Fe-based t7 peptide targeting brain glioma small molecule magnetic resonance contrast agent and its preparation and application

Abstract

This invention discloses a Fe-based T7 peptide-targeting small-molecule magnetic resonance imaging (MRI) contrast agent for gliomas, its synthesis method, and its applications. The small-molecule MRI contrast agent comprises a targeting peptide and an iron-coordinated pyridine ring contrast agent, linked by a click reaction. The T7 peptide portion of this small-molecule MRI contrast agent can bind to transferrin receptors, allowing for more efficient delivery to the brain for imaging. Compared to traditional Gd-based contrast agents, the MRI contrast agent of this invention exhibits higher specificity and lower toxicity, while also demonstrating better stability, enabling efficient imaging of in situ gliomas.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging technology, specifically relating to a Fe-based T7 peptide-targeting small molecule magnetic resonance contrast agent for gliomas, its synthesis method, and its application. Background Technology

[0002] According to the UK's 2023 five-year cancer survival rate statistics, the five-year survival rate for brain tumors is significantly lower than that of other common cancers, such as lung cancer, breast cancer, and stomach cancer. The five-year survival rate for brain tumor patients is only 12.8%, and gliomas, as diffuse and highly malignant brain tumors, are receiving increasing attention. Clinically, gliomas are classified into four grades based on their malignancy, with WHO grade 4 glioblastoma being the most malignant, having a five-year survival rate of less than 3%.

[0003] Magnetic resonance imaging (MRI) is currently the most commonly used and effective clinical examination method. It has unique advantages such as no radiation damage and the ability to visualize vascular structures, making it applicable to various diseases in almost all systems of the body. Its imaging capabilities for the brain are superior to CT scans. Furthermore, it possesses excellent soft tissue imaging capabilities, sensitively detecting changes in water content within tissue components, allowing for earlier and more effective detection of lesions than CT scans. Therefore, it is the most crucial technology in the diagnosis and treatment of gliomas.

[0004] Currently, the contrast agents commonly used in glioma examination are mostly Gd-based magnetic resonance imaging (MRI) contrast agents, such as Magnevist, Dotarem, and Prohance. However, these contrast agents face challenges such as difficulty crossing the blood-brain barrier and metabolic issues. In patients with kidney disease, the use of certain Gd chelates can lead to a condition called renal systemic fibrosis. Therefore, we are seeking a novel contrast agent as an alternative to Gd-based contrast agents.

[0005] Iron has been extensively studied as a coordinating metal, and many iron-coordinating contrast agents such as Fe-CDTA have been developed. However, these iron-coordinating contrast agents, like Gd-based contrast agents, lack specificity, resulting in a short imaging window for gliomas and poor imaging performance. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a Fe-based T7 peptide-targeting small-molecule magnetic resonance imaging (MRI) contrast agent for gliomas, its synthesis method, and its applications. The MRI contrast agent of this invention comprises a targeting peptide and an iron-coordinated pyridine ring contrast agent, linked by a click reaction. The T7 targeting peptide can bind to transferrin receptors, allowing for more efficient delivery to the brain for imaging. Compared to traditional Gd-based contrast agents, the MRI contrast agent of this invention exhibits higher specificity and lower toxicity. Furthermore, the targeting probe demonstrates good stability, enabling efficient imaging of gliomas in situ.

[0007] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0008] A Fe-based T7 peptide-targeting small molecule magnetic resonance imaging agent for gliomas, with the following structural formula:

[0009]

[0010] A method for synthesizing a Fe-based T7 peptide-targeting small molecule magnetic resonance imaging contrast agent for gliomas, comprising the following steps:

[0011] S1. Under alkaline conditions, N-Boc cyclohexanediamine undergoes a substitution reaction with benzyl bromide to synthesize compound (I), the reaction formula of which is as follows:

[0012]

[0013] S2. Under acidic conditions, compound (I) undergoes ester hydrolysis, removing the Boc protecting group to generate compound (II), as shown in the following reaction formula:

[0014]

[0015] S3. Under alkaline conditions, compound (II) undergoes a substitution reaction with tert-butyl bromoacetate to produce compound (III), as shown in the following reaction equation:

[0016]

[0017] S4. In the presence of a catalyst, compound (III) undergoes a hydrogenation reaction to remove the benzyl group, yielding compound (IV), as shown in the following reaction formula:

[0018]

[0019] S5. Under alkaline conditions, compound (IV) undergoes a substitution reaction with 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine to produce compound (V), as shown in the following reaction formula:

[0020]

