CN107629016B

CN107629016B - Evans blue complex and preparation method and application thereof

Info

Publication number: CN107629016B
Application number: CN201711110358.XA
Authority: CN
Inventors: 陈小元; 郎立新; 牛刚; 田蕊
Original assignee: Shamu Shanghai Biotechnology Co ltd
Current assignee: Shamu Shanghai Biotechnology Co ltd
Priority date: 2017-11-12
Filing date: 2017-11-12
Publication date: 2020-05-22
Anticipated expiration: 2037-11-12
Also published as: CN107629016A

Abstract

The invention provides an Evans blue derivative, which is shown as the following formula (I), and the structure of the Evans blue derivative contains two serum albumin binding groups and a macrocyclic polyamine chelating group NOTA. The invention also provides a labeled complex obtained by labeling the Evans blue derivative with a radionuclide. The labeled complex is simple in labeling and good in biological performance; the biological test result shows that the sentinel lymph node and the secondary lymph node are distinguished. The invention also relates to a preparation method of the complex and application of the complex in preparing human or animal organ and tissue imaging agents.

Description

Evans blue complex and preparation method and application thereof

Technical Field

The invention relates to a radioactive complex, a preparation method thereof and application of the complex as sentinel lymph node imaging agent and blood pool imaging agent.

Background

Metastasis and spread of solid tumors usually occur through lymphatic vessels, and tumor cells secrete lymphatic growth factors (such as VEGF-C and VEGF-D) to stimulate the formation of new lymphatic vessels at the margins and inside of the tumor, thereby promoting the spread of tumor cells through the lymphatic vessels to the lymph nodes. The primary Lymph Nodes, Sentinel Lymph Nodes (SLNs), are the first sites of metastasis and spread of tumors, and therefore, the malignancy of tumors and the corresponding therapeutic measures are clinically determined by detecting whether Sentinel Lymph Nodes (SLNs) have metastasized. At present, SLNs biopsy results are the gold criteria for determining tumor metastasis, which has become an important basis for deciding the surgical plan of some tumor patients, including breast cancer, melanoma, colorectal cancer, prostate cancer, and the like. Currently, the most commonly used sentinel lymph node detection method is to inject the radionuclide 99mTc labeled sulfur colloid first, preliminarily determine the approximate location of the SLNs using a gamma detector, and then inject the vital dye (such as patent blue vital dye) several hours later, because the macroscopic vital dye is concentrated in the SLNs site, so that the biopsy can be conducted by removing the SLNs intraoperatively. However, this approach has more limitations. First, the need to inject different probes in two separate injections increases the complexity of clinical diagnosis; secondly, the gamma detector has low relative sensitivity and low local resolution when positioning the 99 mTc; third, blue dyes are generally impermeable to skin and adipose tissue, and therefore must be stripped from the surrounding adipose tissue to accurately remove SLNs; fourthly, the colloid is enriched at the lymph node part to cause inflammatory reaction, and the degradability is lower. Meanwhile, the four clinical test data of the SLNB are comprehensively displayed by applying the current detection means, and the false negative rate (omission ratio) is 4.6-16.7%. In addition, the surgical area may be stained blue by the dye injection. There are also advances in the development of probes for SLNs, for example 99mTc-EB can be injected simultaneously in conjunction with radioactive or colored signals, or near infrared fluorescent dyes such as ICG alone or in conjunction with nanoparticle imaging for the detection of SLNs. Wherein ultra-small silicon nanoparticles having a diameter of about 7nm, Corneldots (C-dots), have been approved by the FDA for use in optical detection or optical-PET dual-modality detection platforms. Because the requirements for both pre-operative evaluation and monitoring during the surgical procedure are met, different imaging modes are generally adopted for joint detection. Thus, these methods still have major limitations. In addition, the existing probes have common problems, the tumor in-situ injection can rapidly migrate to the primary and secondary lymph nodes through lymphatic vessels, and the probe signals of the secondary lymph nodes can interfere the positioning and confirmation of sentinel lymph nodes in sentinel lymph node biopsy operation, thereby increasing the operation difficulty.

