PDA-modified cycleanine coordination compound as well as synthesis method and application thereof

Abstract

The invention discloses a PDA modified cycleanine coordination compound and a synthesis method and application thereof, the coordination compound has the capability of amplifying Zn (II) signals, that is, in normal tissues, the MRI signals of the compound are weak, and in Zn (II) distribution tissues, the compound can be easily combined with HSA so as to detect and amplify the Zn (II) signals. The coordination compound has high responsiveness and sensitivity to Zn (II) detection, can be used as an MRI probe to detect Zn (II) in living cells, and is suitable for the fields of T2ex imaging and detection of Zn (II) in tumors or pathological tissues and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic resonance imaging, and in particular relates to a class of PDA-modified cyclopentane coordination compounds and a synthesis method and application thereof, which amplify the sensitivity of the response to Zn(II) by changing the water exchange rate. Background Art

[0002] Divalent zinc (Zn(II)) is abundant in mammalian tissues. As the second most abundant transition metal, it plays a key role in the structure, catalysis and signal transduction of biological cells. Almost all tissues require Zn(II) for signal transduction, and its concentration is particularly high in the pancreas, prostate, and brain. It mainly exists in the form of protein chelates in the blood. Zn(II) is strictly regulated by a variety of different transporters. The imbalance of Zn(II) content in these different tissues is related to diabetes, Alzheimer's disease and prostate cancer. Therefore, the detection of Zn(II) content in the body has certain value for the diagnosis and treatment of diseases.

[0003] For more than two decades, people have devoted themselves to developing and studying various optical instruments and detection methods for detecting free Zn(II) ions, such as common atomic absorption spectroscopy, spectrophotometry, and fluorescence spectroscopy. Although these detection methods have attracted much attention due to their advantages of high selectivity, convenience, and rapidity, their applicability in monitoring Zn(II) levels in vivo is limited. Therefore, optical-based detectors are largely unable to detect the content of Zn(II) at the cellular level. Magnetic resonance imaging (MRI) has become a method for physiological imaging of in vivo tissues because it is not affected by tissue penetration. However, the sensitivity of magnetic resonance itself is lower than that of optical imaging, and it must be detected indirectly through abundant water protons. The resolution of MRI images is mainly related to the water content in the tissue, but the difference in water molecule content between some tissues and organs in the human body and diseased tissues is not large. Therefore, contrast agents (CA) need to be added to change the relaxation rate of water protons in local tissues in the body, thereby improving the imaging contrast between the diseased area and the normal area.

[0004] After contrast agent (CA) enters the human body, both T1 and T2 will change, but different contrast agents have different effects. Some contrast agents mainly shorten T2 and make the lesion darker. This type is called T2 contrast agent, also called negative contrast agent, including ferromagnetic and supermagnetic contrast agents. According to the Swift-connick equation It can be seen that the factors affecting T2 include: the chemical shift of bound water (Δω), the exchange rate of water molecules (K ex), magnetic field strength (B0), where the only variable is the exchange rate of water molecules. Therefore, the contrast effect of T2 can be affected by changing the exchange rate of water molecules. Similarly, contrast agents that shorten T1 and brighten the lesion are called T1 contrast agents, also known as positive contrast agents. T1 contrast agents are commonly used in clinical practice, and T2 contrast agents are rarely reported. However, the effect of T1 contrast agents will inevitably change with changes in concentration, so the development of T2 contrast agents has received increasing attention. Dysprosium contrast agents have gradually entered the research field of view due to their stability and low toxicity. This type of contrast agent itself does not generate signals, and achieves contrast effects by changing the relaxation time of surrounding water molecules.

[0005] The structure of N,N-di(2-pyridylmethyl)ethylenediamine is closely related to Zn(II). It has been widely studied as a specific recognition site for zinc ions. For example, Gd-DOTA-diBPEN designed by Sherry et al. cannot bind to HSA (human serum albumin) in the absence of Zn(II), but can easily bind to HSA in the presence of Zn(II), effectively increasing the rotational correlation time (T R ) greatly increases the relaxation efficiency.

[0006] In summary, the design and development of a T 2ex It is very necessary to develop a highly sensitive and responsive DPA-modified Zn(II)-responsive contrast agent for early imaging diagnosis of diseases with imbalanced Zn(II) content, such as diabetes, Alzheimer's disease, and prostate cancer. Summary of the invention

[0007] Based on the above prior art, the present invention provides a class of PDA-modified cyclopentane coordination compounds and their synthesis methods and applications. The coordination compounds have the ability to amplify Zn(II) signals, that is, in normal tissues, the compounds themselves have no MRI signals, but in tissues where Zn(II) is distributed, the compounds can easily bind to HSA to detect and amplify Zn(II) signals. The coordination compounds have high responsiveness and sensitivity to Zn(II) detection, can be used as MRI probes to detect Zn(II) in living cells, and are suitable for the detection of Zn(II) in tumors or diseased tissues. 2ex Imaging and detection, etc.