[0021] S6. Under the condition of an acidic catalyst, compound (V) undergoes ester hydrolysis to remove the tert-butyl group, generating compound (VI), as shown in the following reaction formula:

[0022]

[0023] S7. In the presence of a reducing agent and a divalent copper salt, compound (VI) undergoes a click chemical reaction with a T7 peptide linked to an azide group to generate compound (VII), as shown in the following reaction equation:

[0024]

[0025] S8. Under weakly acidic conditions, compound (VII) undergoes a coordination reaction with a ferric salt to form compound (VIII), as shown in the following reaction equation:

[0026]

[0027] Furthermore, in step S1, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 20-40℃, the reaction time is 8-12 hours, and the molar ratio of N-Boc cyclohexanediamine, benzyl bromide and potassium carbonate is 1.0-1.2:7.0-7.4:3.0-3.3.

[0028] Furthermore, in step S2, the acid is hydrochloric acid, the reaction solvent is ethyl acetate, the reaction temperature is 20-40℃, and the reaction time is 8-12 hours.

[0029] Furthermore, in step S3, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 65℃-75℃, the reaction time is 8-12 hours, and the molar ratio of compound (II), tert-butyl bromoacetate and potassium carbonate is 1.0-1.2:3.3-3.5:1.0-1.3.

[0030] Furthermore, in step S4, the catalyst is 10% Pd / C, the reaction solvent is methanol, the reaction temperature is 20℃-30℃, the reaction pressure is 0.5-1.5MPa, and the reaction time is 8-12 hours.

[0031] Furthermore, in step S5, the base is a mixture of potassium carbonate and potassium iodide, the reaction solvent is N,N-dimethylformamide, the reaction temperature is 65℃-75℃, the reaction time is 8-12 hours, and the molar ratio of compound (IV), 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine, potassium carbonate, and potassium iodide is 1.0-1.1:2.0-2.2:5.0-5.5:2.0-2.2.

[0032] Furthermore, in step S6, the acidic catalyst is trifluoroacetic acid, the reaction temperature is 20℃-25℃, and the reaction time is 8-12 hours.

[0033] Furthermore, in step S7, the reducing agent is sodium ascorbate, the divalent copper salt is copper sulfate, the reaction temperature is 20℃-40℃, the reaction time is 6-8 hours, and the molar ratio of compound (VI), T7-N3 peptide, sodium ascorbate and copper sulfate is 1.0-1.1:1.0-1.1:1.0-1.1:1.5-1.7.

[0034] Furthermore, in step S8, the pH of the reaction system is 5.5-6.0, the ferric salt is ferric chloride or its hydrate, the reaction temperature is 20-40℃, and the time is 6-8 hours. The compound of formula (VII) reacts with Fe... 3+ The molar ratio is 1.0-1.1:1.50-1.75.

[0035] Application of a Fe-based T7 peptide-targeting small molecule magnetic resonance contrast agent for gliomas in the preparation of magnetic resonance contrast agents.

[0036] Furthermore, the magnetic resonance imaging contrast agent can target gliomas.

[0037] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0038] 1. The contrast agent of the present invention has high stability. We characterized a series of properties by testing the relaxation rate of the molecule, including the relaxation rate at different pH levels. Through the above experiments, we have confirmed that the contrast agent of this molecule has strong stability below pH=9.

[0039] 2. Experiments show that the contrast agent of the present invention has a certain ability to cross the blood-brain barrier. An in vitro blood-brain barrier model was established by transwell, and the ability of the contrast agent to cross the blood-brain barrier was confirmed after incubation.

[0040] 3. Experiments show that the contrast agent of this invention can image in situ gliomas. Compared with the imaging ability without the added targeting peptide, we found that the introduction of the targeting peptide can prolong the imaging window to a certain extent, and the imaging effect is superior to Magnevis. Simultaneously, the signal in the tumor region can be enhanced by 62%, which can delineate the tumor boundary and provide information about the tumor's locating position.

[0041] 4. The contrast agent of the present invention has good biocompatibility and good water dispersibility, making it suitable for use in vivo MRI, which can better evaluate the treatment effect in the early stage of tumors.

[0042] 5. The contrast agent preparation method of the present invention is simple, the raw materials are cheap and readily available, the synthesis conditions are relatively simple, the synthesis cost is relatively low, the yield is high, and it is suitable for large-scale production. Attached Figure Description

[0043] Figure 1 The graph shows the variation of the longitudinal relaxation rate r1 of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 at different pH values.

