Saturated alkyl double-chain modified magnetic resonance imaging contrast agent as well as preparation method and application thereof

Abstract

The invention relates to a saturated alkyl double-chain modified magnetic resonance imaging contrast agent as well as a preparation method and application thereof, and belongs to the technical field of preparation of magnetic resonance imaging contrast agents. The contrast agent provided by the invention not only retains the high dynamic stability and low gadolinium leakage risk of an annular contrast agent, but also significantly improves the uptake rate of Gd-DOTA in the liver through introduction of saturated alkyl double chains, and effectively solves the problem of cytotoxicity possibly generated by long-chain single-chain modification. Animal experiment results show that the contrast agent provides a clear magnetic resonance imaging signal in a living body, and provides a clearer and more accurate image support for diagnosis of liver diseases by optimizing the length and the structure of an lipid chain. While the imaging effect is improved, the safety and stability of the contrast agent are ensured, and the contrast agent has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of magnetic resonance imaging contrast agent preparation, and particularly relates to a saturated alkyl double-chain modified magnetic resonance imaging contrast agent and a preparation method and application thereof. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is a widely used non-invasive imaging technique in medical diagnosis and biomedical research. It is based on the principle of nuclear magnetic resonance, which uses radio frequency pulses to excite the spins of hydrogen nuclei in water molecules, and distinguishes between diseased and normal tissues by relaxation signals in the magnetic field, providing important reference information for clinical diagnosis. However, the sensitivity of MRI is relatively low, and when the lesion tissue is similar to the normal tissue structure, it is difficult to distinguish the contrast between the two by relying solely on the water molecule signal. Therefore, magnetic resonance imaging contrast agents are usually used to change the relaxation time of water molecules in the target tissue, thereby improving the imaging contrast between the lesion tissue and the surrounding tissue.

[0003] Magnetic resonance imaging contrast agents are mainly divided into T1 contrast agents and T2 contrast agents, which play a role by shortening the longitudinal relaxation time (T1) and transverse relaxation time (T2) of water molecules, respectively. Gadolinium-based small molecule T1 contrast agents mainly shorten the longitudinal relaxation time of water molecules, enhancing image brightness, and are the main commercial products for clinical application. Among them, linear contrast agents represented by gadolinium diethylenetriamine pentaacetate (hereinafter referred to as Gd-DTPA) chelate (such as Magnevist) and cyclic contrast agents represented by 1,4,7,10-tetraazacyclododecane-, 4,7,10-tetraacetic acid (hereinafter referred to as Gd-DOTA) chelate (such as Dotarem) are widely used. In liver diagnosis, gadoxetic acid disodium (Gd-EOB-DTPA) can be taken up by normal hepatocytes through the specific expression of organic anion transport polypeptides (OATP1B1 and OATP1B3) channels in hepatocytes, and shows significant effect in liver disease detection and early diagnosis of liver cancer. However, gadoxetic acid disodium as a linear chelate has the problem of low molecular structure dynamic stability, which may lead to leakage of gadolinium ions, causing local osmotic pressure disorder and cell damage, causing irreversible harm to patients, and there is a certain safety risk. In contrast, cyclic contrast agents represented by Gd-DOTA have higher kinetic stability and smaller risk of gadolinium leakage, but their liver uptake rate is much lower than that of gadoxetic acid disodium. Therefore, no commercial gadolinium-based contrast agent with high liver uptake rate and high kinetic stability has been developed in the prior art. SUMMARY

[0004] In view of the above problems, the present application provides a saturated alkyl double-chain modified magnetic resonance imaging contrast agent and a preparation method and application thereof.

[0005] The first object of the present application provides a saturated alkyl double-chain modified magnetic resonance imaging contrast agent, and the structural formula of the contrast agent is shown as follows:

[0006] wherein R1 and R2 are the same, both are saturated alkyl chains, and the number n of carbon atoms in each saturated alkyl chain satisfies 5≤n≤8.

[0007] In specific embodiments of the present application, the number n of carbon atoms in each saturated alkyl chain is 6 or 7.

[0008] When n is 6, the molecular structural formula of the contrast agent is shown as follows.

[0009]

[0010] When n is 7, the molecular structural formula of the contrast agent is shown as follows.

[0011]

[0012] In the present application, the saturated alkyl double-chain modified magnetic resonance imaging contrast agent significantly enhances the uptake capacity of hepatocytes while maintaining good water solubility. Compared with single-chain modified contrast agents with similar total number of carbon atoms, double-chain modification significantly reduces cytotoxicity, thereby improving its biological safety. At the same time, the cyclic contrast agent chelated by DOTA has higher kinetic stability than the contrast agent chelated by DTPA (such as Gd-EOB-DTPA), effectively reducing the risk of gadolinium ion leakage.

[0013] The second object of the present application provides a preparation method of a saturated alkyl double-chain modified magnetic resonance imaging contrast agent, comprising: tricarboxyl-protected tetraazacyclotetraazidyl acetic acid and R1R2CH-NH2 are subjected to amino and carboxyl reaction to obtain an intermediate product; the intermediate product is subjected to deprotection to obtain a precursor compound; the precursor compound is reacted with a gadolinium compound to obtain a saturated alkyl double-chain modified magnetic resonance imaging contrast agent; wherein R1 and R2 are the same, both are saturated alkyl chains, and the number n of carbon atoms in each saturated alkyl chain satisfies 5≤n≤8.

