Fe-based T7 peptide targeted glioma micromolecular magnetic resonance contrast agent and preparation and synthesis method and application thereof

Abstract

The invention discloses a Fe-based T7 peptide targeted glioma micromolecular magnetic resonance contrast agent and a synthesis method and application thereof.The micromolecular magnetic resonance contrast agent is characterized in that one part of the micromolecular magnetic resonance contrast agent is targeted peptide, the other part of the micromolecular magnetic resonance contrast agent is an iron-coordinated pyridine ring contrast agent, and the targeted peptide and the iron-coordinated pyridine ring contrast agent are connected through click reaction. The T7 peptide part in the micromolecule magnetic resonance contrast agent can be combined with a transferrin receptor and can be more efficiently conveyed to the brain for imaging. Compared with a traditional Gd-based contrast agent, the magnetic resonance contrast agent disclosed by the invention has higher specificity and lower toxicity, and meanwhile, the contrast agent has better stability and can be used for efficiently imaging in-situ brain glioma.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic resonance imaging, and in particular relates to an Fe-based T7 peptide-targeted brain glioma small molecule magnetic resonance contrast agent and a synthesis method and application thereof. Background Art

[0002] According to the UK's 2023 five-year cancer survival rate statistics, the five-year survival rate of brain tumors is much lower than that of other high-incidence cancers, such as lung cancer, breast cancer, gastric cancer, etc. The five-year survival rate of brain tumor patients is only 12.8%. Among them, gliomas, as diffuse and highly malignant brain tumors, have attracted more and more attention. Clinically, gliomas are divided into 4 grades according to their malignancy, among which WHO grade 4 glioblastomas are the most malignant, with a 5-year survival rate of less than 3%.

[0003] Magnetic resonance imaging is currently the most commonly used and effective clinical examination method. It has unique advantages such as no radiation damage and the ability to display vascular structure. It is applicable to almost all diseases of various systems in the body. It is better than CT in imaging the brain. It also has excellent soft tissue imaging capabilities and can sensitively detect changes in water content in tissue components. It can detect lesions earlier and more effectively than CT. Therefore, it is the most critical technology in the diagnosis and treatment of gliomas.

[0004] At present, the contrast agents commonly used in glioma examinations are mostly Gd-based magnetic resonance contrast agents, such as Magnevist, dotarem, prohance, etc., but the problems faced by these contrast agents are difficulty in crossing the blood-brain barrier and metabolic problems. In patients with kidney disease, the use of some Gd chelates can lead to a disease called nephrogenic systemic fibrosis. Therefore, we are looking for a new contrast agent to use as an alternative to Gd-based contrast agents.

[0005] Iron as a coordination metal has been studied in depth, and many iron-coordinated contrast agents such as Fe-CDTA have been developed. However, these iron-coordinated contrast agents, like Gd-based contrast agents, are not specific, resulting in a short glioma imaging window and poor imaging effects. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a Fe-based T7 peptide-targeted glioma small molecule magnetic resonance contrast agent and its synthesis method and application. The magnetic resonance contrast agent of the present invention is a part of a targeting peptide, and the other part is an iron-coordinated pyridine ring contrast agent, and the two are connected by a click reaction. The T7 targeting peptide part can bind to the transferrin receptor and can be more efficiently transported to the brain for imaging. Compared with traditional Gd-based contrast agents, the magnetic resonance contrast agent of the present invention has higher specificity and lower toxicity, and the targeting probe has better stability, and can efficiently image in situ glioma.

[0007] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0008] An Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for brain glioma, the structural formula of which is as follows:

[0009]

[0010] A method for synthesizing an Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma comprises the following steps:

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

[0012]

[0013] S2. Under acidic conditions, the compound of formula (I) undergoes ester hydrolysis reaction to remove the Boc protecting group to generate a compound of formula (II), and the reaction formula is as follows:

[0014]

[0015] S3. Under alkaline conditions, the compound of formula (II) undergoes a substitution reaction with tert-butyl bromoacetate to generate a compound of formula (III), and the reaction formula is as follows:

[0016]

[0017] S4. In the presence of a catalyst, the compound of formula (III) undergoes a hydrogenation reaction to remove the benzyl group to generate a compound of formula (IV), and the reaction formula is as follows:

[0018]

[0019] S5. Under alkaline conditions, the compound of formula (IV) undergoes a substitution reaction with 2-(chloromethyl)-5-(prop-2-ynyloxy)pyridine to generate a compound of formula (V), and the reaction formula is as follows:

[0020]

[0021] S6. In the presence of an acidic catalyst, the compound of formula (V) undergoes an ester hydrolysis reaction to remove the tert-butyl group to generate a compound of formula (VI), and the reaction formula is as follows:

[0022]

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

[0024]

[0025] S8. Under weakly acidic conditions, the compound of formula (VII) undergoes a coordination reaction with a trivalent iron salt to generate a compound of formula (VIII), and the reaction formula is as follows:

[0026]

[0027] Furthermore, in step S1, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 20-40° C., 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° C., 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°C-75°C, the reaction time is 8-12 hours, and the molar ratio of the compound of formula (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° C.-30° C., the reaction pressure is 0.5-1.5 MPa, 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°C-75°C, the reaction time is 8-12 hours, and the molar ratio of the compound of formula (IV), 2-(chloromethyl)-5-(prop-2-ynyloxy)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° C.-25° C., 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°C-40°C, the reaction time is 6-8 hours, and the molar ratio of the compound of formula (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] Further, in step S8, the pH value of the reaction system is 5.5-6.0, the trivalent iron salt is ferric chloride or its hydrate, the reaction temperature is 20-40°C, the reaction time is 6-8h, and the compound of formula (VII) and Fe 3+ The molar ratio is 1.0-1.1:1.50-1.75.

[0035] Application of an Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for brain glioma in the preparation of magnetic resonance contrast agents.

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

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

[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 under different pH values. Through the above experiments, we confirmed that the molecular contrast agent has strong stability below pH = 9.

[0039] 2. Experiments have shown that the contrast agent of the present invention has a certain ability to cross the blood-brain barrier. An in vitro blood-brain barrier crossing model was established by transwell. After incubating the contrast agent, the blood-brain barrier crossing ability of the contrast agent was confirmed.

[0040] 3. The experiment shows that the contrast agent of the present invention can image glioma in situ, and compared with the imaging ability without the targeting peptide molecule, we found that the introduction of the targeting peptide can extend the imaging window to a certain extent, and the imaging effect is better than Magnevist. At the same time, the signal in the tumor area can be enhanced by 62%, which can outline the tumor boundary and provide tumor space information.

[0041] 4. The contrast agent of the present invention has good biosafety and water dispersibility, is suitable for in vivo MRI, and can better evaluate the therapeutic effect of early tumors.

[0042] 5. The contrast agent preparation method of the present invention is simple, the raw materials are cheap and easily 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph showing the change in 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 2This is a graph showing the change in longitudinal relaxation rate r1 of the Fe-based T7 peptide-targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 under the influence of ZnCl2, wherein the red color is the standard curve of the magnetic resonance contrast agent without ZnCl2, and the black color is the standard curve of the magnetic resonance contrast agent under the condition of ZnCl2.

[0045] Figure 3 This is a graph showing the cytotoxicity test results of the Fc-based T7 peptide-targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1.

[0046] Figure 4 In vivo in situ brain glioma of the Fe-based T7 peptide targeted brain glioma small molecule magnetic resonance contrast agent prepared in Example 1 1 H MRI image. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to specific embodiments.

[0048] Example 1

[0049] 1. Synthesis of compound of formula (I)

[0050] Weigh (1S,2S)-1-(Boc-amino)-2-aminocyclohexane (CAS No.: 180683-64-1) (5.0 g, 23.3 mmol) in 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 drop benzyl bromide (4.0 g, 171.0 mmol), after the addition is complete, stir at room temperature overnight. After the reaction is completed by TLC and LC-MS monitoring, filter, concentrate the filtrate, and purify the residue by silica gel flash column chromatography (dichloromethane / methanol 50:1, v / v) to obtain a transparent 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 the compound of formula (II)

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

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

[0057] 3. Synthesis of compound of formula (III)

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

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

[0060] 4. Synthesis of the compound of formula (IV)

[0061] Dissolve the yellow transparent oily liquid (3.48 g, 6.37 mmol) in methanol (15 mL), add 10% Pd / C (500 mg), seal in a high-pressure reactor, and pass hydrogen to protect the reaction (25°C, 1 MPa) for 8 hours. After the reaction is completed, filter and concentrate the filtrate to obtain a yellow oily liquid compound, which is directly used in the next step without purification.