[0044] Figure 2The graph shows the change in longitudinal relaxation rate r1 of the Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma prepared in Example 1 under the influence of ZnCl2. In the graph, red represents the standard curve of the magnetic resonance contrast agent without ZnCl2, and black represents the standard curve of the magnetic resonance contrast agent under ZnCl2.

[0045] Figure 3 The image shows the cytotoxicity test results of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1.

[0046] Figure 4 The Fe-based T7 peptide-targeting small molecule magnetic resonance imaging agent for gliomas prepared in Example 1 was used for in vivo in situ glioma treatment. 1 H MRI image. Detailed Implementation

[0047] The present invention will now be described in detail with reference to specific embodiments.

[0048] Example 1

[0049] 1. Synthesis of compound (I)

[0050] Weigh (1S,2S)-1-(Boc-amino)-2-aminocyclohexane (CAS No.: 180683-64-1) (5.0 g, 23.3 mmol) into a round-bottom flask, add K2CO3 (6.7 g, 69.9 mmol) and acetonitrile (50 mL), stir at room temperature for 10 min, then slowly add benzyl bromide (4.0 g, 171.0 mmol). After the addition is complete, stir overnight at room temperature. After the reaction is completed by TLC and LC-MS monitoring, filter, concentrate the filtrate, and purify the residue by silica gel column chromatography (dichloromethane / methanol 50:1, v / v) to obtain a clear oil (2.6 g, yield 36.9%).

[0051] 1 H NMR (500MHz, CDCl3) δ7.35–7.27(m,4H),7.26–7.21(m,1H),3.90(d,J=13.2Hz,1H),3.68(d,J=13.2Hz,1H),2.26(td,J=9.9,3. 9Hz,1H),2.14–2.06(m,2H),1.68(dd,J=16.4,13.6Hz,3H),1.45(s,9H),1.29(ddd,J=12.9,8.0,3.1Hz,1H),1.21–1.08(m,3H).

[0052] 13C NMR (126MHz, CDCl3) δ156.20,141.01,128.46,128.22,126.94,79.33,60.69,54.38,50.59,33.05,31.75,28.55,25.00,24.79.

[0053] HRMS: m / z = 305.2228 [M+H] + .

[0054] 2. Synthesis of compound (II)

[0055] A transparent oily substance (2.6 g, 8.54 mmol) was dissolved in a 5 M HCl / EA mixed solvent (30 mL), and then stirred overnight at room temperature. After the reaction was completed by TLC and LC-MS monitoring, the resulting mixture was concentrated to obtain an oily crude product (1.6 g, 95%).

[0056] HRMS: m / z = 205.1708 [M+H] +

[0057] 3. Synthesis of compound (III)

[0058] The crude oily product (1.275 g) and potassium carbonate (3.8 g, 6.0 mmol) were added to acetonitrile (50 mL) and stirred at room temperature for 10 minutes. Then, tert-butyl bromoacetate (2.4 mL, 16.1 mmol) was added dropwise. After the addition was complete, the resulting reaction solution was heated to 65 °C and reacted overnight. After the reaction was monitored by LC-MS, the mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol 50:1, v / v) to give a yellow transparent oily liquid (1.78 g, yield 71%).

[0059] HRMS: m / z = 537.3744 [M+H] + .

[0060] 4. Synthesis of compound (Ⅳ)

[0061] A yellow, transparent, oily liquid (3.48 g, 6.37 mmol) was dissolved in methanol (15 mL), and 10% Pd / C (500 mg) was added. The mixture was then sealed in a high-pressure reactor and subjected to hydrogen protection (25 °C, 1 MPa) for 8 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated to obtain a yellow, oily liquid compound. This compound did not require purification and was used directly in the next step.

[0062] HRMS: m / z = 457.31276 [M+H] +

[0063] 5. Synthesis of 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine

[0064]

[0065] 10 g of 6-methyl-3-hydroxypyridine (91.63 mmol) was dissolved in DMF and stirred at 0 °C for 10 minutes. Then, 7.33 g of NaH (183.26 mmol) was slowly added and stirred for another 30 minutes. Next, 8.71 mL of bromopropyne (100.80 mmol) was slowly added dropwise and stirred for another 4 hours. After the reaction was complete, the resulting mixture was concentrated to give a brown oil (compound b, 2-methyl-5-(prop-2-yne-1-oxy)pyridine) (11.04 g, yield 82.4%).

[0066] 1 H NMR (500MHz, CDCl3) δ 8.24 (d, J = 2.9 Hz, 1H), 7.19 (dd, J = 8.5, 3.0 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 4.68 (d, J = 2.4 Hz, 2H), 2.53 (t, J = 2.3 Hz, 1H), 2.48 (s, 3H).