[0008] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0009] A type of PDA-modified cyclopentane coordination compound has the following structural formula:

[0010]

[0011] A method for synthesizing a PDA-modified cyclopentane coordination compound comprises the following steps:

[0012] S1, 1,4,7,10-tetraazacyclododecane and benzyl chloroformate undergo nucleophilic substitution reaction to generate a compound of formula (I). The reaction formula is as follows:

[0013]

[0014] S2. In the presence of a base, the compound of formula (I) undergoes a nucleophilic substitution reaction with tert-butyl bromoacetate to generate a compound of formula (II), and the reaction formula is as follows:

[0015]

[0016] Or S2', in the presence of a base, the compound of formula (I) first undergoes a nucleophilic substitution reaction with ethyl bromoacetate to generate an intermediate compound I, and the intermediate compound I then undergoes a nucleophilic substitution reaction with ethyl bromoacetylglycine to generate a compound of formula (II), and the reaction formula is as follows:

[0017]

[0018] Or S2", in the presence of a base, the compound of formula (I) undergoes a nucleophilic substitution reaction with ethyl bromoacetyl glycine to generate a compound of formula (II), and the reaction formula is as follows:

[0019]

[0020] S3. Under a hydrogen atmosphere, the compound of formula (II) undergoes a hydrogenation reduction reaction under the action of a catalyst to generate a compound of formula (III), and the reaction formula is as follows:

[0021]

[0022] S4. In the presence of a base, N-tert-butyloxycarbonyl-1,2-ethylenediamine and 2-chloromethylpyridine or a salt thereof undergo a nucleophilic substitution reaction to generate tert-butyl-(2-(di(pyridin-2-ylmethyl)amino)ethyl)carbamate, the reaction formula of which is as follows:

[0023]

[0024] S5, tert-butyl-(2-(di(pyridine-2-ylmethyl)amino)ethyl)carbamate is deprotected by the tert-butyloxycarbonyl group to generate N1,N1-di(pyridine-2-methyl)ethane-1,2-diamine, and the reaction formula is as follows:

[0025]

[0026] S6. In the presence of a base, N1,N1-di(pyridine-2-methyl)ethane-1,2-diamine reacts with chloroacetyl chloride to undergo a nucleophilic substitution reaction to generate N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide, and the reaction formula is as follows:

[0027]

[0028] S7. In the presence of a base, the compound of formula (III) undergoes a nucleophilic substitution reaction with N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide to generate a compound of formula (IV), and the reaction formula is as follows:

[0029]

[0030] S8. The compound of formula (IV) is first hydrolyzed to generate an intermediate compound II. The intermediate compound II undergoes a coordination reaction with a trivalent dysprosium ion to generate the PDA-modified cyclopentane coordination compound. The reaction formula is as follows:

[0031]

[0032]

[0033] Furthermore, the nucleophilic substitution reaction in step S1 is carried out at room temperature, with a stirring time of 4-12 hours, and a molar ratio of 1,4,7,10-tetraazacyclododecane to benzyl chloroformate of 0.48-0.5:1.0.

[0034] Furthermore, the base is selected from at least one of potassium carbonate or potassium bicarbonate.

[0035] Furthermore, the reaction temperature of all nucleophilic substitution reactions in step S2 is 65-75° C., and the reaction time is 8-10 h.

[0036] Further, in the step S2, the molar ratio of the compound of formula (I) to tert-butyl bromoacetate is 1.0:2.0-2.2; in the step S2', the molar ratio of the compound of formula (I) to ethyl bromoacetate is 1.0:1.0-1.2, and the molar ratio of the compound of formula (I) to ethyl bromoacetate is 1.0:1.0-1.2; in the step S2", the molar ratio of the compound of formula (I) to ethyl bromoacetyl glycine is 1.0:2.0-2.2.

[0037] Furthermore, in step S3, the catalyst is a palladium-carbon catalyst, the hydrogenation reduction reaction is carried out at room temperature, and the reaction time is 8-10 hours.

[0038] Furthermore, the reaction temperature of the nucleophilic substitution reaction in step S4 is 80-110° C., the reaction time is 8-12 h, and the molar ratio of N-tert-butyloxycarbonyl-1,2-ethylenediamine to 2-chloromethylpyridine or its salt is 0.9-1.0:3.0-3.5.

[0039] Furthermore, the reaction temperature of the nucleophilic substitution reaction in step S6 is -5-5°C, the reaction time is 3-5h, and the molar ratio of N-tert-butyloxycarbonyl-1,2-ethylenediamine to chloroacetyl chloride is 0.9-1:1.

[0040] Furthermore, the nucleophilic substitution reaction in step S7 is carried out at 65-75° C., the reaction time is 8-10 h, and the molar ratio of the compound of formula (III) to N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide is 1.0:2.0-2.2.

[0041] Furthermore, in step S8, the coordination reaction is carried out at room temperature, the reaction time is 6-8 hours, and the molar ratio of the compound of formula (IV) to the trivalent dysprosium ion is 1.0:0.9-1.0.

[0042] Furthermore, the trivalent dysprosium ion is selected from dysprosium trifluoromethanesulfonate.

[0043] Application of a PDA-modified cyclopentane coordination compound in the preparation of a divalent zinc ion-responsive T2 contrast agent.

[0044] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0045] 1. The coordination compound of the present invention contains an N,N-di(2-pyridylmethyl)ethylenediamine structure. In the tissue where Zn(II) is distributed, the N,N-di(2-pyridylmethyl)ethylenediamine structure can be easily combined with HSA. At the same time, Zn(II) can form a coordination bond with the two pyridine structures on the N,N-di(2-pyridylmethyl)ethylenediamine structure. HSA and the presence of Zn(II) will change the relaxation environment of the dysprosium coordination compound, causing the dysprosium coordination compound r2 to change, thereby detecting and amplifying the signal of Zn(II), thereby realizing signal amplification and detection of specific tissues, especially Zn(II) imbalance areas.

[0046] 2. The series of coordination compounds of the present invention are connected with different numbers of glycine, so that the coordination compounds exhibit different water molecule exchange rates. The T of the contrast agent is determined by testing the change in its transverse relaxation. 2exImaging effect. Due to the uneven distribution of Zn(II) in various tissues, the original detection method is prone to misjudgment, that is, the molar relaxation rate value will change with the increase of concentration. Therefore, by detecting r1 and r2 at the same time, and then comparing the two, the r2 / r1 ratio is used to reflect the change of r2, so as to know T 2ex This new quantitative method is particularly important in conditions where abnormal Zn(II) is produced, such as cancer, diabetes, etc.