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

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

[0064]

[0065] Weigh 6-methyl-3-hydroxypyridine (10 g, 91.63 mmol) and dissolve it in DMF. Stir at 0°C for 10 minutes, then slowly add NaH (7.33 g, 183.26 mmol), continue stirring for 30 minutes, then slowly drop propargyl bromide (8.71 mL, 100.80 mmol), continue stirring and react for 4 hours. After the reaction is completed, the obtained mixed product is concentrated to obtain a brown oil (compound b, 2-methyl-5-(prop-2-yn-1-oxy)pyridine) (11.04 g, yield 82.4%).

[0066] 1 H NMR (500MHz, CDCl3) δ8.24 (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] The brown oil (16.7 g, 113.56 mmol) was added to chloroform (250 mL), stirred on an ice bath for 30 minutes, and then m-CPBA (29.4 g, 170.34 mmol) was added, and the reaction was continued to stir for 1 hour. After the reaction was completed by TLC monitoring, a saturated sodium citrate solution was added to the obtained mixed product, followed by extraction with dichloromethane (20 mL×2), the organic phases were combined, the combined organic phases were washed with saturated brine, and then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product, which was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate 1:1, v / v) to obtain a white solid (intermediate product compound c), and then the intermediate product white solid (13.5 g, 113.56 mmol) was weighed and dissolved in acetic anhydride (21 mL, 227.12 mmol), heated to 120 ° C for reaction. After the reaction was completed under TLC monitoring, a saturated sodium citrate solution was added to the obtained mixed product, followed by extraction with dichloromethane (20 mL × 2), the organic phases were combined, and the combined organic phases were washed with saturated brine, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product, which was purified by silica gel flash column chromatography (dichloromethane / methanol 50:1, v / v) to obtain 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] The white solid (compound d) (100 mg, 0.61 mmol) was dissolved in chloroform (5 mL), stirred at 0°C for 15 minutes, then slowly added with thionyl chloride (1.5 mL), and stirred at 0°C for 8 hours. After the reaction was completed, the obtained mixed product was freed from the solvent by a rotary evaporator, and the residue was a yellow oily liquid. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate 1:1, v / v) to obtain a white solid (compound e, 2-(chloromethyl)-5-(prop-2-ynyloxy)pyridine) (90 mg, yield 81.1%).

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

[0078] The compound of formula (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), stirred at room temperature for 30 minutes, then 2-(chloromethyl)-5-(prop-2-ynyloxy)pyridine (90 mg, 0.5 mmol) was added, heated to 100°C, and reacted at 100°C overnight. After the reaction was completed by LC-MS monitoring, the obtained mixed product was filtered, the filtrate was concentrated, and the residue was purified by silica gel flash column chromatography (dichloromethane / methanol 100:1, v / v) to obtain a transparent oily liquid (99 mg, yield 66%).

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

[0080] 7. Synthesis of the compound of formula (VI)

[0081] Weigh a transparent oily liquid (300 mg, 0.5 mmol) into a 50 mL round-bottom flask, then add 10 mL of trifluoroacetic acid, and stir the reaction at room temperature and pressure overnight. After the reaction is completed, remove the solvent from the obtained mixed product under reduced pressure, and dissolve the residue in pure acetonitrile, perform preparative liquid chromatography separation (mobile phase: phase A: water, phase B: acetonitrile), and 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 the compound of formula (VII)

[0085] 8.1. Weigh dichlororesin (250 mg, 0.25 mmol) into a peptide synthesis tube, add 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide, and swell at room temperature and pressure for 2 hours. After draining the above solvents, the resin remains in the peptide synthesis tube. Add Fmoc-His(Trt)-OH (532.6 mg, 1 mmol), HATU (380.4 mg, 1 mmol) and DIPEA (368 μL, 2 mmol) into the peptide synthesis tube, then add 10 mL of N,N-dimethylformamide to dissolve, and then react by nitrogen blowing. After 2 hours of reaction, the reaction is terminated. After draining the solution, wash the resin with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide in turn. Add 20 wt% piperidine solution (solvent is N,N-dimethylformamide) into the peptide synthesis tube, and react by nitrogen blowing. After 0.5 hours, the reaction is terminated, the solution is drained, and the resin is washed with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide in turn.