[0014] In specific embodiments of the present application, the carboxyl protecting group in the tricarboxyl-protected polyazacyclotetraazidyl acetic acid is a tert-butyl group.

[0015] In specific embodiments of the present application, preferably, the tricarboxyl-protected polyazacyclotetraazidyl acetic acid is tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0016] In the specific embodiments of the present application, the molar ratio of the tricarboxy-protected tetraazacyclotetraazadiacetic acid and R1R2CH-NH2 is 1:1-1.5.

[0017] In the specific embodiments of the present application, the reaction of the amino group and the carboxyl group is carried out in the presence of a first organic solvent and a condensation reagent.

[0018] In the specific embodiments of the present application, the first organic solvent is acetonitrile, dichloromethane or tetrahydrofuran, preferably acetonitrile.

[0019] In the specific embodiments of the present application, the condensation reagent is a commonly used condensation reagent for the reaction of amino group and carboxyl group. As non-exhaustive examples, these condensing agents can be selected from one or two of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1-hydroxybenzotriazole (HOBT), N,N'-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIPEA), N-methylmorpholine (NMM) or triethylamine. The specific selection is easily determined by those skilled in the art according to actual needs.

[0020] In the specific embodiments of the present application, after the reaction of the intermediate product is stopped, a post-treatment step is carried out, and the post-treatment uses extraction method to separate the intermediate product, unreacted reaction substrate and by-product. For example, the extraction solvent can be selected as a saturated citric acid solution to remove unreacted R1R2CH-NH2, and a sodium bicarbonate solution can be used to remove the by-product.

[0021] In the specific embodiments of the present application, according to the specific type of the carboxyl protecting group in the tricarboxy-protected polyazacyclotetraazadiacetic acid, a subsequent deprotection reaction is selected. Specifically, the protecting group is tert-butyl, and an acid solution commonly used in the technical field is used to remove the tert-butyl group, such as trifluoroacetic acid, and the reaction is carried out at room temperature to remove the tert-butyl group.

[0022] In the specific embodiments of the present application, the reaction of the amino group and the carboxyl group is carried out at a temperature of 10-60°C for 2-24h.

[0023] In the specific embodiments of the present application, the gadolinium compound is gadolinium chloride or gadolinium acetate. Gadolinium chloride or gadolinium acetate has better reaction performance, and at the same time makes the structure of the obtained ligand more stable.

[0024] In the specific embodiments of the present application, the molar ratio of the tricarboxyl-protected tetraazacyclotetraazadiacetic acid to the gadolinium compound is 1:1.05-1.5. The gadolinium compound needs to be appropriately excessive during the reaction to ensure that the reaction proceeds fully.

[0025] In the specific embodiments of the present application, after the reaction of the precursor compound with the gadolinium compound is stopped, post-treatment is needed, and the post-treatment is performed by adding a base solution to adjust the pH value. The base can be selected from one of NaOH, KOH, LiOH, or NH4OH. Ca(OH)2, Na2CO3, and the like are not selected as the base because they can easily produce precipitates during the reaction.

[0026] A third object of the present application is to provide the use of the above-mentioned saturated alkyl double-chain modified magnetic resonance imaging contrast agent in magnetic resonance imaging.

[0027] The beneficial effects of the present application are as follows: The saturated alkyl double-chain modified Gd-DOTA contrast agent provided by the present application not only retains the higher kinetic stability and smaller gadolinium leakage risk of the cyclic contrast agent, but also improves the low uptake rate of Gd-DOTA in the liver by introducing the saturated alkyl double chain, and effectively solves the cytotoxicity that may be caused by long-chain single-chain modification. The subsequent animal experiment results show that clear magnetic resonance imaging (hereinafter referred to as MRI) signals are provided in vivo, indicating that the contrast agent of the present application significantly improves the imaging contrast of liver tissue by optimizing the length and structure of the lipid chain, providing clear and accurate image support for the diagnosis of liver diseases.

[0028] The preparation method of the saturated alkyl double-chain modified magnetic resonance imaging contrast agent of the present application has relatively simple steps and is easy to operate, and can realize large-scale preparation.

[0029] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application can be achieved and obtained by the structures indicated in the specification, claims, and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without any creative labor.

[0031] Figure 1 A synthesis route diagram of a saturated alkyl double-chain modified magnetic resonance imaging contrast agent according to an embodiment of the present application is shown; Figure 2 A plot showing the kinetic stability evaluation of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths in 1 M HC1 and 10 mM Zn 2+ A plot showing the kinetic stability evaluation of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths in 1 M HC1 and 10 mM Zn Figure 3 A plot showing the relaxivity of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths in Test Example 2 according to embodiments of the present application; Figure 4 A plot showing the cytotoxicity characterization of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths in Test Example 3 according to embodiments of the present application; Figure 5 A plot showing the magnetic resonance imaging of liver tissue of normal female C57 black mice at different time points after injection of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths respectively in Test Example 4 according to embodiments of the present application.

[0032] Figure 6 A plot showing the contrast agent distribution in each major tissue of normal female C57 black mice after injection of alkyl-modified Gd-DOTA contrast agents with different lipid chain lengths respectively in Test Example 5 according to embodiments of the present application. DETAILED DESCRIPTION

[0033] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely explain the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0034] The currently commercially available liver-specific magnetic resonance imaging contrast agent is gadopentetic acid disodium, which is a linear chelate of DTPA, and its structural formula is shown as follows.