Human Serum Albumin (HSA) is the most abundant protein in Human plasma. Due to the long half-life in vivo, the polypeptide can be reversibly combined with the property of carrying small molecules and other substances, and is often used as a drug carrier for a drug delivery platform for diabetes, tumors, rheumatoid arthritis, infectious diseases and the like. HSA is also directly used as an imaging probe, labeled with a fluorescent dye for optical imaging, labeled with a radionuclide for PET imaging, and labeled with Gd³⁺For magnetic resonance imaging. In the early stage of the studyIn the present invention, the applicant creatively modifies Evans Blue (EB) by using the high affinity between evans blue and serum albumin to obtain evans blue derivative (NEB), which has excellent biological properties as a blood pool imaging agent, and has the characteristics of simple preparation method and low cost, thus having a broad application prospect (chinese patent 201310157168.9). In addition, the mixed EB and EB were used in a blood pool developer¹⁸The F-AlF-NEB is injected locally, and the SLNs can be accurately and clearly positioned by using PET detection, so that positioning reference is provided for the operation. Meanwhile, EB emits fluorescence (the emission wavelength is about 670nm) after being combined with albumin in vivo, so that the detection and the positioning can be carried out by fluorescence imaging at the same time. In addition, the EB dye is blue, and the target lymph node is dyed blue during the operation process, so as to confirm the accuracy of the operation. The PET, fluorescence and naked eye visible three-mode SLNs imaging mode is verified in an animal model to have excellent imaging effect, and has the characteristics of higher safety, easy degradation and metabolism, no need of two injections and the like, so that the SLNs imaging mode has more advantages than the traditional SLNs detection mode. Provides a new simple, safe and effective method for detecting SLNs (In vivo album laboratory labeling. Proc Natl Acad Sci U S A.2015; 112(1): 208-13). However, since NEB diffuses at a high rate and spreads to sentinel lymph nodes and secondary lymph nodes within a short time after local injection, the secondary lymph nodes interfere with the sentinel lymph node signals during surgery, and the difficulty of the operating physician in distinguishing the sentinel lymph nodes increases. Meanwhile, the problem is a common problem of other SLNs imaging methods at present.

Disclosure of Invention

Based on the background, the invention provides a novel Evans blue derivative which is characterized by having two HSA binding groups, having stronger affinity with HSA and being capable of simultaneously binding two HSA molecules to form HSA-N (EB)₂HSA dimer, whereby it has a longer half-life in peripheral blood, slower migration in lymphatic vessels, allows differentiation between sentinel and secondary lymph nodes, and higher uptake enrichment in tumors, with more of a blood pool imaging agent and a sentinel lymph node imaging agentGood biological performance and is expected to be popularized and applied clinically.

The above object of the present invention is achieved by the following technical solutions:

firstly, providing a kind of radioactive labeled Evans blue complex with excellent imaging performance;

it is another object of the present invention to provide a method for preparing said radiolabeled evans blue complex;

the invention also aims to provide the application of the complex in preparing sentinel lymph node, cardiovascular blood pool and tumor blood pool imaging agents.

The technical scheme for realizing the above primary object of the invention has the following aspects of ligand synthesis and radioactive labeling.

In one aspect, the present invention provides an Evans blue derivative (Compound N (EB))₂) The compound N (EB)₂The structure is shown as the following formula (I):

wherein R can be a linking group having the structure shown in formula (II),

x in the formula (II) is selected from C atom or N atom; n is₁、n₂And n₃Can be the same or different and is an integer of 0-3; said Y₁、Y₂And Y₃Can be the same or different and are selected from any one of the following structures:

in the scheme of the invention, when X in the formula (II) is a C atom, Y is₁Preference is given to

Said Y₂And Y₃Are all preferred

N is₁Preferably 1, said n₂And n₃Are all preferably 0; namely, the structure of the R is shown as the following formula (III):

thus, when X in formula (II) is a C atom, a preferred compound of the invention is N (EB)₂The structure is shown as the following formula (IV):

in the scheme of the invention, when X in the formula (II) is an N atom, Y is₁And Y₂Are all preferred

Said Y₃Preference is given to

N is₁、n₂And n₃Are all preferably 1; namely, the structure of R is shown as the following formula (V):

thus, when X in formula (II) is an N atom, another preferred compound of the invention is N (EB)₂The structure is shown as the following formula (VI):

on the basis, the invention further provides a method for preparing the compound N (EB) shown as the formula (IV)₂The method comprises the following steps:

① introducing tert-butyloxycarbonyl (Boc) group to the amino group at one end of 3,3' -dimethylbenzidine, diazotizing the amino group at the other end to generate diazonium salt, coupling with 1-amino-8-naphthol-2, 4-disulfonic acid monosodium salt, removing Boc protecting group, reacting deprotected amino group with ammonia water and carbon disulfide to generate intermediate compound A with thiocyano group;

② 6 Fmoc-amino (Fmoc) -2-tert-butyloxycarbonyl (Boc) aminobutyric acid and the coupling reaction product in the step ① are acylated and condensed, then Fmoc protecting group is removed, and the removed amino and NOTA-OBu^tCarrying out acylation condensation reaction, and then removing Boc protecting groups in the condensation product to obtain an intermediate compound B with NOTA groups;