[0047] 3. The coordination compound of the present invention has good biosafety and excellent water dispersibility, is suitable for in vivo MRI, and has good application prospects in the early diagnosis of diabetes, Alzheimer's disease, and prostate cancer.

[0048] 4. The coordination compound of the present invention is simple, the raw materials are cheap and easily available, the synthesis conditions are relatively simple, and the synthesis cost is relatively low. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a graph showing the changes in the molar transverse relaxation rate and the molar longitudinal relaxation rate of the cyclopentane coordination compound Dy-1 prepared in Example 1 in response to Zn(II).

[0050] Figure 2 This is a graph showing the changes in the molar transverse relaxation rate and the molar longitudinal relaxation rate of the cyclopentane coordination compound Dy-2 prepared in Example 2 in response to Zn(II).

[0051] Figure 3 This is a graph showing the changes in the molar transverse relaxation rate and the molar longitudinal relaxation rate of the cyclopentane coordination compound Dy-3 prepared in Example 3 in response to Zn(II).

[0052] Figure 4 This is a graph showing the toxicity test of the cyclopentane coordination compound Dy-1 prepared in Example 1 on human breast cancer cells MCF-7.

[0053] Figure 5 This is a graph showing the toxicity test of the cyclopentane coordination compound Dy-2 prepared in Example 2 on human breast cancer cells MCF-7.

[0054] Figure 6 This is a graph showing the toxicity test of the cyclopentane coordination compound Dy-3 prepared in Example 3 on human breast cancer cells MCF-7.

[0055] Figure 7 This is a graph showing the competitive test results of the cyclopentane coordination compound Dy-1 prepared in Example 1 against different metal ions.

[0056] Figure 8 This is a graph showing the competitive test results of the cyclopentane coordination compound Dy-2 prepared in Example 2 against different metal ions.

[0057] Fig. 9 This is a graph showing the competitive test results of the cyclopentane coordination compound Dy-3 prepared in Example 3 against different metal ions. DETAILED DESCRIPTION

[0058] The present invention is described in detail below with reference to specific embodiments.

[0059] Example 1

[0060]

[0061] 1. Synthesis of 1,4,7,10-tetraazacyclododecane-1,7-dicarboxylic acid dibenzyl ester hydrochloride (1b)

[0062] 1,4,7,10-Tetraazacyclododecane 1a (14.0 g, 81.3 mmol) was dissolved in 300 mL of dichloromethane and cooled to 0°C. Then, benzyl chloroformate (24.0 mL, 170.7 mmol) was slowly added dropwise. The mixture was heated to room temperature and stirred for 4 hours at room temperature. The reaction was monitored by TLC until completion. The mixed product was concentrated and diethyl ether (400 mL) was added to the residue. The mixture was stirred for 1 hour and filtered. The filter cake was repeatedly washed with diethyl ether and dried to obtain a white solid 1b (38.3 g, yield 92%).

[0063] HRMS: m / z=441.2505[M+H] + .

[0064] Synthesis of 2,4,10-bis(2-(tert-butoxy)-carbonylethyl)-1,4,7,10-tetraazacyclododecane-1,7-dicarboxylic acid dibenzyl ester (1c)

[0065] White solid 1b (6.0 g, 11.7 mmol) and potassium carbonate (8.0 g, 58.5 mmol) were added into acetonitrile (50 mL), stirred at room temperature for 10 minutes, and then tert-butyl bromoacetate (2.9 mL, 25.7 mmol) was added dropwise. The mixture was heated to 65°C and reacted overnight. After the reaction was completed as monitored by LC-MS, the mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol 50:1, v / v) to obtain a yellow transparent oily liquid 1c (5.0 g, yield 64%).

[0066] HRMS: m / z=669.3866[M+H] + .

[0067] 1H NMR (400MHz, CDCl3) δ7.40–7.27(m,10H),5.12(s,4H),3.36(d,J=45.4Hz,12H),2.87(s,6H),2.04(d,J=0.4Hz,2H),1.43(s,18H).

[0068] 3. Synthesis of 1,7-tert-butyl diacetate-1,4,7,10-tetraazacyclododecane (1d)

[0069] The yellow transparent oily liquid 1c (2.2 g, 3.3 mmol) was dissolved in methanol (15 mL), 10% Pd / C (200 mg) was added, and the mixture was transferred into a sealed high-pressure reactor. Hydrogen was introduced and reacted for 8 hours. After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain a yellow oily liquid 1d, which was directly used in the next step without purification.

[0070] HRMS: m / z = 401.3126 [M+H] + .

[0071] 1 H NMR (400MHz, CDCl3) δ3.41–3.21 (m, 4H), 3.05–2.56 (m, 16H), 1.42 (d, J = 6.4Hz, 18H).

[0072] 4. Synthesis of N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide (1f)

[0073] Dissolve N-tert-butoxycarbonyl-1,2-ethylenediamine (1.4 mL, 8.7 mmol), sodium carbonate (4.6 g, 43.5 mmol) and 2-chloromethylpyridine hydrochloride (4.3 g, 26.1 mmol) in ethanol (50 mL), and react at 110°C overnight. After the reaction is completed as monitored by TLC, filter and concentrate the filtrate. The residue is purified by flash column chromatography on a silica gel column (dichloromethane / methanol 50:1, v / v) to obtain a yellow oil (2.7 g, 8.0 mmol).

[0074] The yellow oil was added into 4M hydrogen chloride dioxane solution (15 mL) and reacted at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a yellow solid crude product.

