subtypes are selected from one or more of G12C, G12D, and G12V.

5. A kit for targeting pan-KRAS mutant protein, characterized in that: The radiotracer targeting the pan-KRAS mutant protein as described in claim 1.

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