③ step ① provides intermediate compound A which reacts with intermediate compound B of step ② to form a thiourea bond by condensation of the thiocyano group with the amino group, providing a N (EB) according to formula (IV) of the present invention₂。

The process for the preparation of the compound of formula (IV) preferably comprises in particular the following steps:

1) dropwise adding di-tert-butyl dicarbonate into a compound 1(3,3' -dimethylbenzidine) solution of dichloromethane for reaction and purifying to obtain a light yellow compound 2; under the ice-bath condition, HCl and NaNO are sequentially dripped into the acetonitrile solution of the compound 2₂Reacting to generate yellow diazonium salt compound 3 solution;

2) in NaHCO₃And 1-amino-8-naphthol-2, 4-disodium disulfonate ice water solution, dropwise adding the compound 3 solution obtained in the step 1), and reacting to obtain a compound 4; further mixing the compound 4 with trifluoroacetic acid for reaction to obtain a compound 5; dissolving the compound 5 in DMF, and dropwise adding ammonia water for reacting overnight; adding CS₂Reacting to obtain a crude product of a compound 6; compound 6 and Pb (NO) in acetonitrile solution₃)₂Reacting and purifying to obtain a compound 7;

the synthetic route of the steps is as follows:

3) in DMF solution, compound 5 is reacted with 6-fluorenylmethoxycarbonylamino group (A)Fmoc) -2-tert-butyloxycarbonyl (Boc) aminobutyric acid (compound 8), HATU and DIPEA are mixed, reacted and purified to obtain a compound 9; further, in a DMF solution, adding piperidine dropwise into the compound 9 for reaction and purifying to obtain a compound 10; continuing in DMF solution, Compound 10, NOTA-OBu^tMixing HATU and DIPEA for reaction and purifying to obtain a compound 11; reacting the compound 11 with trifluoroacetic acid and purifying to obtain a compound 12; the specific synthetic route is as follows:

4) mixing the compound 7 obtained in the step 2) and the compound 12 obtained in the step 3) with HATU and DIPEA for reaction and purifying to obtain a target compound 13; the specific synthetic route is as follows:

other N (EB) in the scheme of the invention₂The preparation of the compound is similar to the preparation of compound 13 and can be prepared essentially by reference to the synthetic route for compound 13.

In a second aspect, the present invention further provides a radiolabelled Evans blue complex which is a compound of formula (I) as defined in the present invention N (EB)₂As a ligand, a complex obtained by labeling a radionuclide.

In the radiolabeled Evans blue complex of the present invention, the radionuclide may be selected from¹⁸F、⁶⁴Cu、⁶⁸Ga、⁶²Cu、⁶⁷Cu、⁸⁶Y、⁸⁹Zr、⁹⁰Y is or¹⁷⁷Lu, etc., preferably¹⁸F or⁶⁴Cu。

The invention further provides a method for preparing the radiolabeled evan blue complex, which comprises the following steps: i.e. said radionuclide and the compound of formula (I) N (EB)₂Reacting under proper conditions to obtain the radiolabeled Evans blue complex.

The preferred preparation method is the following wet or freeze drying method:

in a preferred embodiment of the present invention, the compound 13 represented by the formula (IV) is used as a ligand to wet-label a radioactive substance¹⁸F, comprising the following steps:

A1) dissolving a proper amount of compound 13 in a buffer solution or deionized water to obtain a solution a;

A2) dissolving aluminum chloride in a buffer solution or deionized water to obtain a solution b;

A3) mixing solution a and solution b, adding acetonitrile or ethanol and fresh solution¹⁸F, sealing the aqueous solution of ions, reacting for 5-30 min at 70-120 ℃, and cooling;

A4) diluting the reaction solution obtained in the step A3) with water, separating and purifying by Sep-Pak C18 chromatographic column, washing the chromatographic column with buffer solution or water to remove unreacted¹⁸Eluting F ions with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline, and sterile filtering to obtain the final product¹⁸F-labeled evans blue derivative injection.