[0075] The crude yellow solid product (2.3 g) was dissolved in dichloromethane (50 mL), cooled to 0°C, and then potassium bicarbonate (4 g, 40 mmol) was added, stirred for 30 minutes, and then a dichloromethane solution (10 mL) containing chloroacetyl chloride (716 μL, 9.0 mmol) was added dropwise. After the addition was completed, the mixture was reacted at 0°C for 4 hours. After the reaction was completed as monitored by LC-MS, the mixture was concentrated under reduced pressure. The residue was purified by silica gel normal phase flash column chromatography (dichloromethane / methanol 20:1, v / v) to obtain a brown oil 1f (2.1 g, yield 84%).

[0076] HRMS: m / z=319.1329[M+H] + .

[0077] 1 H NMR(500MHz, CDCl3)δ8.56–8.50(m,2H),7.61(td,J=7.7,1.7Hz,2H),7.37(d,J=7.8Hz ,2H),7.14(dd,J=6.9,5.4Hz,2H),4.02(s,2H),3.86(s,4H),3.51–3.31(m,2H),2.81–

[0078] 2.72 (m, 2H).

[0079] 6. Synthesis of 1,7-tert-butyl diacetate-4,10-bis(2,2'-bis(2-methylpyridine)aminoethylamino-1,4,7,10-tetraazacyclododecane (1e)

[0080] Yellow oily liquid 1d (1.2 g, 2.9 mmol) and potassium carbonate (2.0 g, 14.5 mmol) were dissolved in acetonitrile (50 mL), heated to 65°C, brown oil 1f (1.9 g, 6.0 mmol) was added, stirred and reacted overnight, and after the reaction was completed by LC-MS monitoring, the reaction was filtered and the filtrate was concentrated. The residue was purified by neutral alumina flash column chromatography (dichloromethane / methanol 20:1, v / v), and the obtained crude product was subjected to reverse phase preparation to obtain yellow oil 1e (587.3 mg, yield 21%).

[0081] HRMS: m / z=965.6086[M+H] + .

[0082] 1H NMR(500MHz,CD3OD)δ8.88(d,J=5.6Hz,4H),8.51(t,J=7.8Hz,4H),8.18–8.05(m,4H),7.96(d,J=6.7Hz,4 H), 4.36 (s, 8H), 4.17 (d, J = 5.7Hz, 4H), 3.56–3.44 (m, 14H), 3.26–2.73 (m, 15H), 1.41 (s, 9H), 1.34 (s, 9H).

[0083] 7. Synthesis of Cyclotrimethylenetetramine Complex Dy-1

[0084] 5 M ethyl acetate solution (15 mL) of hydrogen chloride was added dropwise to the yellow oil 1e (530.2 mg, 0.55 mmol). After the addition was complete, the mixture was reacted at room temperature for 2 hours. After the reaction was completed as monitored by LC-MS, the mixture was concentrated to obtain a yellow oily ligand.

[0085] After the yellow oily ligand is dissolved in 20 mL of water, 1 M sodium hydroxide solution is slowly added dropwise to adjust the pH of the system to 5.5-6.0. During this period, 1 M hydrochloric acid solution can be used to adjust the pH of the system to 5.5-6.0. Dysprosium trifluoromethanesulfonate (301.8 mg, 0.495 mmol) is added while stirring, and then stirring is continued for 6 hours. During the reaction, the reaction is monitored by xylenol orange test until the color of the solution stabilizes in light yellow. 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%. The molecular weight of the final product identified by LC-MS is 1015.1990 [M+H]

[0086] Example 2

[0087]

[0088] 1. Synthesis of 1,7-dibenzyl dicarboxylate-4-ethyl acetate-1,4,7,10-tetraazacyclododecane (2a)

[0089] White solid 1b (6.0 g, 11.7 mmol) and potassium carbonate (8.0 g, 58.5 mmol) were dissolved in acetonitrile (50 mL), stirred at room temperature for 10 minutes, heated to 65°C, and then a solution of ethyl bromoacetate (1.5 mL, 12.9 mmol) in acetonitrile was added dropwise. After the addition was complete, the reaction was stirred overnight. After the reaction was completed under the monitoring of TLC and LC-MS, the mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol 20:1, v / v) to obtain a pale yellow solid 2a crude product, which was directly used for the next step.

[0090] HRMS: m / z=567.2869[M+H] + .

[0091] 2. Synthesis of ethyl bromoacetylglycine (2f)

[0092] Glycine ethyl hydrochloride (10.0 g, 71.9 mmol) and potassium bicarbonate (30 g, 217.4 mmol) were dissolved in dichloromethane (300 mL), cooled to 0°C and stirred for 30 minutes, then a dichloromethane solution (20 mL) containing bromoacetyl bromide (6.3 mL, 72 mmol) was added dropwise, the mixture was heated to room temperature, and the mixture was reacted for 8 hours at room temperature. After the reaction was completed by TLC detection, a saturated sodium citrate solution was added to the mixed product, followed by dichloromethane extraction (20 mL×2), the organic phases were combined, the organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate 30:1, v / v) to give a white solid 2f (12.1 g, yield 81.3%).

[0093] HRMS: m / z=209.9765[M+H] + .

[0094] 1 H NMR (500MHz, CDCl3) δ4.24 (q, J = 7.1Hz, 2H), 4.06 (t, J = 6.8Hz, 2H), 3.91 (s, 2H), 1.29 (t, J = 7.2Hz, 3H).

[0095] 3. Synthesis of 1,7-dibenzyl dicarboxylate-4-ethyl acetate-10-carbonylglycine ethyl ester-1,4,7,10-tetraazacyclododecane (2b)

[0096] The light yellow solid 2a (3.0 g, 5.7 mmol) and potassium carbonate (4.0 g, 28.5 mmol) were dissolved in acetonitrile (35 mL), stirred at room temperature for 10 minutes, and then 2f (2.4 g, 11.4 mmol) was added, followed by heating to 65°C and reacting overnight. After the reaction was completed as monitored by TLC and LC-MS, the reaction was filtered and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol 30:1, v / v) to obtain a yellow oil 2b (2.1 g, yield 54.9%).