[0067] 13 C NMR (126MHz, CDCl3) δ151.82,151.51,137.32,123.39,122.76,78.00,76.29,56.40,23.50.

[0068] HRMS: m / z = 148.0766 [M+H] + .

[0069] A brown oily substance (16.7 g, 113.56 mmol) was added to 250 mL of chloroform and stirred on an ice bath for 30 minutes. Then, m-CPBA (29.4 g, 170.34 mmol) was added, and the reaction was continued with stirring for 1 hour. After the reaction was completed by TLC monitoring, a saturated sodium citrate solution was added to the resulting mixture, followed by extraction with dichloromethane (20 mL × 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate 1:1, v / v) to obtain a white solid (intermediate compound c). The white solid intermediate (13.5 g, 113.56 mmol) was then weighed and dissolved in acetic anhydride (21... The mixture was heated to 120 °C in a solution of dichloromethane (227.12 mmol / L). After the reaction was completed by TLC monitoring, a saturated sodium citrate solution was added to the resulting mixture, followed by extraction with dichloromethane (20 mL × 2). The organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol 50:1, v / v) to give a white solid (compound d) (20.63 g, yield 67.1%).

[0070] 1 H NMR (500MHz, CDCl3) δ8.33 (d, J = 2.5 Hz, 1H), 7.45–7.28 (m, 1H), 7.23 (d, J = 8.6 Hz, 1H), 4.90–4.63 (m, 4H), 2.62–2.47 (m, 1H).

[0071] 13 C NMR (126MHz, CDCl3) δ153.13,152.83,152.18,136.55,123.58,121.18,77.67,76.70,63.98,56.53.

[0072] HRMS: m / z = 164.0713 [M+H] + .

[0073] A white solid (compound d) (100 mg, 0.61 mmol) was dissolved in chloroform (5 mL) and stirred at 0 °C for 15 minutes. Then, thionyl chloride (1.5 mL) was slowly added dropwise, and the mixture was stirred at 0 °C for 8 hours. After the reaction was complete, the solvent was removed from the resulting mixture using a rotary evaporator. The residue was a yellow oily liquid, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate 1:1, v / v) to give a white solid (compound e, 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine) (90 mg, yield 81.1%).

[0074] 1 H NMR (500MHz, CDCl3) δ8.32 (d, J = 2.8 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 (dd, J = 8.6, 2.9 Hz, 1H), 4.74 (d, J = 2.4 Hz, 2H), 4.64 (s, 2H), 2.56 (t, J = 2.4 Hz, 1H).

[0075] 13 C NMR (126MHz, CDCl3) δ153.27,149.37,137.67,123.54,122.75,56.27,46.21.

[0076] HRMS: m / z = 182.0374 [M+H] + .

[0077] 6. Synthesis of compound (V)

[0078] Compound (IV) (114 mg, 0.25 mmol), potassium carbonate (172.5 mg, 1.25 mmol), and potassium iodide (83 mg, 0.5 mmol) were added to DMF (10 mL) and stirred at room temperature for 30 minutes. Then, 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine (90 mg, 0.5 mmol) was added, and the mixture was heated to 100 °C and reacted overnight at 100 °C. After the reaction was completed as monitored by LC-MS, the resulting mixture was filtered, the filtrate was concentrated, and the residue was purified by rapid silica gel column chromatography (dichloromethane / methanol 100:1, v / v) to give a clear oily liquid (99 mg, 66% yield).

[0079] HRMS: m / z = 602.3806 [M+H] + .

[0080] 7. Synthesis of compound (VI)

[0081] Weigh 300 mg (0.5 mmol) of a transparent oily liquid into a 50 mL round-bottom flask, then add 10 mL of trifluoroacetic acid. Stir and react overnight at room temperature and pressure. After the reaction is complete, remove the solvent from the resulting mixture under reduced pressure. Dissolve the residue in pure acetonitrile and separate by preparative liquid chromatography (mobile phase: A phase: water, B phase: acetonitrile). Freeze-dry to obtain a white solid (197 mg, yield 91.9%).

[0082] 1 H NMR(500MHz, DMSO)δ8.30(d,J=2.8Hz,1H),7.66–7.47(m,2H),4.92(d,J=2.2Hz,2H),4.52–4.26(m,2H),4.16–3.86(m,2H), 3.66(t,J=2.3Hz,1H),3.62–2.99(m,6H),2.21–2.00(m,2H),1.72(t,J=19.2Hz,2H),1.53–1.34(m,1H),1.32–1.14(m,3H).