In another preferred embodiment of the present invention, the compound 13 represented by the formula (IV) is used as a ligand, and the radioactivity is labeled by lyophilization¹⁸F, comprising the following steps:

B1) dissolving a proper amount of compound 13 in a buffer solution or deionized water to obtain a solution a;

B2) dissolving aluminum chloride in a buffer solution or deionized water to obtain a solution b;

B3) mixing solution a and solution b, adding acetonitrile or ethanol and fresh solution¹⁸F, sealing the aqueous solution of ions, reacting for 5-30 min at 70-120 ℃, and cooling;

B4) sterile filtering the solution obtained in the step B3), subpackaging in containers, freeze-drying, plugging and sealing, and waiting for a freeze-drying medicine box; the containers to be dispensed are preferably tube-type antibiotic bottles;

B5) adding an appropriate amount of acetic acid solution or buffer solution into the kit obtained in the step B4), dissolving, and addingAdding acetonitrile or ethanol and freshly prepared¹⁸F, sealing the aqueous solution of ions, reacting for 5-30 min at 70-120 ℃, and cooling;

B6) diluting the reaction solution obtained in the step B5) with water, separating and purifying by Sep-Pak C18 chromatographic column, and washing the chromatographic column with buffer solution or water to remove unreacted¹⁸Eluting F ions with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline, and sterile filtering to obtain the final product¹⁸F-labeled evans blue derivative injection.

In another preferred embodiment of the present invention, the compound 13 represented by the formula (IV) is used as a ligand to wet-label a radioactive substance⁶⁴Cu, comprising the steps of:

C1) dissolving a proper amount of compound 13 in a buffer solution or deionized water to obtain a solution a;

C2) adding into the solution a⁶⁴Cu²⁺(CuCl₂) Sealing the sodium acetate solution, reacting at 40-80 ℃ for 5-100 min, and cooling;

C3) separating and purifying the solution obtained in the step C2) by semi-preparative HPLC (high performance liquid chromatography), removing the solvent by rotary evaporation, dissolving the residue with Phosphate Buffer Solution (PBS) or water, and performing sterile filtration to obtain the final product⁶⁴Cu-labelled evans blue derivative injection.

In another preferred embodiment of the present invention, the compound 13 represented by the formula (IV) is used as a ligand, and the radioactivity is labeled by lyophilization⁶⁴Cu, comprising the steps of:

D1) dissolving a proper amount of compound 13 in a buffer solution or deionized water to obtain a solution a;

D2) sterile filtering the solution a obtained in the step D1), subpackaging in containers, freeze-drying, plugging and sealing to obtain a freeze-dried medicine box; the containers to be dispensed are preferably tube-type antibiotic bottles; excipients such as mannitol, ascorbic acid and the like can be added into the medicine box according to the forming condition of the freeze-dried powder of the medicine box, the dosage of the compound 13 and the excipients can be adjusted, and the forming of the medicine box is optimal;

D3) adding an appropriate amount of acetic acid solution or buffer solution into the medicine box obtained in the step D2), dissolving, and adding⁶⁴Cu²⁺(CuCl₂) VinegarSealing the sodium acid solution, reacting for 5-30 min at 70-120 ℃, and cooling;

D4) separating and purifying the solution obtained in the step D3) by semi-preparative HPLC (high performance liquid chromatography), removing the solvent by rotary evaporation, dissolving the residue with Phosphate Buffer Solution (PBS) or water, and performing sterile filtration to obtain the final product⁶⁴Cu-labelled evans blue derivative injection.

Other chemicals used in the above synthesis steps are commercially available.

In the above method, the radionuclide is part of nuclear medicine imaging, except¹⁸F and⁶⁴in addition to Cu, may also be⁶⁷Ga、⁶⁸Ga、⁶²Cu、⁶⁷Cu、⁸⁶Y、⁸⁹Zr、⁹⁰Y is or¹⁷⁷Lu, etc.; the buffer solution is a substance for stabilizing the pH value of the reaction solution, and can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, a mixture thereof and the like.

The radioactive-labeled Evans blue complex provided by the invention is simple to label and has good biological performance. Biological test results show that the sentinel lymph node imaging agent has the performance of distinguishing sentinel lymph nodes from secondary lymph nodes, the novel performance is not possessed by other current lymph node imaging agents, and the sentinel lymph node imaging agent is suitable for sentinel lymph node biopsy operations. In addition, it has high uptake in blood and long circulation time, and is also suitable for use as blood pool imaging agent.

Drawings

Figure 1 atomic force microscopy characterizes the binding of compound 13 to HSA;

figure 2 comparison of PET images of compound 13 and NEB at different times after normal mouse tail vein injection, and 60min time-radioactivity curves after administration to the heart blood pool and bladder, respectively;

FIG. 3 comparison of PET primary and secondary lymph node images, and lymph node time-radioactivity plot, and primary and secondary lymph node imaging time interval, respectively, of Compound 13 and NEB at different times after injection of normal mouse footpads.

FIG. 4 comparison of fluorescence images of primary and secondary lymph nodes at different times after injection of compound 13 and NEB, respectively, into the footpads of normal mice.