[0086] 8.2. According to the operation and method of step 1.1, 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) were connected respectively.

[0087] 8.3. After the amino acid connection is completed, azidoacetic acid (100.0 mg, 1 mmol), HATU (380.4 mg, 1 mmol) and DIPEA (368 μL, 2 mmol) are added to the peptide synthesis tube, and then 10 mL of N, N-dimethylformamide is added for dissolution. The reaction is continued for 2 hours. After the reaction is completed, the solution is drained, and the resin is washed with 5 mL of dichloromethane and 5 mL of N, N-dimethylformamide in sequence. Then, 10 mL of 1 wt% TFA trifluoroacetic acid in dichloromethane solution is added to the resin to free the synthesized peptide from the resin. After the resin is filtered and the solvent is dried, a yellow oily liquid is obtained, and petroleum ether is added for precipitation to precipitate 227 mg of a white solid.

[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 and react at room temperature for 6 hours. After the reaction, separate the obtained mixed product by preparative liquid chromatography (mobile phase: phase A: water, phase B: acetonitrile), and freeze-dry to obtain a white solid (113 mg, yield 45.9%).

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

[0092] 9. Synthesis of the compound of formula (VIII)

[0093] Weigh the white solid (113 mg, 0.08 mmol) and dissolve it in water (5 mL), then slowly drip 1M sodium hydroxide solution, adjust the system pH to 5.5-6.0, add ferric chloride hexahydrate (3.8 mg, 0.14 mmol) while stirring, and then continue stirring and reacting for 6 hours. During this period, the system pH can be adjusted to 5.5-6.0 with 1M hydrochloric acid solution. After the reaction is completed by LC-MS monitoring, it is purified by high-performance liquid preparative chromatography (mobile phase: phase A is water, phase B is acetonitrile) to obtain the final product. The purity of the final product is analyzed by high-performance liquid analytical chromatography, and the purity is 95%. LC-MS identifies the molecular weight of the final product as 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

[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. The contrast agent solution was divided into 9 portions, and the pH of the system was adjusted to 1, 2, 3, 4, 5, 6, 7, 8, and 9 with 1 M hydrochloric acid and 1 M sodium hydroxide solution, respectively, to obtain 9 portions of contrast agent mother solutions with different pH values.

[0098] 3. Treat each contrast agent mother solution with different pH values ​​as follows:

[0099] The contrast agent mother solution was gradiently diluted with PBS to 100uM, 80uM, 40uM, 20uM, and 10uM contrast agent working solutions. Each portion of the contrast agent working solution was placed in a 5mm nuclear magnetic resonance tube, and the longitudinal relaxation time T1 was tested using the saturation inversion recovery method (instrument parameters: DS=4, NS=8, D1=1s, D20=150μs, L20=15000) on a Bruker 21MHz nuclear magnetic resonance spectrometer. The concentration of the contrast agent working solution of different concentrations was used as the abscissa, and the reciprocal of the longitudinal relaxation time T1 obtained by testing the contrast agent working solution of different concentrations was used as the ordinate, and a plot was drawn to obtain a standard curve by fitting, and the slope of the standard curve was the longitudinal relaxation rate r1 of the contrast agent mother solution.

[0100] 4. Plot the pH value of the contrast agent mother solution as the horizontal axis and the longitudinal relaxation rate r1 of the contrast agent mother solution at the pH value as the vertical axis to obtain the relaxation rate diagram of the iron-based glioma targeting probe at different pH values.

[0101] Test results:

[0102] The relaxation rate stability of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 can be evaluated by testing its longitudinal relaxation rate r1, such as Figure 1 As shown by Figure 1 It can be seen that the relaxation rate r1 of the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example 1 is relatively stable between pH 1 and 7, and the relaxation rate gradually decreases when the pH is 8 to 9. This shows 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 targeting 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 a contrast agent mother solution, and dilute the contrast agent mother solution in a gradient manner to form contrast agent working solutions of 190 μM, 380 μM, 570 μM, and 760 μM, respectively.