[0083] 13 C NMR (126MHz, DMSO) δ159.04,158.77,158.50,158.23,153.95,137.82,125.88,123.20,1 20.38,118.03,115.68,113.33,79.58,78.84,64.25,56.51,25.30,24.39,24.30,24.19.

[0084] 8. Synthesis of compound (VII)

[0085] 8.1 Weigh 250 mg, 0.25 mmol of dichloromethane resin into a polypeptide synthesis tube, add 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide, and allow it to swell for 2 hours at room temperature and pressure. After the solvent is removed, the resin remains in the polypeptide synthesis tube. Add Fmoc-His(Trt)-OH (532.6 mg, 1 mmol), HATU (380.4 mg, 1 mmol), and DIPEA (368 μL, 2 mmol) to the polypeptide synthesis tube, followed by the addition of 10 mL of N,N-dimethylformamide for dissolution. Then, the reaction is carried out by nitrogen purging. After 2 hours, the reaction is stopped. After the solution is removed, the resin is washed once with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide. Add 20 wt% piperidine solution (solvent N,N-dimethylformamide) to the polypeptide synthesis tube, and carry out the reaction by nitrogen purging. After 0.5 hours, stop the reaction, drain the solution, and wash the resin once with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide.

[0086] 8.2. Following the procedures and methods in step 1.1, perform the following amino acid linkages: the second amino acid Fmoc-Arg(Pbf)-OH (644.7 mg, 1 mmol), the third amino acid Fmoc-Pro-OH (337.5 mg, 1 mmol), the fourth amino acid Fmoc-Tyr(tbu)-OH (460.9 mg, 1 mmol), and the fifth amino acid Fmoc-Ala-OH (312.5 mg, 1 mmol).

[0087] 8.3 After completing the amino acid linkage, azidoacetic acid (100.0 mg, 1 mmol), HATU (380.4 mg, 1 mmol), and DIPEA (368 μL, 2 mmol) were added to the peptide synthesis tube. Then, 10 mL of N,N-dimethylformamide was added for dissolution, and the reaction was continued for 2 hours. After the reaction was completed, the solution was dried under vacuum, and the resin was washed once with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide. Then, 10 mL of a 1 wt% TFA trifluoroacetic acid solution in dichloromethane was added to the resin to release the synthesized peptide from the resin. After filtering the resin to remove the solvent, the solvent was evaporated to obtain a yellow oily liquid. Petroleum ether was added to precipitate the liquid, and 227 mg of white solid was precipitated.

[0088] 1H NMR(500MHz,DMSO)δ8.96(d,J=9.6Hz,2H),8.35(dd,J=31.9,7.7Hz,3H),8.07(dd,J=15.9,7.8Hz,2H),7.95(d,J=9.0Hz,1H),7.72(t,J=5.5Hz,1H),7.36(t,J=9.9Hz,4H),7.04(d,J=8.4Hz,3H),6.61(d,J=8.3Hz,2H),4.67–4.52(m,3H),4.35–4.28(m,2H),4.23–4.14(m,2H),3.84(d,J=2.9Hz,3H),3.67–3.61(m,3H),3.47(d,J=5.8Hz,2H),3.20–3.13(m,2H),3.12–3.07(m,2H),3.05(d,J=4.8Hz,1H),2.99(d,J=7.4Hz,1H),2.91–2.83(m,1H),2.65(dd,J=14.1,9.2Hz,1H),2.03–1.94(m,1H),1.84–1.74(m,2H),1.72–1.60(m,2H),1.52(dd,J=12.8,6.9Hz,2H),1.32(dd,J=9.9,7.5Hz,1H),1.18(d,J=7.0Hz,3H),1.03–0.93(m,1H),0.79–0.68(m,6H)。

[0089] 13C NMR (126MHz, DMSO) δ172.75,172.35,171.88,171.82,171.55,171.50,171.43,171.10,170.57,170.13,169.93,169.22,169. 13,167.52,159.13,158.87,158.61,158.36,156.87,156.13,155.86,133.79,130.31,130.07,129.35,128.93,127.64,126. 24,120.53,120.46,118.10,117.22,116.97,115.73,115.26,114.93,113.36,59.32,58.83,56.88,52.27,52.12,51.30,51.02,50.58,48.48,46.92,40.44,39.52,36.91,35.83,29.08,28.76,27.46,26.23,25.03,24.51,24.04,17.76,15.27,11.06.