Figure 5 comparison of PET imaging images at different times after tail vein injection of compound 13 and NEB, respectively, in U87 nude mouse tumor model, and tumor uptake at different time points.

Detailed Description

The invention will be more clearly illustrated by the following specific examples.

Example 1 preparation of Evans blue derivative ligand (Compound 13)

Synthesis of Compound 2:

a250 mL flask was charged with compound 1(3,3' -dimethylbenzidine, 4.25g,20.0mmol) and 50mL of dried dichloromethane, respectively, to give a pale yellow solution. A solution of 20mL of di-tert-butyl dicarbonate (4.36g,20.0mmol) in dichloromethane was slowly added dropwise to the flask. Stirring at room temperature, reacting for 24h, and spin-drying the solvent to obtain a yellow solid. The residue was purified over silica gel column (petroleum ether/ethyl acetate ═ 1: 3) to give compound 2 as pale yellow in 50% yield.¹H NMR(300MHz,CDCl₃)δ7.79(d,J＝8.0Hz,1H),7.35(d,J＝8.5Hz,1H),7.31(s,1H),7.24(d,J＝3.7Hz,1H),6.71(d,J＝7.9Hz,1H),6.27(s,1H),3.63(s,2H),2.28(s,3H),2.21(s,3H),1.53(s,9H).

Synthesis of Compound 3:

15mL of ice in 2.0M HCl was added dropwise to 40mL of compound 2(3.12g,10.0mmol) in acetonitrile under ice-bath conditions. Stirring for 15min, adding 20mL NaNO₂(2.07g,30.0mmol) of an ice water solution was slowly added dropwise to the flask, and after the dropwise addition was completed, stirring was continued for 30min to produce a yellow diazonium salt compound 3 solution for further use.

Synthesis of Compound 4:

NaHCO is added₃(3.36g,40.0mmol) and 1-amino-8-naphthol-2, 4-disulfonic acid monosodium hydrate (3.19g,10.0mmol) were dissolved in 20mL of ice water, and then a new solution of diazonium compound 3 was slowly added dropwise, and stirring was continued for 60min under ice bath to give a violet solution. The purple solution is frozen and dried to obtain the purple solid compound 4 with the yield of 60 percent.¹H NMR(300MHz,MeOD)δ8.71(s,1H),8.00(dd,J＝9.2,6.9Hz,2H),7.60(d,J＝8.6Hz,1H),7.53(d,J＝18.0Hz,2H),7.47(s,2H),7.18(d,J＝9.9Hz,1H),2.56(s,3H),2.32(s,3H),1.53(s,9H).MS(LC-MS):calcd.For For C₂₉H₃₀N₄O₉S₂642.1；found 641.1[M-H]^-.

Synthesis of Compound 5:

compound 4(3.22g,5.0mmol) was added in portions to a mixed solution of 20mL of trifluoroacetic acid, and stirred at room temperature for 60 min. Trifluoroacetic acid was removed under reduced pressure to give compound 5 as a crude product in 50% yield.¹H NMR(300MHz,MeOD)δ8.67(s,1H),7.96(d,J＝9.9Hz,1H),7.85(d,J＝8.5Hz,1H),7.49(d,J＝8.7Hz,1H),7.43(s,1H),7.28(s,1H),7.24(d,J＝8.3Hz,1H),7.15(d,J＝9.9Hz,1H),6.70(d,J＝8.2Hz,1H),2.47(s,3H),2.18(s,3H).MS(LC-MS):calcd.For C₂₄H₂₂N₄O₇S₂542.1；found 541.1[M-H]^-.

Synthesis of Compound 6:

compound 5(1.08g,2.0mmol) was dissolved in DMF, and 1ml of aqueous ammonia was added dropwise thereto, followed by reaction overnight with stirring at room temperature. To connect CS₂(0.74g, 10.0mmol) was added to the reaction mixture, and the mixture was stirred at 40 ℃ for 8 hours. The solvent was removed by rotary evaporation to give the crude compound 6. Purification on a reverse phase column and freeze drying gave pure compound 6 in 80% yield.

Synthesis of compound 7:

to 50mL of a solution of Compound 6(0.58g,1.0mmol) in acetonitrile was added Pb (NO)₃)₂(0.66g,2.0mmol), stirring overnight at room temperature, filtering, and spin-drying the filtrate to give the crude product. Purification on a reverse phase column and freeze drying gave pure compound 7 in 95% yield.

Synthesis of compound 9:

compound 5(0.54g,1.0mmol), 6-fluorenylmethoxycarbonylamino (Fmoc) -2-tert-butoxycarbonyl (Boc) aminobutyric acid (compound 8) (0.43g,1.0mmol), HATU (0.38g,1.0mmol) and DIPEA (0.26g,2.0mmol) were sequentially charged to 20mL of DMF. The reaction mixture was stirred for 24 hours, and the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column and lyophilized to give pure compound 9 in 80% yield.