[0097] HRMS: m / z=670.3456[M+H] + .

[0098] 1H NMR(500MHz, CDCl3)δ7.31–7.15(m,10H),5.03(s,4H),4.11–4.01(m,4H), 3.85(s,2H),3.50–2.95(m,12H),2.89–2.54(m,8H),1.17(t,J=7.2Hz,6H).

[0099] 4. Synthesis of 1-ethyl acetate-7-carbonylglycine ethyl ester-1,4,7,10-tetraazacyclododecane (2c)

[0100] The yellow oil 2b (2.5 g, 3.8 mmol) was dissolved in ethanol (15 mL), and 10% Pd / C (300 mg) was added. The mixture was transferred into a sealed autoclave, and hydrogen was introduced for 8 hours. After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain a yellow oily liquid 2c, which was used directly in the next step without purification.

[0101] HRMS: m / z=402.2719[M+H] + .

[0102] 1 H NMR (500MHz, CDCl3) δ7.98 (s, 1H), 4.06-3.95 (m, 6H), 3.29 (s, 2H), 3.17 (s, 2H), 2.81-2.53 (m, 16H), 1.13 (td, J = 7.2, 4.4Hz, 6H).

[0103] 5. Synthesis of 1-ethyl acetate-7-carbonylglycine ethyl ester-4,10-bis(2,2'-bis(2-methylpyridine)aminoethylamino-1,4,7,10-tetraazacyclododecane (2d)

[0104] The yellow oily liquid 2c (1 g, 2.5 mmol) and potassium carbonate (1.7 g, 12.5 mmol) were dissolved in acetonitrile (20 mL), and the temperature was raised to 65°C. Then, a brown oily substance 1f (1.9 g, 6 mmol) was added, and the reaction was stirred overnight. After the reaction was completed by LC-MS monitoring, the mixture was filtered, and the filtrate was concentrated. The residue was purified by neutral alumina flash column chromatography (dichloromethane / methanol 20:1, v / v). The obtained crude product was subjected to reverse phase preparation to obtain a yellow oily substance 2d (256 mg, yield 10.6%).

[0105] HRMS: m / z=966.5677[M+H] + .

[0106] 1H NMR (600MHz, CDCl3) δ9.82-9.17(m,4H),8.64–8.26(m,8H),8.12(d,J=7.7Hz,2H),8.00–7.74(m,4H),7.71–7.56(m,4H),7.42– 7.28(m,4H),7.13(dd,J=29.7,14.0Hz,4H),6.55–5.98(m,4H),4.29–3.63(m,22H),3.53–3.26(m,6H),2.83(d,J=59.1Hz,6H).

[0107] 6. Synthesis of Cyclotrimethylenetetramine Complex Dy-2

[0108] The yellow oil 2d (300 mg, 0.31 mmol) was dissolved in water, 1M sodium hydroxide solution was added dropwise, the pH of the system was adjusted to 14, and the mixture was stirred overnight. After the reaction was completed by LC-MS monitoring, the pH of the mixture was adjusted to 5.5-6.0 with 1M hydrochloric acid solution, and dysprosium trifluoromethanesulfonate (170 mg, 0.279 mmol) was added dropwise while stirring. After the addition was completed, the reaction was stirred at room temperature overnight, and the pH value was monitored at any time. The pH value was adjusted every 2 hours. During the reaction, the reaction was monitored by xylenol orange test until the solution color stabilized in light yellow. After the reaction was completed by LC-MS monitoring, the reaction was 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 was analyzed by high performance liquid analytical chromatography, and the purity was 98%. The molecular weight of the final product identified by LC-MS was 1072.1190 [M+H]. Example 3

[0109]

[0110] 1. Synthesis of 1,7-dibenzyl dicarboxylate-4,10-di(carbonylglycine ethyl ester)-1,4,7,10-tetraazacyclododecane (3a)

[0111] White solid 1b (6.0 g, 11.7 mmol) and potassium carbonate (8.0 g, 58.5 mmol) were dissolved in acetonitrile (100 mL), stirred at room temperature for 10 minutes, then white solid 2f (5.3 g, 25.7 mmol) was added, and then heated to 65 ° C. and reacted overnight. After the reaction was completed under LC-MS monitoring, the reaction was filtered and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol 20:1, v / v) to obtain a yellow oil 3a (6.1 g, yield 72%).

[0112] HRMS: m / z=727.3666[M+H] + .

[0113] 1 H NMR (400MHz, CDCl3) δ7.37–7.27(m,10H),5.09(s,4H),4.15(q,J=7.1Hz,4H),3.92 (s,4H),3.48(s,8H),3.21(s,4H),2.83(s,8H),1.68(s,2H),1.25(t,J=7.1Hz,6H).

[0114] 2. Synthesis of 1,7-di(carbonylglycine ethyl ester)-1,4,7,10-tetraazacyclododecane (3b)

[0115] The yellow oil 3a (5.0 g, 6.9 mmol) was dissolved in ethanol (40 mL), and 10% Pd / C (500 mg) was added. The mixture was transferred into a sealed autoclave, and hydrogen was introduced for 8 hours. After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain a yellow oily liquid 3b, which was used directly in the next step without purification.

[0116] HRMS: m / z=459.2933[M+H] + .

[0117] 1 H NMR (400MHz, CDCl3) δ4.21 (q, J = 7.1Hz, 4H), 4.06 (s, 4H), 3.72 (q, J = 7.0Hz, 2H), 3.34 (d, J = 19.8Hz, 4H), 2.94–2.68 (m, 16H), 1.31 (t, J = 7.1Hz, 5H).