[0035]

[0036] A method for preparing a saturated alkyl double-chain modified magnetic resonance imaging contrast agent according to some embodiments of the present application, comprising: The intermediate product is obtained by reacting tricarboxyl-protected tetraazacyclotetraazatetraacetic acid and R1R2CH-NH2 through amino and carboxyl reactions; The intermediate product is deprotected to obtain a precursor compound; The precursor compound is reacted with a gadolinium compound to obtain a saturated alkyl double-chain modified magnetic resonance imaging contrast agent; wherein R1 and R2 are the same, both are saturated alkyl chains, and the number of carbon atoms n in the saturated alkyl chain satisfies: 5≤n≤8.

[0037] In some embodiments of the present application, the tricarboxyl-protected tetraazacyclotetraacetic acid is tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and when n=6, the synthesis route of the saturated alkyl chain-modified magnetic resonance imaging contrast agent is as shown in Figure 1 .

[0038] By selecting the gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd-DOTA) with a saturated alkyl chain of medium length, the hepatocyte uptake of the magnetic resonance imaging contrast agent can be enhanced, and the image contrast and clarity can be improved. At the same time, compared with a single chain structure with a similar total number of carbon atoms, they have lower cytotoxicity and exhibit good biocompatibility.

[0039] Example 1 According to the synthesis route as shown in Figure 1 , a tridecane-7-amine-modified Gd-DOTA (hereinafter referred to as Gd-C13) is prepared, which specifically includes: 572.74 mg (1 mmol) of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-tBu) (purchased from Ardrich, batch number T162093) and 394 mg (1.04 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (purchased from Ardrich, batch number H106174) were dissolved in 10 mL of acetonitrile (purchased from Ardrich, batch number A104443), then 181 μL (1.04 mmol) of N,N-diisopropylethylamine (DIPEA) (purchased from sigma-aldrich, batch number 550043), 260 μL (1.04 mmol) of tridecane-7-amine (purchased from TCI, batch number T3591) were added to the solution, and the reaction was stirred at room temperature for 3 hours. After removing the acetonitrile solution using a rotary evaporator, the product was redissolved in 30 mL of ethyl acetate (purchased from National Pharmaceutical Group, batch number 20230620), washed once with 30 mL of saturated citric acid, 30 mL of pure water, and 30 mL of 5% sodium bicarbonate solution, respectively, and the upper organic phase was retained. The ethyl acetate was removed using a rotary evaporator, and the product was dissolved in 10 mL of 95% trifluoroacetic acid (purchased from Macklin, batch number T818778) at room temperature and reacted for 12 hours. After removing the trifluoroacetic acid and the byproduct tert-butyl alcohol using a rotary evaporator, tridecane-7-amine modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-C13) was obtained after vacuum drying for 12 hours. 1 HNMR (500 MHz, DMSO, δ): 0.88 (t, CH3, 6H), 1.23 (m, CH2, 16H), 1.42 (m, CH3, 4H), 2.92-3.08 (m, CH2-N, 16H), 3.33 (s, CH-NH, 1H), 3.59 (m, CH2, 6H), 3.71 (s, CH2, 2H), 8.00 (s, NH, 1H); the molecular formula of the resulting compound is C 29 H 56 N5O7[(M+H) + ]: 586.41, mass spectrum m / z: 586.43.

[0040] The above prepared intermediate was re-dissolved in 200 mL of deionized water, 334.5 mg (0.9 mmol) of gadolinium chloride hexahydrate (purchased from Aldrich, batch number G119152) was added, the pH was adjusted to between 6-7 by slowly adding 1 mol / L sodium hydroxide solution, and the reaction was continuously stirred at 90 °C for 24 hours. After the solution was completely cooled, it was centrifuged at 3500 rpm for 10 minutes using a centrifuge, the white precipitate was removed, and the supernatant solution was freeze-dried and then separated and purified by high performance liquid chromatography. The product had a mass spectrum molecular formula of C 29 H 53 GdN5O7[(M+H) + ]: 741.31, mass spectrum m / z: 741.32, and it was determined that the final product Gd-C13 was prepared.

[0041] The molecular structure of the Gd-C13 is shown below.

[0042]

[0043] Example 2 Preparation of 8-pentadecylamine modified Gd-DOTA (Gd-C15) 572.74 mg (1 mmol) of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-tBu) (purchased from Aldrich, batch number T162093) and 394 mg (1.04 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (purchased from Aldrich, batch number H106174) were dissolved in 10 mL of acetonitrile (purchased from Aldrich, batch number A104443), followed by the addition of 181 μL (1.04 mmol) of N,N-diisopropylethylamine (DIPEA) (purchased from sigma-aldrich, batch number 550043), 304 μL (1.04 mmol) of 8-pentadecylamine (purchased from TCI, batch number P2543) to the solution, and the reaction was stirred at room temperature for 3 hours. After the acetonitrile solution was removed using a rotary evaporator, the product was re-dissolved in 30 mL of ethyl acetate (purchased from Sinopharm, batch number 20230620), washed once with 30 mL of saturated citric acid, 30 mL of pure water, and 30 mL of 5% sodium bicarbonate solution, respectively, and the upper organic phase was retained. The ethyl acetate was removed using a rotary evaporator, and the product was dissolved in 10 mL of 95% trifluoroacetic acid (purchased from Macklin, batch number T818778) and reacted at room temperature for 12 hours. After the trifluoroacetic acid and the byproduct tert-butyl alcohol were removed using a rotary evaporator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid modified with 8-pentadecylamine (DOTA-C13) was obtained after vacuum drying for 12 hours.1 HNMR (500 MHz, DMSO, δ): 0.85 (t, CH3, 6H), 1.23 (m, CH2, 20H), 1.40 (m, CH3, 4H), 2.92-3.08 (m, CH2-N, 16H), 3.32 (s, CH-NH, 1H), 3.59 (m, CH2, 6H), 3.71 (s, CH2, 2H), 7.97 (s, NH, 1H); the molecular formula of the resulting compound is C 31 H 60 N5O7[(M+H) + ]: 614.44, mass spectrum m / z: 614.46.