Synthesis of compound 10:

compound 9(0.48g,0.5mmol) was dissolved in 10mL of DMF, and piperidine (2mL) was added dropwise while cooling on ice. After dropping, the temperature is raised to room temperature, and stirring is continued for 2 hours. And distilling the reaction mixed solution under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by reverse phase column and lyophilized to give pure compound 10 in 99% yield.

Synthesis of compound 11:

compound 10(0.37g,0.5mmol), NOTA-OBu^t(0.25g,0.5mmol), HATU (0.19g,0.5mmol) and DIPEA (0.13g,1.0mmol) were charged sequentially to 5mL of DMF. The reaction mixture was stirred overnight and the solvent was distilled off under reduced pressure to give the crude product. The crude product was purified by reverse phase column and lyophilized to give pure compound 11 in 75% yield.

Synthesis of compound 12:

compound 11(0.24g,0.2mmol) was added to a mixed solution of 20mL of trifluoroacetic acid and stirred at room temperature for 2 hours. Trifluoroacetic acid was removed under reduced pressure to give crude compound 12. The crude product was purified by reverse phase column and lyophilized to give pure compound 12 in 80% yield.

Synthesis of compound 13:

compound 7(0.06g,0.1mmol), compound 12(0.10g,0.1mmol), HATU (0.38g,0.1mmol) and DIPEA (0.04g,0.2mmol) were charged sequentially to 2mL of DMF. The reaction mixture was stirred overnight and the solvent was distilled off under reduced pressure to give the crude product. The crude product was purified by reverse phase column and lyophilized to give pure compound 13 in 60% yield.

Examples 2 to 20

N (EB) of examples 2 to 20₂The structure of the compound is shown as the following formula (I), wherein R can be a connecting group shown as the following formula (II), each part group in the formula (II) is selected and listed in the following table 1, and the preparation methods can be all referred to the example 1:

TABLE 1

Example 21 radioactivity¹⁸Preparation of F-labeled Evans blue complex

1. Radioactivity¹⁸Preparation of lyophilized kit for marker F (example of 100 pieces)

10mg of compound 13 prepared in example 1 was dissolved in 10mL of 0.5mol/L acetic acid-sodium acetate buffer solution (pH 4), and 0.1mg of aluminum chloride (AlCl) was added₃) Dissolved in 10mL of 0.5mol/L acetic acid-sodium acetate buffer solution (pH 4), and the two solutions were mixed well. Sterile filtering, subpackaging in 100-branch antibiotic vials, freeze-drying in a freeze-drying machine for 24 hours, plugging and sealing to obtain the freeze-dried medicine box. According to the different requirements of the output of the medicine box and the content of the components in each medicine box, the dosage of the compound 13 and the aluminum chloride can be adjusted to ensure that the mass ratio of the compound 13 to the aluminum chloride is 20-100: 1, in the above range.

2.¹⁸Preparation of F-labeled Evans blue complex

1) And (2) wet method: to a 1mL plastic tube, 3. mu.L of 2mM AlCl was added₃Acetic acid-sodium acetate buffer solution (0.5mol/L, pH 4) and 6 μ L of 3mM of compound 13 prepared in the example acetic acid-sodium acetate buffer solution (0.5mol/L, pH 4) were then added 0.13mL of acetonitrile and about 370 megabytes (MBq)¹⁸And F, fully mixing the aqueous solution, and then putting the mixture into boiling water for reaction for 10 minutes. The reaction mixture was cooled, diluted with water to a volume of 10mL, separated and purified by a Varian Bond Elut C18 column (100mg), and the column was washed with 10mL of water to remove unreacted water¹⁸Eluting with 0.3mL 80% aqueous ethanol (containing 1mM HCl), spinning off the ethanol under argon, dissolving the final product in phosphate buffer (0.5mol/L, pH 7.4) and sterile filtering to obtain¹⁸F-labeled evans blue complex, identified using analytical HPLC.

2) The freeze-drying method comprises the following steps: 0.5mL of 0.5mol/L acetic acid-sodium acetate buffer solution (pH 4) was added to the lyophilized kit, and the whole solution was dissolvedAdding 37-3700 MBq after the solution¹⁸F acetonitrile leacheate (obtained from anion trap column QMA), reacting for 5min at the temperature of 120 ℃ in a closed manner, and cooling; diluting with water, separating and purifying with Sep-Pak C18 chromatographic column, washing the chromatographic column with 0.5mol/L phosphate buffer solution to remove unreacted¹⁸Eluting F ions with hydrochloric acid ethanol solution or ethanol solution, diluting with normal saline, and sterile filtering to obtain the final product¹⁸F-labeled evans blue derivative injection.