[0090] 8.4. Dissolve the white solid (75 mg, 0.174 mmol), copper sulfate (43.5 mg, 0.174 mmol), sodium ascorbate (51.7 mg, 0.261 mmol), and T7-N3 peptide (170 mg, 0.174 mmol) in water (3 mL). Stir the mixture at room temperature for 6 hours. After the reaction is complete, separate the resulting mixture by preparative liquid chromatography (mobile phase: A phase: water, B phase: acetonitrile), freeze-dry, and obtain a white solid (113 mg, yield 45.9%).

[0091] HRMS: m / z = 1464.2825 [M+H] + .

[0092] 9. Synthesis of compound (VIII)

[0093] A white solid (113 mg, 0.08 mmol) was weighed and dissolved in 5 mL of water. Then, 1 M sodium hydroxide solution was slowly added dropwise to adjust the pH to 5.5-6.0. While stirring, 3.8 mg (0.14 mmol) of ferric chloride hexahydrate was added, and the reaction was continued with stirring for 6 hours. During this time, the pH was adjusted to 5.5-6.0 using 1 M hydrochloric acid solution. After the reaction was completed, the product was purified by preparative high-performance liquid chromatography (mobile phase: water in phase A, acetonitrile in phase B) to obtain the final product. The purity of the final product was analyzed by analytical high-performance liquid chromatography, and the purity was 95%. The molecular weight of the final product was determined by LC-MS to be 1477.5938 [M+H].

[0094] Experiment 1: Relaxation test of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent of the present invention at different pH levels.

[0095] Test method:

[0096] 1. Weigh 10 mg of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 and dissolve it in 1 mL of PBS to prepare a 6 mM contrast agent solution.

[0097] 2. Divide the contrast agent solution into 9 portions, and adjust the pH of the system to 1, 2, 3, 4, 5, 6, 7, 8, and 9 respectively using 1M hydrochloric acid and 1M sodium hydroxide solution to obtain 9 contrast agent stock solutions with different pH values.

[0098] 3. Each sample of contrast agent stock solution with different pH values ​​was processed as follows:

[0099] The contrast agent stock solution was serially diluted with PBS to prepare working solutions of 100 μM, 80 μM, 40 μM, 20 μM, and 10 μM. Each working solution was placed in a 5 mm NMR tube, and the longitudinal relaxation time T1 was measured using the saturation inversion recovery method on a Bruker 21 MHz NMR spectrometer (instrument parameters: DS = 4, NS = 8, D1 = 1 s, D20 = 150 μs, L20 = 15000). A standard curve was plotted with the concentration of different concentrations of the working solution on the x-axis and the reciprocal of the longitudinal relaxation time T1 obtained from the test of different concentrations of the working solution on the y-axis. The slope of this standard curve is the longitudinal relaxation rate r1 of the contrast agent stock solution.

[0100] 4. Plot the pH value of the contrast agent stock solution on the x-axis and the longitudinal relaxation rate r1 of the contrast agent stock solution at that pH value on the y-axis to obtain the relaxation rate map of the iron-based glioma targeting probe at different pH values.

[0101] Experimental results:

[0102] The Fe-based T7 peptide targeted glioma small molecule magnetic resonance imaging agent prepared in Example 1 can be evaluated for its relaxation rate stability by testing its longitudinal relaxation rate r1. Figure 1 As shown, by Figure 1 It can be seen that the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 has a relatively stable relaxation rate r1 between pH 1 and 7, while the relaxation rate gradually decreases at pH 8 and 9. This indicates that the small molecule magnetic resonance contrast agent is stable under physiological conditions.

[0103] Experiment 2: Relaxation test method of the Fe-based T7 peptide-targeted glioma small molecule magnetic resonance contrast agent of the present invention under the influence of zinc salt:

[0104] 1. Weigh 10 mg of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 and dissolve it in 1 mL of PBS to prepare a 6 mM contrast agent solution.

[0105] 2. Add ten times the equivalent of ZnCl2 solution to the contrast agent solution to obtain the contrast agent stock solution. Perform serial dilution of the contrast agent stock solution to prepare contrast agent working solutions of 190μM, 380μM, 570μM, and 760μM, respectively.

[0106] 3. Place each probe working solution in a 5 mm NMR tube and test the longitudinal relaxation time T1 using the saturation inversion recovery method on a Bruker 21 MHz NMR spectrometer (instrument parameters: DS = 4, NS = 8, D1 = 1 s, D20 = 150 μs, L20 = 15000). Plot the longitudinal relaxation time T1 obtained from different concentrations of contrast agent working solution on the x-axis and the reciprocal of the longitudinal relaxation time T1 obtained from different concentrations of contrast agent working solution on the y-axis. Fit the plot to obtain a standard curve. The slope of the standard curve is the longitudinal relaxation rate r1 of the contrast agent stock solution.