[0044] The above prepared intermediate was re-dissolved in 200 mL of deionized water, 334.5 mg (0.9 mmol) of gadolinium chloride hexahydrate (purchased from Aldrich, batch number G119152) was added, 1 mol / L sodium hydroxide solution was slowly added to adjust the pH to between 6 and 7, and the reaction was continuously stirred at 90 °C for 24 hours. After the solution was completely cooled, a centrifuge was used to centrifuge at a speed of 3500 rpm for 10 minutes, the white precipitate was removed, and the supernatant solution was freeze-dried and then separated and purified by high performance liquid chromatography. The product mass spectrum molecular formula is C 31 H 57 GdN5O7[(M+H) + ]: 769.34, mass spectrum m / z: 769.35, the final product Gd-C15 was determined to be prepared.

[0045] The molecular structure of the Gd-C15 is shown below.

[0046]

[0047] Example 3 Preparation of n-hexylamine modified Gd-DOTA (Gd-C6) 572.74 mg (1 mmol) of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-tBu) (purchased from Macrocyclics, batch number T823757) and 394 mg (1.04 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) purchased from sigma-aldrich, batch number 12804) were dissolved in 10 mL of acetonitrile, followed by the addition of 181 μL (1.04 mmol) of N,N-diisopropylethylamine (DIPEA) (purchased from sigma-aldrich, batch number 550043), 137 μL (1.04 mmol) of n-hexylamine (purchased from Macrocyclics, batch number H810936) to the solution, stirring at room temperature for 3 hours, after removing the acetonitrile solution using a rotary evaporator, the product was redissolved in 30 mL of ethyl acetate, extracted with 30 mL of saturated citric acid, 30 mL of pure water and 30 mL of 5% sodium bicarbonate solution respectively, the upper organic phase was retained, ethyl acetate was removed using a rotary evaporator, and the product was dissolved in 10 mL of 95% trifluoroacetic acid (purchased from Macrocyclics, batch number T818778) after vacuum drying at room temperature for 12 hours, after reacting at room temperature for 12 hours, trifluoroacetic acid and the byproduct tert-butyl alcohol were removed using a rotary evaporator, and n-hexylamine modified tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid was obtained after vacuum drying for 12 hours. 1 H NMR (500 MHz, DMSO, δ): 0.87 (t, CH3, 3H), 1.27 (m, CH2, 6H), 1.43 (m, CH2, 2H), 2.84-3.47 (m, CH2-N, 16H), 3.22 (s, CH2-NH, 2H), 3.57 (m, CH2, 4H), 3.88 (s, CH2, 2H), 4.05 (s, CH2, 2H), 8.47 (s, NH, 1 H); the molecular formula of the resulting compound is C 22 H 42 N5O7[(M+H) + ] : 488.30, mass spectrum m / z: 488.45.

[0048] The intermediate prepared above was redissolved in 200 mL of deionized water, 334.5 mg (0.9 mmol) of gadolinium chloride hexahydrate (purchased from Aldrich, batch number G119152) was added, the pH was adjusted to between 6 and 7 by slowly adding 1 mol / L sodium hydroxide solution, and the reaction was continuously stirred at 90 °C for 24 hours. After the solution was completely cooled, it was centrifuged at 3500 rpm for 10 minutes using a centrifuge, the white precipitate was removed, and the supernatant solution was freeze-dried and then separated and purified by high performance liquid chromatography. The product obtained had a mass spectrum molecular formula of C 22 H 39 GdN5O7[(M+H) + ]: 643.20, mass spectrum m / z: 643.21, and it was determined that the final product Gd-C6 was prepared.

[0049] The molecular structure of the Gd-C6 is shown below.

[0050]

[0051] Example 4 Preparation of 1,5-dimethylhexylamine modified Gd-DOTA (Gd-C8 branch) The preparation method was the same as in Example 5, except that n-hexylamine in Example 5 was replaced by 1,5-dimethylhexylamine (purchased from sigma-aldrich, batch number D161292), and the intermediate product obtained was 1,5-dimethylhexylamine modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., Gd-C8 branch): 1 H NMR (500 MHz, DMSO, δ): 0.86 (d, CH3, 6H),1.07(d, CH3, 3H), 1.15(m, CH2, 2H), 1.26 (m,CH2, 2H),1.37 (m, CH2,2H),1.51 (m, CH,1H),2.85-3.49(m, CH2-N,16H), 3.56(s, CH2,4H), 3.77(s, CH-NH, 1H), 3.91(s,CH2, 2H), 4.10 (s,CH2,2H), 8.41(s, NH, 1H);the molecular formula of the intermediate compound obtained was C 24 H 46 N5O7[(M+H) + ]: 516.33, mass spectrum m / z: 516.55. The intermediate compound was reacted with a gadolinium compound to obtain a product with a mass spectrum molecular formula of C 24 H 43 GdN5O7[(M+H)+ ] : 671.23, mass spectrum m / z: 671.50, final product (Gd-C8) was confirmed to be prepared.