Example 22 characterization of the binding Properties of Evans blue Complex (Compound 13) to HSA

Compound 13 prepared in example 1 was mixed with HSA at a molar concentration of 1:1, and then an atomic force microscope observation sample was prepared. Samples were photographed using a Bioscope Catalyst atomic force microscope (Bruker Santa Barbara, CA) coupled to an inverted optical microscope (IX71, Olympus, Japan) and it was observed that compound 13 can be linked to 2 HSAs to form HSA dimers or cross-linked to multimers (figure 1).

Example 23.¹⁸MicroPET imaging of F-labeled Evans blue derivatives in normal mice

Prepared by the method of example 21 to a purity of greater than 95%¹⁸F-labeled Evans blue derivative (Compound 13) was injected into 3.7MBq tail vein of normal FVB mice¹⁸F-AlF-N(EB)₂Or¹⁸F-AlF-NEB was used as a control, and then dynamic MicroPET imaging was performed 0-60 min after administration under isoflurane anesthesia, respectively, and the results are shown in FIG. 2. The results show that Compound 13(N (EB)₂) The mouse has higher uptake in the cardiovascular pool, is obviously improved compared with NEB, and can be used for imaging the cardiovascular pool.

Example 24.¹⁸F-labeled Evans blue derivatives for sentinel lymph node imaging

Prepared by the method of example 21 and with the purity of more than 95 percent¹⁸F-labeled Evans blue derivative (Compound 13) was injected into a pad of the sole of a foot with 10. mu.L of 0.37MBq in normal FVB mice¹⁸F-AlF-N(EB)₂Or¹⁸F-AlF-NEB is used as a control, and then dynamic MicroPET imaging is carried out 0-60 min after administration under isoflurane anesthesia, and 90min andstatic 120min imaging, results are shown in FIG. 3, Compound 13(N (EB)₂) High-quality lymph node PET imaging can be obtained, the lymph node PET imaging has low migration speed in a lymphatic vessel, a sentinel lymph node has a PET signal in a long time, and a secondary lymph node does not exist, so that the two lymph nodes can be effectively distinguished.

Meanwhile, compound 13 can have strong fluorescence emission at 670nm after being combined with HSA, so that compound 13 can also be used for fluorescence imaging of lymph nodes and lymph vessels. Fluorescence imaging was performed at various time points after injection of compound 13 in the ball pad of normal mice, using NEB as control. FIG. 4 shows that compound 13 can clearly visualize lymph nodes and vessels, and at the same time, it has a slower migration speed in the lymph vessels, and can distinguish sentinel lymph nodes from secondary lymph nodes for a longer period of time.

Thus, the bimodal imaging properties of PET imaging and fluorescence imaging after labeling of the radionuclide with compound 13, as well as the ability to distinguish sentinel lymph nodes from secondary lymph nodes, can be used as sentinel lymph node imaging agents.

EXAMPLE 25 Evans blue derivative of the invention Wet-labeling of radioactivity⁶⁴Cu

The method comprises the following steps:

C1) dissolving a proper amount of the compound 13 prepared in example 1 in a buffer solution or deionized water to obtain a solution a;

Example 26.⁶⁴MicroPET imaging of Cu-labeled Evan blue derivatives in U87 nude mice

Prepared by the method of example 25 and with the purity of more than 95 percent⁶⁴Cu-labelled evans blue derivative (compound 13). In the U87 nude mouse subcutaneous tumor model, 3.7MBq was injected into tail vein⁶⁴Cu-N(EB)₂Or⁶⁴Cu-NEB served as a control and MicroPET imaging was performed at 1h,4h,24h and 48h post-dose. As shown in FIG. 5, Compound 13(N (EB)₂) Has higher uptake in tumor, is obviously higher than NEB, and can be used as tumor imaging agent.