[0107] 4. Do not add ZnCl2 solution to the contrast agent solution prepared in step 1. Proceed with the treatment and operation according to steps 2-3.

[0108] Experimental results:

[0109] Adding zinc ions to the magnetic resonance contrast agent of the present invention allows for testing of its metallic stability. The change in longitudinal relaxation rate r1 before and after adding ZnCl2 solution to the contrast agent solution prepared using the Fe-based T7 peptide-targeting glioma small molecule magnetic resonance contrast agent prepared according to the examples is shown below. Figure 2 As shown, by Figure 2 It can be seen that the relaxation rate of the small molecule magnetic resonance contrast agent prepared in the examples did not change significantly after the addition of Zn ions, which indicates that the coordination of Fe in the small molecule magnetic resonance contrast agent of the present invention is relatively stable and has not been replaced by zinc ions.

[0110] Experiment 3: Cytotoxicity test of the Fe-based T7 peptide targeted small molecule magnetic resonance contrast agent for glioma of the present invention.

[0111] Test method:

[0112] The Fe-based T7 peptide-targeting glioma small molecule magnetic resonance imaging agent prepared in the examples is abbreviated as Fe-T7-PyC3A. Human glioma cell line U87-MG was seeded in 96-well plates for 24 hours, and then different Fe... 3+ Fe-T7-PyC3A solution of a certain concentration, Fe-PyC3A solution without T7 peptide probe, and commercial contrast agent Gd-DTPA solution (prepared with PBS buffer, Fe 3+ Cells were incubated at concentrations of 0, 10, 20, 40, 80, 100, 200, and 300 μM for 12 h. Then, 200 μL of 5 mg / mL LTT solution was added to each well, and incubation continued for another 4 h. After removing the solution, the wells were washed with PBS, and the cell viability in each well was measured and calculated. The cell viability of the human glioma cell line U87-MG under different contrast agent treatments was then calculated.

[0113] It should be noted that the synthesis method of the probe Fe-PyC3A without T7 peptide is roughly the same as that of Fe-T7-PyC3A, except that step 8 is missing from Example 1.

[0114] Experimental results:

[0115] Survival rates of human glioma cell line U87-MG treated with different contrast agents, such as Figure 3 As shown, by Figure 3 It was found that the survival rate of the human glioma cell line U87-MG treated with Fe-T7-PyC3A, Fe-PyC3A, and Gd-DTPA was all above 95%, and the effect of increasing contrast agent concentration on cell survival was minimal. Even at a Fe-T7-PyC3A contrast agent concentration of 300 μM, the survival rate of the human glioma cell line U87-MG remained above 95%. Experiment 4: In vivo in situ glioma treatment with the Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent of this invention. 1 H MRI test

[0116] Test method:

[0117] 1. Weigh 10 mg of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 and dissolve it in 500 μL PBS to prepare a contrast agent of 12 mg / mL.

[0118] 2. U87-MG cells (approximately 5 × 10⁶ cells) were subcutaneously injected into the right foreleg of Babl / c nude mice, forming an orthotopic tumor model. 5 (The tumor develops in situ after 2-3 weeks, with individual cells). Mice with subcutaneous tumor models were anesthetized with isoflurane, and 300 μL of contrast agent was injected into the tumor model mice via tail vein. Subsequently, the tumor was detected at different time points (5 min, 15 min, 30 min, 45 min, 60 min, 100 min, and 240 min) using a 9.4T MRI scanner. 1 H MRI images, 1 H MRI images were obtained using the RARE method with the following parameters: TR = 500ms, TE = 6ms, FOV = 4×4cm, slice thickness = 30mm, acquisition time of 1 minute 16 seconds, RARE factor of 4, matrix size of 96*96, and average number of acquisitions of 4.

[0119] Experimental results:

[0120] In Example 1, the small molecule magnetic resonance contrast agent prepared was injected via tail vein into mice with an in situ tumor model. The tumor site in the mice showed... 1 The graph shows the change in H MRI intensity over time with contrast agent injection. Figure 4 As shown, by Figure 4 It can be seen that the magnetic resonance contrast agent prepared in Example 1 had been distributed throughout the mouse body via blood circulation within the initial 5 minutes, and within 30 minutes... 1 The H magnetic resonance signal intensity reached its peak at 45 minutes. 1 The H-magnetic resonance signal is still strong, indicating that the small molecule magnetic resonance contrast agent of the present invention enters the mouse brain and thus achieves imaging. Because the contrast agent has a large molecular weight and a strong blood-brain barrier crossing effect, it prolongs the retention time of the probe in the mouse brain tumor site and the imaging time.