[0106] 3. Each probe working solution was placed in a 5 mm nuclear magnetic resonance tube, and the longitudinal relaxation time T1 was tested using the saturation inversion recovery method (instrument parameters: DS = 4, NS = 8, D1 = 1s, D20 = 150μs, L20 = 15000) on a Bruker 21MHz nuclear magnetic resonance spectrometer. The concentration of the contrast agent working solution with different concentrations was used as the horizontal coordinate, and the reciprocal of the longitudinal test relaxation time T1 obtained by testing the contrast agent working solution with different concentrations was used as the vertical coordinate. A plot was drawn and fitted to obtain a standard curve. The slope of the standard curve was the longitudinal relaxation rate r1 of the contrast agent mother solution.

[0107] 4. Do not add ZnCl2 solution to the contrast agent solution prepared in step 1, and process and operate according to the method of steps 2-3.

[0108] Test results:

[0109] Adding zinc ions to the magnetic resonance contrast agent of the present invention can test the stability of the metal. The change of the longitudinal relaxation rate r1 before and after adding ZnCl2 solution to the contrast agent solution prepared by the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent prepared in Example is shown in FIG. Figure 2 As shown by Figure 2 It can be seen that after adding Zn ions, the relaxation rate of the small molecule magnetic resonance contrast agent prepared in the example does not change significantly, which shows that the coordination of Fe in the small molecule magnetic resonance contrast agent of the present invention is relatively stable and is not replaced by zinc ions.

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

[0111] Test method:

[0112] The Fe-based T7 peptide-targeted glioma small molecule magnetic resonance contrast agent prepared in the example is abbreviated as Fe-T7-PyC3A. The human glioma cell line U87-MG was inoculated in a 96-well plate for 24 hours, and then different Fe 3+ The Fe-T7-PyC3A solution with different concentrations, the Fe-PyC3A solution without T7 peptide, and the commercial contrast agent Gd-DTPA solution (prepared with PBS buffer, Fe 3+ The concentrations were 0, 10, 20, 40, 80, 100, 200, and 300 μM, respectively, and incubated for 12 h. Subsequently, 200 uL of 5 mg / mL MTT solution was added to each well and incubated for another 4 h. After removing the solution, the wells were washed with PBS, and the cell survival in each well was tested and calculated, and then the cell survival rate of the human glioma cell line U87-MG under different contrast agent solutions was calculated.

[0113] It should be noted here 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 based on Example 1.

[0114] Test results:

[0115] The survival rate of human glioma cell line U87-MG treated with different contrast agents Figure 3 As shown by Figure 3 It can be seen that the survival rates of human glioma cell line U87-MG treated with Fe-T7-PyC3A, Fe-PyC3A, and Gd-DTPA are all above 95%, and the effect on cell survival rate is not significant as the concentration of the contrast agent solution increases. When the concentration of Fe-T7-PyC3A contrast agent is 300 μM, the survival rate of human glioma cell line U87-MG cells is still above 95%. Experiment 4: In vivo in situ brain glioma using the Fe-based T7 peptide targeted glioma small molecule magnetic resonance contrast agent of the present 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 12 mg / mL contrast agent.

[0118] 2. Take the orthotopic tumor model mice (Babl / c nude mice) and inject U87-MG cells subcutaneously in the right foreleg, about 5×10 5 The subcutaneous tumor model mice were anesthetized with isoflurane, and 300 μL of contrast agent was injected into the orthotopic tumor model mice through the tail vein. Then, the 9.4T nuclear magnetic resonance imaging instrument was used to detect the tumor at different time points (5min, 15min, 30min, 45min, 60min, 100min, and 240min). 1 H MRI images, 1 H MRI images were acquired by the RARE method with the following parameters: TR = 500 ms, TE = 6 ms, FOV = 4 × 4 cm, slice thickness = 30 mm, acquisition time was 1 minute 16 seconds, RARE factor was 4, matrix size was 96*96, and the number of averages was 4.