Claims

1. An evans blue derivative, the structure of which is shown as the following formula (IV):

2. a process for preparing a compound of formula (IV) according to claim 1, comprising the steps of:

① introducing tert-butyloxycarbonyl group to the amino group at one end of 3,3' -dimethylbenzidine, diazotizing the amino group at the other end to generate diazonium salt, then carrying out coupling reaction with 1-amino-8-naphthol-2, 4-disulfonic acid, removing tert-butyloxycarbonyl group, reacting the deprotected amino group with ammonia water and carbon disulfide to generate an intermediate compound A with thiocyano group;

② 6-fluorenylmethoxycarbonylamino-2-tert-butyloxycarbonylaminobutyric acid is acylated and condensed with the coupling reaction product in the step ① to remove fluorenylmethoxycarbonylamino, the deprotected amino is subjected to acylation condensation reaction with 1,4, 7-triazacyclononane-1, 4-bis-tert-butyl acetate-7-acetic acid, and then tert-butyloxycarbonyl and tert-butyl in the condensation product are removed to obtain an intermediate compound B;

③ reaction of intermediate compound A obtained in step ① with intermediate compound B obtained in step ② to form a thiourea bond by condensation reaction of the thiocyano group with an amino group to obtain a compound represented by the formula (IV) in claim 1.

3. A radiolabeled evans blue complex obtained by labeling a radionuclide with the compound of formula (IV) according to claim 1 as a ligand.

4. The radiolabeled evans blue complex of claim 3The method is characterized in that: the radionuclide is selected from¹⁸F、⁶⁴Cu、⁶⁸Ga、⁶²Cu、⁶⁷Cu、⁸⁶Y、⁸⁹Zr、⁹⁰Y is or¹⁷⁷Any one of Lu.

5. The radiolabeled evans blue complex according to claim 3, wherein: the radionuclide is selected from¹⁸F or⁶⁴Cu。

6. A process for preparing a radiolabelled evan blue complex according to claim 3, characterised in that a compound of formula (IV) according to claim 1 is used as ligand, and the radiolabel is carried out by wet-labelling¹⁸F, comprising the following steps:

A1) dissolving a proper amount of a compound shown as (IV) in a buffer solution or deionized water to obtain a solution a;

7. A process for the preparation of a radiolabelled evan blue complex according to claim 3, characterised in that a compound of formula (IV) according to claim 1 is used as ligand, labelling the radiolabel by lyophilization¹⁸F, comprising the following steps:

B1) dissolving a proper amount of a compound shown as (IV) in a buffer solution or deionized water to obtain a solution a;

B5) adding appropriate amount of acetic acid solution or buffer solution into the kit obtained in step B4), dissolving, adding acetonitrile or ethanol, and fresh¹⁸F, sealing the aqueous solution of ions, reacting for 5-30 min at 70-120 ℃, and cooling;

8. A process for preparing a radiolabelled evan blue complex according to claim 3, characterised in that a compound of formula (IV) according to claim 1 is used as ligand, and the radiolabel is carried out by wet-labelling⁶⁴Cu, comprising the steps of:

C1) dissolving a proper amount of a compound shown as (IV) in a buffer solution or deionized water to obtain a solution a;

C2) adding into the solution a⁶⁴Cu²⁺The sodium acetate solution is sealed and reacted for 5-100 min at the temperature of 40-80 ℃, and then cooled;

C3) separating and purifying the solution obtained in the step C2) by semi-preparative HPLC, removing the solvent by rotary evaporation, dissolving the remainder with phosphate buffer solution or water, and performing sterile filtration to obtain the final product⁶⁴Cu-labelled evans blue derivative injection.

9. A process for the preparation of a radiolabelled evan blue complex according to claim 3, characterised in that a compound of formula (IV) according to claim 1 is used as ligand, labelling the radiolabel by lyophilization⁶⁴Cu, comprising the steps of:

D1) dissolving a proper amount of a compound shown as (IV) in a buffer solution or deionized water to obtain a solution a;

D2) sterile filtering the solution a obtained in the step D1), subpackaging in containers, freeze-drying, plugging and sealing to obtain a freeze-dried medicine box;

D3) adding an appropriate amount of acetic acid solution or buffer solution into the medicine box obtained in the step D2), dissolving, and adding⁶⁴Cu²⁺The sodium acetate solution is sealed and reacted for 5-30 min at 70-120 ℃, and then cooled;

D4) separating and purifying the solution obtained in the step D3) by semi-preparative HPLC, removing the solvent by rotary evaporation, dissolving the residue with phosphate buffer solution or water, and performing sterile filtration to obtain the final product⁶⁴Cu-labelled evans blue derivative injection.

10. The method of claim 9, wherein: D2) adding an excipient into the freeze-dried medicine box, and adjusting the dosage of the compound shown in the formula (IV) and the excipient to enable the medicine box to be optimally formed; the excipient is selected from mannitol or ascorbic acid.

11. Use of the radiolabeled evans blue complex according to claim 3 for the preparation of a nuclear medicine imaging agent.

12. The use of claim 11, wherein: the nuclear medicine developer is sentinel lymph node developer, cardiac blood pool developer or tumor blood pool developer.