[0118] 3. Synthesis of 1,7-bis(carbonylglycine ethyl ester)-4,10-bis(2,2'-bis(2-methylpyridine)aminoethylamino-1,4,7,10-tetraazacyclododecane (3c)

[0119] Yellow oily liquid 3b (2.0 g, 4.4 mmol) and potassium carbonate (3.0 g, 22.0 mmol) were dissolved in acetonitrile (25 mL), and the temperature was raised to 65°C. Then, brown oil 1f (2.9 g, 9.0 mmol) was added, and the reaction was stirred overnight. After the reaction was completed by LC-MS monitoring, the reaction was filtered and the filtrate was concentrated. The residue was purified by neutral alumina flash column chromatography (dichloromethane / methanol 20:1, v / v). The obtained crude product was subjected to reverse phase preparation to obtain yellow oil 3c (566.6 mg, yield 12.6%).

[0120] HRMS: m / z=1023.5894[M+H] +

[0121] 1 H NMR (600MHz, CDCl3) δ8.60 (s, 1H), 8.57 (s, 1H), 8.53 (s, 4H), 7.65 (t, J = 7.4Hz, 4H), 7.35 (dd, J = 18.5, 9.1Hz, 4H), 7.17 (d, J = 5. 8Hz,4H),4.21–3.99(m,3H),3.97–3.71(m,16H),3.39–3.04(m,12H),2.77–2.30(m,20H),1.28–1.15(m,3H),1.12–0.93(m,3H).

[0122] 4. Synthesis of Cyclotrimethylenetetramine Complex Dy-3

[0123] The yellow oil 3c (296.4 mg, 0.29 mmol) was dissolved in water, and 1 M sodium hydroxide solution was added dropwise to adjust the pH of the system to 14. The mixture was stirred overnight. After the ethyl ester hydrolysis reaction was completed by LC-MS monitoring, the pH value of the obtained mixed product was adjusted to 5.5-6.0 with 1 M hydrochloric acid. Dysprosium trifluoromethanesulfonate (159.1 mg, 0.26 mmol) was added dropwise while stirring. After the addition was completed, the reaction was stirred at room temperature overnight, and the pH value was monitored at any time. The pH value was adjusted every 2 hours. The reaction was stirred at room temperature overnight. During the reaction, the reaction was monitored by xylenol orange test until the solution color stabilized in light yellow. After the reaction was completed by LC-MS monitoring, it was 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 was analyzed by high performance liquid analytical chromatography, and the purity was 97%. The molecular weight of the final product identified by LC-MS was 1129.4420 [M+H].

[0124] Test 1: Test for determination of longitudinal and molar transverse relaxation rates of the cyclopentane coordination compound of the present invention

[0125] Test method:

[0126] 1. Dissolve Dy-1 in distilled water and prepare Dy 3+ Dy-1 solution with concentration of 1.0, 2.0, 3.0, 4.0 mmol / L. Dissolve Dy-2 in distilled water to prepare Dy 3+ Dy-2 solution with concentration of 1.0, 2.0, 3.0, 4.0 mmol / L. Dissolve Dy-3 in distilled water to prepare Dy 3+ Dy-3 solution with concentrations of 1.0, 2.0, 3.0, and 4.0 mmol / L.

[0127] 2. Each group of different Dy 3+The Dy-1 solution was treated as follows: 0.5 equivalent (0.5 equivalent refers to the Zn in the ZnCl2 solution) was added. 2+ The concentration of Dy in Dy-1 solution 3+ Four samples were obtained from a ZnCl2 solution (distilled water as solvent) with a concentration ratio of 0.5. The four samples were respectively transferred into 4 mm NMR tubes, and the T1 values ​​of the four samples were tested on a Bruker 400M NMR spectrometer using the saturation inversion recovery method (instrument parameters: DS=4, NS=8, D1=1s, D20=50μs, L20=15000). The molar longitudinal relaxation rate r1 was calculated by computer fitting.

[0128] The T2 values ​​of the four samples were tested using a spin echo method (instrument parameters: DS=4, NS=8) on a Bruker 400M nuclear magnetic resonance spectrometer, and the molar transverse relaxation rate r2 was calculated by computer fitting.

[0129] 3. Each group of different Dy 3+ The Dy-1 solution with different concentrations was mixed with 0 (distilled water), 1, 1.5, 2, and 2.5 equivalents of ZnCl2 solution, and the test treatment was performed according to the method in step 2.

[0130] 4. Treat each group of different Dy according to the method of steps 2 and 3. 3+ Concentration Dy-2 solution and each group of different Dy 3+ Concentration Dy-3 solution.

[0131] Test results:

[0132] 1. Dy-1 solution to Zn 2+ The changes of molar transverse relaxation rate and molar longitudinal relaxation rate under response are shown in the figure Figure 1 As shown by Figure 1 It can be seen that the molar longitudinal relaxation rate r1 of the coordination compound Dy-1 increases with the Zn 2+ The concentration does not change significantly, but the molar transverse relaxation rate r2 increases with Zn 2+ As the concentration increases, it gradually increases. T1, which does not change significantly, can be used as a control to show the degree of change in T2. ​​That is, T2 / T1 decreases as the concentration increases, and the range of change is 7-11mM -1 s -1 between.

[0133] 2. Dy-2 solution to Zn 2+ The changes of molar transverse relaxation rate and molar longitudinal relaxation rate under response are shown in the figure Figure 2 As shown in the figure, the coordination compound Dy-2 introduces a glycine structure based on the coordination compound Dy-1. Figure 2It can be seen that the molar longitudinal relaxation rate r1 of the coordination compound Dy-2 increases with the Zn 2+ The concentration does not change significantly, but the molar transverse relaxation rate r2 increases with Zn 2+ The concentration increases and the molar transverse relaxation rate r2 increases by 2-3 times compared with the molar relaxation rate r2 of the coordination compound Dy-1. The T1 with no obvious change can be used as a control to show the degree of change of T2. T2 / T1 decreases with the increase of concentration, and the range of change is 12-18mM -1 s -1 That is, the T2 / T1 value increased by 2-3 times, showing better T2 responsiveness.