[0119] Test results:

[0120] After the small molecule magnetic resonance contrast agent prepared in Example 1 was injected into the tail vein of the orthotopic tumor model mice, the tumor site of the orthotopic tumor model mice 1 The change of H MRI intensity with contrast agent injection time is shown in the figure Figure 4 As shown by Figure 4 It can be seen that the magnetic resonance contrast agent prepared in Example 1 has spread throughout the mouse body through the blood circulation at the initial time of 5 minutes, and at 30 minutes 1 The H magnetic resonance signal intensity reaches a peak at 45 min. 1 The H magnetic resonance signal is still strong, which shows that the small molecule magnetic resonance contrast agent of the present invention enters the mouse brain and achieves imaging. Since the contrast agent has a large molecular weight and a strong blood-brain barrier penetration effect, the retention time and imaging time of the probe in the mouse brain tumor site are prolonged.

Claims

1. An Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma, characterized in that Its structural formula is as follows:

2. A method for synthesizing the Fe-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma according to claim 1, characterized in that The steps include: S1. Under alkaline conditions, N-Boc cyclohexanediamine and benzyl bromide undergo substitution reaction to generate a compound of formula (I), and the reaction formula is as follows: S2. Under acidic conditions, the compound of formula (I) undergoes ester hydrolysis reaction to remove the Boc protecting group to generate a compound of formula (II), and the reaction formula is as follows: S3. Under alkaline conditions, the compound of formula (II) undergoes a substitution reaction with tert-butyl bromoacetate to generate a compound of formula (III), and the reaction formula is as follows: S4. In the presence of a catalyst, the compound of formula (III) undergoes a hydrogenation reaction to remove the benzyl group to generate a compound of formula (IV), and the reaction formula is as follows: S5. Under alkaline conditions, the compound of formula (IV) undergoes a substitution reaction with 2-(chloromethyl)-5-(prop-2-ynyloxy)pyridine to generate a compound of formula (V), and the reaction formula is as follows: S6. In the presence of an acidic catalyst, the compound of formula (V) undergoes an ester hydrolysis reaction to remove the tert-butyl group to generate a compound of formula (VI), and the reaction formula is as follows: S7. In the presence of a reducing agent and a divalent copper salt, the compound of formula (VI) undergoes a click chemical reaction with the T7 peptide connected to an azide group to generate a compound of formula (VII), and the reaction formula is as follows: S8. Under weakly acidic conditions, the compound of formula (VII) undergoes a coordination reaction with a trivalent iron salt to generate a compound of formula (VIII), and the reaction formula is as follows:

3. The method for synthesizing the Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma according to claim 2, characterized in that: In the step S1, the base is potassium carbonate, the reaction solvent is acetonitrile, the reaction temperature is 20-40° C., 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 Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma 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° C., and the reaction time is 8-12 hours.

5. The method for synthesizing the Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma 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° C.-75° C., the reaction time is 8-12 hours, and the molar ratio of the compound of formula (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 Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma 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° C.-30° C., the reaction pressure is 0.5-1.5 MPa, and the reaction time is 8-12 hours.

7. The method for synthesizing the Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma 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°C-75°C, the reaction time is 8-12 hours, and the molar ratio of the compound of formula (IV), 2-(chloromethyl)-5-(prop-2-ynyloxy)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 Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma according to claim 2, characterized in that: In step S6, the acidic catalyst is trifluoroacetic acid, the reaction temperature is 20° C.-25° C., and the reaction time is 8-12 hours.

9. The method for synthesizing the Fc-based T7 peptide-targeted small molecule magnetic resonance contrast 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°C-40°C, the reaction time is 6-8 hours, and the molar ratio of the compound of formula (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 Fc-based T7 peptide-targeted small molecule magnetic resonance contrast agent for glioma according to claim 2, characterized in that: In step S8, the pH value of the reaction system is 5.5-6.0, the trivalent iron salt is ferric chloride or its hydrate, the reaction temperature is 20-40°C, the reaction time is 6-8h, and the compound of formula (VII) is reacted with Fe 3+ The molar ratio is 1.0-1.1:1.50-1.

75.

11. Use of the Fe-based T7 peptide targeted brain glioma small molecule magnetic resonance contrast agent according to claim 1 in the preparation of a magnetic resonance contrast agent.

12. The use of the Fc-based T7 peptide targeted brain glioma small molecule magnetic resonance contrast agent according to claim 11, characterized in that: The magnetic resonance contrast agent can target brain glioma.

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