Claims

1. A Fe-based T7 peptide-targeting small molecule magnetic resonance imaging agent for gliomas, characterized in that... Its structural formula is as follows: 。 2. A method for synthesizing the Fe-based T7 peptide-targeting small molecule magnetic resonance imaging agent for gliomas as described in claim 1, characterized in that... Includes the following steps: S1. Under alkaline conditions, N-Boc cyclohexanediamine undergoes a substitution reaction with benzyl bromide to produce compound (I), the reaction formula of which is as follows: ； S2. Under acidic conditions, compound (I) undergoes ester hydrolysis, removing the Boc protecting group to generate compound (II), as shown in the following reaction formula: ； S3. Under alkaline conditions, compound (II) undergoes a substitution reaction with tert-butyl bromoacetate to produce compound (III), as shown in the following reaction formula: ； S4. In the presence of a catalyst, compound (III) undergoes a hydrogenation reaction to remove the benzyl group, yielding compound (IV), as shown in the following reaction formula: ； S5. Under alkaline conditions, compound (IV) undergoes a substitution reaction with 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine to produce compound (V), the reaction formula of which is as follows: ； S6. Under the condition of an acidic catalyst, compound (V) undergoes ester hydrolysis to remove the tert-butyl group, generating compound (VI), as shown in the following reaction formula: ； S7. In the presence of a reducing agent and a divalent copper salt, compound (VI) undergoes a click chemical reaction with a T7 peptide linked to an azide group to generate compound (VII), as shown in the following reaction equation: ； S8. Under weakly acidic conditions, compound (VII) undergoes a coordination reaction with a ferric salt to form compound (VIII), as shown in the following reaction equation: 。 3. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging agent for gliomas according to claim 2, characterized in that: In step S1, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 20-40℃, the reaction time is 8-12 hours, and the molar ratio of N-Boc cyclohexanediamine, benzyl bromide and potassium carbonate is 1.0-1.2:7.0-7.4:3.0-3.

3.

4. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging contrast agent for gliomas according to claim 2, characterized in that: In step S2, the acid is hydrochloric acid, the reaction solvent is ethyl acetate, the reaction temperature is 20-40℃, and the reaction time is 8-12 hours.

5. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging agent for gliomas according to claim 2, characterized in that: In step S3, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 65℃-75℃, the reaction time is 8-12 hours, and the molar ratio of compound (II), tert-butyl bromoacetate and potassium carbonate is 1.0-1.2 : 3.3-3.5 : 1.0-1.

3.

6. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging contrast agent for gliomas according to claim 2, characterized in that: In step S4, the catalyst is 10% Pd / C, the reaction solvent is methanol, the reaction temperature is 20℃-30℃, the reaction pressure is 0.5-1.5MPa, and the reaction time is 8-12 hours.

7. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging contrast agent for gliomas according to claim 2, characterized in that: In step S5, the base is a mixture of potassium carbonate and potassium iodide, the reaction solvent is N,N-dimethylformamide, the reaction temperature is 65℃-75℃, the reaction time is 8-12 hours, and the molar ratio of compound (IV), 2-(chloromethyl)-5-(prop-2-alkynyloxy)pyridine, potassium carbonate and potassium iodide is 1.0-1.1:2.0-2.2:5.0-5.5:2.0-2.

2.

8. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging agent for gliomas according to claim 2, characterized in that: In step S6, the acidic catalyst is trifluoroacetic acid, the reaction temperature is 20℃-25℃, and the reaction time is 8-12 hours.

9. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging agent for glioma according to claim 2, characterized in that: In step S7, the reducing agent is sodium ascorbate, the divalent copper salt is copper sulfate, the reaction temperature is 20℃-40℃, the reaction time is 6-8 hours, and the molar ratio of compound (VI), T7-N3 peptide, sodium ascorbate and copper sulfate is 1.0-1.1:1.0-1.1:1.0-1.1:1.5-1.

7.

10. The method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance imaging contrast agent for gliomas according to claim 2, characterized in that: In step S8, the pH of the reaction system is 5.5-6.0, the ferric salt is ferric chloride or its hydrate, the reaction temperature is 20-40℃, and the time is 6-8 h. Compound (VII) reacts with Fe... 3+ The molar ratio is 1.0-1.1:1.50-1.

75.

11. The application of the Fe-based T7 peptide-targeting glioma small molecule magnetic resonance contrast agent according to claim 1 in the preparation of magnetic resonance contrast agents.

Images