[0134] 3. Dy-3 solution to Zn 2+ The changes of molar transverse relaxation rate and molar longitudinal relaxation rate under response are shown in the figure Figure 3 As shown in the figure, the coordination compound Dy-3 introduces two glycine structures based on the coordination compound Dy-1. Figure 3 It can be seen that the molar longitudinal relaxation rate r1 of the coordination compound Dy-3 increases with the Zn 2+ The concentration does not change significantly, but the molar transverse relaxation rate r2 increases with Zn 2+ As the concentration increases, it gradually decreases. T1, which does not change significantly, can be used as a control to show the degree of change in T2. ​​That is, T2 / T1 decreases with increasing concentration, and the range of change is 2-5mM -1 s -1 between.

[0135] Test 2: Cytotoxicity test of the cyclopentane coordination compound of the present invention

[0136] Test method:

[0137] Human breast cancer cells MCF-7 were inoculated in 96-well plates for 24 h, and then different Dy 3+ The cells were incubated with a basal medium solution of Dy-1 / Dy-2 / Dy-3 (0, 25, 50, 100, 300, 600 μg / mL) and incubated for 24 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 in the wells, the wells were washed with PBS, and the survival rate of human breast cancer MCF-7 cells was calculated based on the average value of 6 parallel wells.

[0138] Test results:

[0139] The survival of human breast cancer cells MCF-7 treated with different concentrations of the cyclopentane ligand Dy-1 Figure 4 As shown in Figure 2, the survival of human breast cancer cells MCF-7 treated with different concentrations of the cyclopentane coordination compound Dy-2 is shown in Figure 2. Figure 5As shown in Figure 2, the survival of human breast cancer cells MCF-7 treated with different concentrations of the cyclopentane coordination compound Dy-3 is shown in Figure 2. Figure 6 As shown by Figure 4-6 As shown in the figure, after Dy-1, Dy-2, and Dy-3 were treated with human breast cancer cells MCF-7, their survival rates were all above 95%, and as the concentration of the cyclopentane complex increased, the effect on the survival rate of breast cancer cells MCF-7 was not significant. When the concentration of the cyclopentane complex was 600 μg / mL, the survival rate of breast cancer cells MCF-7 was still above 95%.

[0140] Experiment 3: Competitive test of the invented cyclopentane coordination compound for metal ions

[0141] Test method:

[0142] 1. Prepare 50mM stock solutions of MgCl2, FeCl3, CaCl2, CuCl2, CuCl and ZnCl2 respectively with distilled water;

[0143] 2. Prepare Dy 3+ Dy-1 / Dy-2 / Dy-3 solution with a concentration of 50 μM;

[0144] 3. Treat the Dy-1 solution as follows: add 0 and 0.5 (0.5 equivalent refers to the Mg in the MgCl2 solution) 2+ The concentration of Dy in Dy-1 solution 3+ The MgCl2 solutions with the concentration ratios of 0.5, 1.0, 1.5 and 2.0 were prepared to obtain 5 samples to be tested. The 5 samples to be tested were respectively transferred into 4 mm NMR tubes. The T2 values ​​of the 5 samples to be tested were tested on a Bruker 400M NMR spectrometer using the spin echo method (instrument parameters: DS=4, NS=8). The molar transverse relaxation rate r2 was calculated by computer fitting.

[0145] 4. The competitive test of the coordination compound Dy-1 with FeCl3, CaCl2, CuCl2, CuCl and ZnCl2 was carried out according to step 3.

[0146] 5. According to the methods of steps 3 and 4, the competitive tests of the coordination compound Dy-2 and the coordination compound Dy-3 with MgCl2, FeCl3, CaCl2, CuCl2, CuCl and ZnCl2 were tested.

[0147] Test results:

[0148] 1. The competitive results of the coordination compound Dy-1 for 6 metal ions: Mg, Fe, Ca, Cu, Cu and Zn are as follows: Figure 7 As shown by Figure 7It can be seen that the molar transverse relaxation rate r2 of the coordination compound Dy-1 in the presence of the other five metal ions ranges from 7 to 8 mM. -1 s -1 Between, and in Zn 2+ The molar transverse relaxation rate r 2ex 2-3mM higher -1 s -1 , that is, the coordination compound Dy-1 to Zn 2+ The transverse relaxation time T2 is small, indicating that the coordination compound Dy-1 has a strong affinity to Zn 2+ The response sensitivity is better than that of the other five metal ions.

[0149] 2. The competitive results of the coordination compound Dy-2 for 6 metal ions including Mg, Fe, Ca, Cu, Cu and Zn are as follows: Figure 8 As shown by Figure 8 It can be seen that the molar transverse relaxation rate r2 of the coordination compound Dy-2 in the presence of 6 metal ions increases, and the molar transverse relaxation rate r2 of the coordination compound Dy-2 in the presence of the other 5 metal ions ranges from 16 to 18 mM. -1 s -1 Between Zn 2+ The molar transverse relaxation rate r2 in the presence of Zn is lower than that in the presence of the other five metals, that is, the coordination compound Dy-2 has a lower molar transverse relaxation rate r2 for Zn 2+ The transverse relaxation time T2 is larger, indicating that the coordination compound Dy-2 has a strong affinity to Zn 2+ The response sensitivity is low.