[0052] The molecular structure of the Gd-C8 is shown below.

[0053]

[0054] Example 5 Preparation of n-decylamine modified Gd-DOTA (Gd-C10) 572.74 mg (1 mmol) of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-tBu) (purchased from Aldrich, batch number T162093) and 394 mg (1.04 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (purchased from Aldrich, batch number H106174) were dissolved in 10 mL of acetonitrile (purchased from Aldrich, batch number A104443), then 181 μL (1.04 mmol) of N,N-diisopropylethylamine (DIPEA) (purchased from sigma-aldrich, batch number 550043), 219 μL (1.04 mmol) of n-decylamine (purchased from Genview, batch number D21130) were added to the solution, and the reaction was stirred at room temperature for 3 hours. After removing the acetonitrile solution using a rotary evaporator, the product was redissolved in 30 mL of ethyl acetate (purchased from Sinopharm, batch number 20230620), washed once with 30 mL of saturated citric acid, 30 mL of pure water, and 30 mL of 5% sodium bicarbonate solution, respectively, and the upper organic phase was retained. The ethyl acetate was removed using a rotary evaporator, and the product was dissolved in 10 mL of 95% trifluoroacetic acid (purchased from Macklin, batch number T818778) and reacted at room temperature for 12 hours. After removing the trifluoroacetic acid and the byproduct tert-butyl alcohol using a rotary evaporator, n-decylamine modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-C10) was obtained after vacuum drying for 12 hours. 1 H NMR (500 MHz, DMSO, δ): 0.86 (t, CH3, 3H), 1.25 (m, CH2, 14H), 1.43 (m, CH3, 2H), 2.89-3.02 (m, CH2-N, 16H), 3.08 (s, CH2-NH, 2H), 3.54 (m, CH2, 2H), 3.59 (m, CH2, 6H), 8.20 (s, NH, 1H); the molecular formula of the resulting compound is C 26 H 50N5O7[(M+H) + ] : 544.36, Mass: m / z 544.29.

[0055] The intermediate prepared above was redissolved in 200 mL of deionized water, 334.5 mg (0.9 mmol) of gadolinium chloride hexahydrate (purchased from Aldrich, batch number G119152) was added, 1 mol / L sodium hydroxide solution was slowly added to adjust the pH to 6-7, and the reaction was continuously stirred at 90 °C for 24 hours. After the solution was completely cooled, a centrifuge was used to centrifuge at a speed of 3500 rpm for 10 minutes, the white precipitate was removed, and the supernatant solution was freeze-dried and then separated and purified by high performance liquid chromatography. The product had a mass spectrum molecular formula of C 26 H 47 GdN5O7[(M+H) + ] : 699.26, Mass: m / z 699.17. It was determined that the final product Gd-C10 was prepared.

[0056] The molecular structure of the Gd-C10 is shown below.

[0057]

[0058] Example 6 Preparation of n-tetradecyl modified Gd-DOTA (Gd-C14) The preparation method was the same as in Example 5, and n-hexylamine in Example 5 was replaced with tetradecylamine (purchased from Zhi Ji Biological, batch number T80680). The obtained intermediate tetradecylamine modified tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid: 1 H NMR (500MHz, CDCl3, δ): 0.86 (t, CH3, 3H), 1.24 (m, CH2,22H),1.43(m, CH2, 2H), 2.83-3.47 (m, CH2-N, 16H), 3.28 (s, CH2-NH, 2H), 3.61 (m, CH2,4H), 3.91 (s, CH2, 2H), 4.11 (s, CH2, 2H), 8.48(s, NH, 1H); The molecular formula of the obtained intermediate compound was C 30 H 58 N5O7[(M+H) + ] : 600.43, Mass: m / z 600.44. The intermediate compound was reacted with a gadolinium compound to obtain a product with a mass spectrum molecular formula of C 30 H 55 GdN5O7[(M+H) +] : 755.33, mass spectrometry m / z: 755.34, final product (Gd-C14) was confirmed to be prepared.

[0059] The molecular structure of the Gd-C14 is shown below.

[0060]

[0061] Performance test of magnetic resonance contrast agent for liver diagnosis Test Example 1 Kinetic stability evaluation of contrast agent The dissociation of small molecule gadolinium-based contrast agents is closely related to serious side effects such as nephrogenic systemic fibrosis (NSF) and Gd 3+ deposition in patients' bodies, so it is essential to evaluate its kinetic stability. The attack of endogenous metal ions Zn 2+ is the main cause of Gd 3+ leakage in vivo, and this process is mainly mediated by acid for macrocyclic complexes. To evaluate the kinetic stability of the contrast agent, the relaxation rate of the contrast agent was measured under 1 M HC1 and 10 mM Zn 2+ ion concentration, respectively, with Gd-DOTA and Gd-EOB-DTPA as controls.

[0062] Kinetic stability test under 1 M HC1 conditions.

[0063] Dissolve Gd-DOTA, Gd-C10, Gd-C13, Gd-C14, Gd-C15 in 1 M HC1, prepare a 1 mM concentration of the contrast agent solution, and incubate at 25 °C for 7 days. During this period, the relaxation time (T1) at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 16 h, 20 h, 24 h, 55 h, 71 h, 97 h, 126 h and 170 h was measured using a 9.4T / 400mm MRI device (Bruker BioSpin) relaxation enhancement fast acquisition sequence (RARE), respectively. The parameter settings are: echo time TE = 13 ms, repetition time TR is 0.5, 0.73, 1, 1.3, 1.8, 2.3, 3.3 and 7.5 s, respectively. By plotting the ratio of T1(t) to the initial value T1(0) measured at each time point, the release rate of Gd 3+ was reflected.

[0064] Kinetic stability test under 10 mM Zn 2+ conditions.

[0065] Gd-EOB-DTPA, Gd-DOTA, Gd-C10, Gd-C13, Gd-C14, Gd-C15 were dissolved in PBS solution containing 10 mM Zn 2+ 3+

[0066] The linear chelate Gd-EOB-DTPA is very sensitive to acidic environment, with a half-life (t 1 / 2 ) of less than 5 seconds in 0.1 M HC1 (BioMetals, 2008, 21, 469).

[0067] The results of the kinetic stability test under the above-mentioned 1 M HC1 condition and 10 mM Zn 2+ Figure 2

[0068] Figure 2 The release rate of Gd 3+ under the 1 M HC1 condition is shown in Figure a. The results show that the Gd 3+ release rate of the Gd-DOTA compounds modified with lipids is similar, and is significantly slower than that of Gd-DOTA, indicating that the lipid modification significantly improves the chemical kinetic stability of the contrast agent. Figure 2 The change in relaxivity of Gd-DOTA, Gd-EOB-DTPA, Gd-C10, Gd-C13, Gd-C14 and Gd-C15 and Gd-DOTA under the 10 mM Zn 2+ 2+ The T1 relaxation time of Gd-EOB-DTPA continues to increase, while the relaxivity of Gd-C10, Gd-C13, Gd-C14, Gd-C15 and Gd-DOTA changes little within 7 days of incubation at 37°C, indicating that the Gd-DOTA type chelate is superior to the linear chelate Gd-EOB-DTPA in resisting Zn​​​​​

[0069] In summary, the saturated alkyl single or double chain modified Gd-DOTA contrast agents prepared in the present application show excellent kinetic stability in 1 M HCl and 10 mM Zn 2+ under conditions, significantly better than the commercial contrast agent Gd-EOB-DTPA. By the modification of the lipid chain, the contrast agents of the present application reduce the risk of Gd 3+ leakage and improve the stability under complex physiological conditions, providing an important guarantee for reducing side effects and improving safety in clinical applications.

[0070] Test Example 2: In vitro relaxation performance test of the contrast agents Gd-C10, Gd-C13, Gd-C14 and Gd-C15 were dissolved in 300 μL deionized water, and the concentrations of Gd 3+ were 0.1, 0.2, 0.4, 0.8, 1.6 mM, respectively. At 25 °C, the longitudinal (T1) and transverse (T2) relaxation times of the samples were recorded using a 9.4T / 400mm MRI device (Bruker BioSpin). RARE (rapid acquisition with relaxation enhancement) was used to measure T1, with the following parameter settings: echo time TE = 13 ms, repetition time TR = 0.5, 0.73, 1, 1.3, 1.8, 2.3, 3.3 and 7.5 s, respectively. T2 was measured using a multi-echo spin echo sequence with the following parameter settings: TR = 10 s, TE values from 10 to 300 ms, a total of 30 data points. The in vitro relaxation performance r1 and r2 values of the four synthesized contrast agents were determined by plotting the curves of 1 / T1 or 1 / T2 versus sample concentration and calculating the slope r1 and r2 of the linear fitting curve. Figure 3 The relaxation performance r1 and r2 fitting curves of Gd-C10, Gd-C13, Gd-C14 and Gd-C15 and their values are shown. Compared with Gd-EOB-DTPA, the contrast agents modified by different lengths of lipid chains did not change significantly in relaxation performance. The r1 values of Gd-EOB-DTPA, Gd-C10, Gd-C13, Gd-C14 and Gd-C15 were 4.18, 3.48, 3.96, 3.34 and 3.39 mM -1 s -1 .

[0071] Test Example 3: Cytotoxicity test of the contrast agents Cell culture. Human kidney epithelial cell 293T fibroblasts (purchased from MELON BIOTECH) were cultured using DMEM medium (purchased from GIBCO, batch number 21068028) containing 10% fetal bovine serum, passaged every 2-3 days, and incubated at 37°C in a humidified environment with 5% CO2. Then, the cells were digested using 0.25% trypsin (purchased from GIBCO, batch number 25200072). The day before treatment, the cells were seeded on a 96-well plate at the desired cell density.