[0150] 3. The competitive results of the coordination compound Dy-3 for 6 metal ions including Mg, Fe, Ca, Cu, Cu and Zn are as follows: Fig. 9 As shown by Fig. 9 It can be seen that the molar transverse relaxation rate r2 of the coordination compound Dy-3 in the presence of 6 metal ions increases, and the molar transverse relaxation rate r2 in the presence of the other 5 metal ions ranges from 16 to 18 mM. -1 s -1 Between Zn 2+ The molar transverse relaxation rate r 2ex Compared with the other five metal ions, the molar transverse relaxation rate r2 is lower, that is, the coordination compound Dy-3 has a lower molar transverse relaxation rate r2 for Zn 2+ The transverse relaxation time T2 is large, indicating that the coordination compound Dy-3 has a strong affinity to Zn 2+ The response sensitivity is low.

Claims

1. A PDA-modified cyclopentane coordination compound, characterized in that Its structural formula is as follows:

2. A method for synthesizing a PDA-modified cyclopentane coordination compound, characterized in that The steps include: S1, 1,4,7,10-tetraazacyclododecane and benzyl chloroformate undergo nucleophilic substitution reaction to generate a compound of formula (I). The reaction formula is as follows: S2. In the presence of a base, the compound of formula (I) undergoes a nucleophilic substitution reaction with tert-butyl bromoacetate to generate a compound of formula (II), and the reaction formula is as follows: Or S2', in the presence of a base, the compound of formula (I) first undergoes a nucleophilic substitution reaction with ethyl bromoacetate to generate an intermediate compound I, and the intermediate compound I then undergoes a nucleophilic substitution reaction with ethyl bromoacetylglycine to generate a compound of formula (II), and the reaction formula is as follows: Or S2", in the presence of a base, the compound of formula (I) undergoes a nucleophilic substitution reaction with ethyl bromoacetyl glycine to generate a compound of formula (II), and the reaction formula is as follows: S3. Under a hydrogen atmosphere, the compound of formula (II) undergoes a hydrogenation reduction reaction under the action of a catalyst to generate a compound of formula (III), and the reaction formula is as follows: S4. In the presence of a base, N-tert-butyloxycarbonyl-1,2-ethylenediamine and 2-chloromethylpyridine or a salt thereof undergo a nucleophilic substitution reaction to generate tert-butyl-(2-(di(pyridin-2-ylmethyl)amino)ethyl)carbamate, the reaction formula of which is as follows: S5, tert-butyl-(2-(di(pyridine-2-ylmethyl)amino)ethyl)carbamate is deprotected by the tert-butyloxycarbonyl group to generate N1,N1-di(pyridine-2-methyl)ethane-1,2-diamine, and the reaction formula is as follows: S6. In the presence of a base, N1,N1-di(pyridine-2-methyl)ethane-1,2-diamine reacts with chloroacetyl chloride to undergo a nucleophilic substitution reaction to generate N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide, and the reaction formula is as follows: S7. In the presence of a base, the compound of formula (III) undergoes a nucleophilic substitution reaction with N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide to generate a compound of formula (IV), and the reaction formula is as follows: S8. The compound of formula (IV) is first hydrolyzed to generate an intermediate compound II. The intermediate compound II undergoes a coordination reaction with a trivalent dysprosium ion to generate the PDA-modified cyclopentane coordination compound. The reaction formula is as follows:

3. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The nucleophilic substitution reaction in step S1 is carried out at room temperature, with a stirring time of 4-12 hours, and a molar ratio of 1,4,7,10-tetraazacyclododecane to benzyl chloroformate of 0.48-0.5:1.

0.

4. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The base is selected from at least one of potassium carbonate or potassium bicarbonate.

5. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The reaction temperature of all nucleophilic substitution reactions in step S2 is 65-75° C., and the reaction time is 8-10 h.

6. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: In the step S2, the molar ratio of the compound of formula (I) to tert-butyl bromoacetate is 1.0:2.0-2.2; in the step S2', the molar ratio of the compound of formula (I) to ethyl bromoacetate is 1.0:1.0-1.2, and the molar ratio of the compound of formula (I) to ethyl bromoacetate is 1.0:1.0-1.2; in the step S2", the molar ratio of the compound of formula (I) to ethyl bromoacetyl glycine is 1.0:2.0-2.

2.

7. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: In step S3, the catalyst is a palladium-carbon catalyst, and the hydrogenation reduction reaction is carried out at room temperature for 8-10 hours.

8. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The reaction temperature of the nucleophilic substitution reaction in step S4 is 80-110° C., the reaction time is 8-12 h, and the molar ratio of N-tert-butyloxycarbonyl-1,2-ethylenediamine to 2-chloromethylpyridine or its salt is 0.9-1.0:3.0-3.

5.

9. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The reaction temperature of the nucleophilic substitution reaction in step S6 is -5-5°C, the reaction time is 3-5h, and the molar ratio of N-tert-butyloxycarbonyl-1,2-ethylenediamine to chloroacetyl chloride is 0.9-1:

1.

10. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The nucleophilic substitution reaction in step S7 is carried out at 65-75° C. for 8-10 h, and the molar ratio of the compound of formula (III) to N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-2-chloroacetamide is 1.0:2.0-2.

2.

11. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: In the step S8, the coordination reaction is carried out at room temperature, the reaction time is 6-8 hours, and the molar ratio of the compound of formula (IV) to the trivalent dysprosium ion is 1.0:0.9-1.

0.

12. The method for synthesizing the PDA-modified cyclopentane coordination compound according to claim 2, characterized in that: The trivalent dysprosium ion is selected from dysprosium trifluoromethanesulfonate.

13. Use of the PDA-modified cyclopentane coordination compound according to claim 1 in the preparation of a divalent zinc ion-responsive T2 contrast agent.

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