[0072] Measurement of cell survival rate. 293T cells were seeded on a 96-well plate at a density of 5000 cells / well, cultured for 24 hours, and after the cells adhered, the old culture medium was discarded and washed twice with PBS, and then fresh culture medium containing different concentrations of contrast agent (concentrations were 10, 50, 100, 200 and 500 μM) was added, and then incubated at 37°C for 24 hours. 10 μL of CCK-8 reagent (purchased from MELON BIOTECH, batch number MA0218-Oct-31J) was added to each well, and incubated for 2 hours. After the incubation step, the absorbance at 450 nm wavelength was detected using a microplate reader (Tecan, model: SPARK). The cell survival rate was determined as the percentage of the number of cells surviving in the culture medium containing the contrast agent to the number of cells surviving in the untreated control. By comparing the absorbance of the treated and untreated cells, the cell survival rate was calculated according to formula (1).

[0073]

[0074] In formula (1), F 样本 is the average absorbance of the experimental group (cells incubated with culture medium containing contrast agent), F 阴性对照组 is the average absorbance of the negative control group (no cells, only culture medium containing contrast agent), F 阳性对照组 is the average absorbance of the control group (no cells incubated with culture medium containing contrast agent).

[0075] Figure 4The results of the cytotoxicity experiments of Gd-EOB-DTPA and six kinds of lipid chain modified contrast agents (Gd-C6, Gd-C8, Gd-C10, Gd-C13, Gd-C14, Gd-C15) are shown. The experimental results show that within the concentration range of 10-500 μM of the contrast agents, the cell survival rates of Gd-C6, Gd-C8, Gd-C10, Gd-C13 and Gd-C15 do not decrease significantly, showing excellent biological safety. However, Gd-C14 may have a significant increase in lipophilicity due to the longer length of the single lipid chain, and at a concentration of 500 μM, the cell survival rate decreases to 7% of the initial value, showing obvious cytotoxicity, which is not suitable for in vivo experiments. In contrast, the double-chain modified contrast agents Gd-C13 and Gd-C15 with similar total number of carbon atoms effectively alleviate the problem of excessive binding to cell membrane phospholipids caused by the increase in the total number of carbon atoms by optimizing the molecular structure, thereby significantly reducing the cytotoxicity and further improving the biological safety of the contrast agents.

[0076] Test Example 4 In vivo Liver Magnetic Resonance Contrast Agent Effect of Animal The purchased 6-8 week old female C57BL / 6 black mice (weight 20±2 g) were placed in a laminar flow rack and raised under constant temperature conditions (23±2℃) using sterile feed and sterile water for 2 weeks. After the small black mice adapted to the new environment, they were used for magnetic resonance imaging research.

[0077] In the MRI in vivo experiment, isoflurane (purchased from Hengfengqiang Company) was used to anesthetize the mice (concentration of 3.5% during induction and 1.0-1.5% during maintenance). The animals were placed in a special support in a prone position, and the respiratory rate of the mice was monitored by a physiological detection device. During the entire experiment, the body temperature of the animals was maintained at about 36.5℃ by a heating pad. A 9.4 T / 400 mm MRI device was used to acquire axial images of the mouse at the liver position using a T1-weighted-relaxation enhancement rapid acquisition (RARE) sequence. Five images were continuously acquired before contrast agent injection, with a sampling interval of 1.875 minutes, and then Gd-EOB-DTPA or lipid chain modified Gd-DOTA complex was injected into the tail vein of the mouse (0.1 mmol / kg). Within 120 minutes after injection, a set of contrast agent enhanced images was acquired every 1.875 minutes. The specific parameter settings are as follows: TR = 500 ms, TE = 8 ms, field of view FOV = 30 ×30 mm 2 , matrix size = 150 × 150, slice thickness = 1 mm, number of slices = 10 (with an interval of 0).

[0078] Figure 5The liver region magnetic resonance imaging images of mice at different time points after tail vein injection of Gd-EOB-DTPA and five kinds of lipid chain modified contrast agents (Gd-C6, Gd-C8 branch, Gd-C10, Gd-C13, Gd-C15) are shown. Before injection of the contrast agent, the contrast of liver tissue and surrounding visceral tissue is low and almost indistinguishable. After injection of Gd-EOB-DTPA, the signal intensity of the liver region is significantly enhanced, with an enhancement rate of 192%, and the contrast with the surrounding tissue is significantly improved. Notably, the double lipid chain modified contrast agents Gd-C13 and Gd-C15 show significant advantages, with relative enhancement rates of 167% and 143% respectively, close to the level of Gd-EOB-DTPA, clearly showing the liver region. In contrast, the single chain modified contrast agents Gd-C6, Gd-C8 branch and Gd-C10 have insufficient signal enhancement, with relative enhancement rates of 49%, 97% and 108% respectively. This comparison fully demonstrates that double chain modified contrast agents have a significant advantage in improving the contrast of liver imaging, providing clearer and more accurate image support for the diagnosis of liver diseases.

[0079] Test Example 5 Tissue distribution experiment of contrast agent The above-mentioned 6-8 week old female C57BL / 6 mice (weight 20±2g) were used for the tissue distribution study of the lipid chain modified contrast agent under constant temperature conditions (23±2°C) using sterile feed and sterile water. After the mice adapted to the new environment, they were used for the tissue distribution study of the lipid chain modified contrast agent.

[0080] In the tissue distribution experiment, isoflurane (purchased from Hengfengqiang Company) was used to anesthetize the mice (induction period concentration of 3.5%, maintenance period concentration of 1.0-1.5%). The lipid chain modified Gd-DOTA (0.1 mmol / kg) was injected through the tail vein. At 0.5 hours after injection, the main organ tissues (heart, liver, spleen, kidney and brain) were collected and weighed. The tissue samples were cut into small pieces and heated and digested with concentrated nitric acid at 120°C overnight. The digested residue was diluted with 5 mL of 2% nitric acid solution, and the Gd 3+ concentration in each organ was determined by inductively coupled plasma mass spectrometry (ICP-MS) to evaluate the tissue distribution of lipid single or double chain modified Gd-DOTA.

[0081] Figure 6 The concentration of gadolinium ions in the main organs of C57BL / 6 mice of Gd-DOTA and four kinds of lipid chain modified contrast agents (Gd-C6, Gd-C8 branch, Gd-C10, Gd-C13) is shown. When the carbon chain length increases to more than 10 carbon atoms, the liver becomes the main organ for uptake of the contrast agent, and the uptake rate of the contrast agent in the liver continues to increase with the increase in the number of carbon atoms. From Figure 6It can be seen that the uptake rate value distribution of Gd-DOTA and four kinds of lipid chain modified contrast agents in the liver is: the average uptake rate of Gd-DOTA is 3198.3 ng / g, the uptake rate value of Gd-C6 is 809.6 ng / g, the average uptake rate of Gd-C8 is 2168.3 ng / g, the uptake rate value of Gd-C10 is 15121.5 ng / g, and the uptake rate value of Gd-C13 is 22509.0 ng / g. It can be seen that in the aspect of uptake rate value performance in the liver, compared with the uptake rate of Gd-DOTA, the single chain modified Gd-C6 and the single chain modified Gd-C8 appear a clear downward trend, which cannot solve the problem of low liver absorption of Gd-DOTA. At the same time, the long chain modified Gd-C10 and the double chain modified Gd-C13 both improve the problem of low liver absorption of Gd-DOTA, but the average uptake rate of the double chain modified Gd-C13 is nearly 1.5 times that of the single chain modified Gd-C10, and its enhanced absorption effect is more significant.

[0082] In summary, the saturated alkyl double chain modified contrast agent (Gd-C13 and Gd-C15, especially Gd-C13) has a signal in the liver region equivalent to the commercial contrast agent Gd-EOB-DTPA, and its kinetic stability is significantly better than that of the commercial contrast agent Gd-EOB-DTPA, and it has higher biological safety.

[0083] Although the contrast agents Gd-C10, Gd-C13, Gd-C14 and Gd-C15 all retain the higher kinetic stability and smaller gadolinium leakage risk of Gd-DOTA, the saturated alkyl double chain modified contrast agent (Gd-C13 and Gd-C15) of the present application, by introducing a saturated alkyl double chain, on the one hand, significantly reduces the cytotoxicity (possible reason: the saturated alkyl double chain structure reduces the binding ability of the contrast agent to the cell membrane phospholipid), and on the other hand, significantly improves the low uptake rate of Gd-DOTA in the liver. Compared with the single chain modified Gd-C14 (which has biological safety hazards) and Gd-C10 (which has weak contrast ability), the saturated alkyl double chain modified magnetic resonance imaging contrast agent Gd-C13 and Gd-C15 provided by the present application have higher biological safety and contrast ability, and have broad application prospects in the diagnosis of liver diseases, especially in clinical scenarios that require consideration of imaging effect and safety.

[0084] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A magnetic resonance imaging contrast agent modified with saturated alkyl double chains, characterized in that, The structural formula of the contrast agent is shown below: Among them, R1 and R2 are the same, both being saturated alkyl chains, and the number n of carbon atoms in each saturated alkyl chain satisfies: 5≤n≤8.

2. The magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 1, characterized in that, The number of carbon atoms n in each of the saturated alkyl chains is 6 or 7.

3. A method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 1 or 2, characterized in that, include: Tricarboxylated tetraaza-heterocyclic acetic acid and R1R2CH-NH2 react with the amino and carboxyl groups to obtain an intermediate product; The intermediate product is deprotected to obtain the precursor compound; The precursor compound reacts with the gadolinium compound to obtain a magnetic resonance imaging contrast agent modified with saturated alkyl double chains; Among them, R1 and R2 are the same, both being saturated alkyl chains, and the number n of carbon atoms in each saturated alkyl chain satisfies: 5≤n≤8.

4. The method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 3, characterized in that, The carboxyl protecting group in the tricarboxyl-protected polynitrogen heterocyclic polynitrogen acetic acid is tert-butyl.

5. The method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 3, characterized in that, The molar ratio of the tricarboxylated protected tetrazole heterocyclic tetrazole acetic acid and R1R2CH-NH2 is 1:1-1.

5.

6. The method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 3, characterized in that, The reaction between the amino and carboxyl groups is carried out in the presence of a first organic solvent and a condensing agent.

7. The method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 3, characterized in that, The reaction between the amino and carboxyl groups is carried out at a temperature of 10-60℃ for 2-24 hours.

8. The method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to claim 3, characterized in that, The gadolinium compound is gadolinium chloride or gadolinium acetate.

9. A method for preparing a magnetic resonance imaging contrast agent modified with saturated alkyl double chains according to any one of claims 3-8, characterized in that, The molar ratio of the precursor compound to the gadolinium compound is 1:1.05-1.

5.

10. The application of the saturated alkyl double-chain modified magnetic resonance imaging contrast agent as described in claim 1 or 2 in magnetic resonance imaging.

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