CN106632571B - Molecular targeted imaging agent of receptor class and its preparation method and application - Google Patents

Abstract

The invention discloses molecular targeted imaging agent of receptor class and its preparation method and application, which has a short chain of polyethylene glycol, is connected by " click " reaction with the female alcohol of 17 alpha-acetylenes of targeting estrogen receptor, the imaging agent specific structure general formula is as follows:Wherein, the female alcohol of 17 alpha-acetylenes is as targeting group, polyethylene glycol as hydrophilic radical,¹⁸F is radioactive group.The molecular targeted imaging agent of this receptor class has excellent biological property, the intake with higher in the breast cancer tumour of estrogen receptor positive, estrogen positive receptor and estrogen negative receptors can be distinguished, there is relatively strong specificity, meet the condition for being used as breast cancer receptor developer.

Description

Molecular targeted imaging agent of receptor class and its preparation method and application

Technical field

The invention belongs to Medical Imaging Technology fields, and in particular to a kind of receptor target imaging agent and preparation method thereof.

Background technique

Breast cancer seriously threatens the health and life of women, shows pathogenesis of breast carcinoma according to cancer statistical data in 2015 Rate occupy female malignant first place.Early diagnosis is the key that treatment breast cancer.1978, Lippman et al. passed through experiment The patient with breast cancer for obtaining about 70%-80% can over-express the conclusion of estrogen receptor.Now, the distribution of estrogen receptor It is one of the project of Prognostic Factors of Breast Carcinoma key with level, clinically determines that patient uses according to the situation of estrogen receptor The dosage of anti-estrogens drug.

Currently, clinically the method for main applied pathology detects and quantifies breast cancer acceptor type.This pathology is cut The method of piece needs invasive operation to obtain tissue, brings great pain to patient, and is difficult to obtain transfer stove and answer Send out stove tissue, can not dynamic detection tumor markers expression.

In past more than 20 years, with the continuous development of molecular image technology, PET imaging technique is in detection cancer Using increasing.PET imaging technique has dynamic in real time, polynary accurate, the advantages such as rapid and convenient noninvasive in body, therefore PET Imaging technique is the desirable route for realizing early diagnosing mammary cancer.

Core of the molecular image probe as PET imaging technique, decides the development of PET imaging technique.1984, Kiesewetter et al. is synthesized and 16 α-is marked¹⁸F-17 beta estradiol (¹⁸F-FES),¹⁸F-FES is to estrogen receptor Estradiol molecules with affinity are skeleton, are marked at its C-17¹⁸What F was obtained.1984, the human hairs such as Kiesewetter Table¹⁸The preparation method of F-FES, and demonstrate¹⁸F-FES has good Relative binding capacity to estrogen receptor (RBA), biodistribution experiments have been done to immature rat again later, has found the intake in rat estrogen receptor uterus abundant It is apparently higher than its hetero-organization and organ.1988, Mintun et al. trial pair for the first time¹⁸F-FES carries out clinical research, 13 trouble Person passes through¹⁸The mode of F-FES-PET imaging is diagnosed as breast cancer, it is most important that, it can obviously observe and turn from imaging figure The case where moving stove.Up to the present,¹⁸F-FES has become most successful estrogen receptor imaging agent, and has entered the clinic II phase Experiment.But¹⁸There are also limitations by F-FES, i.e., due to¹⁸The fat-soluble height of F-FES is discharged mainly through hepatic metabolism through kidney In vitro, metabolic rate quickly, so being difficult effectively to be enriched in target tissue and organ.

In order to improve¹⁸The metabolic condition of F-FES in vivo, makes it preferably act on targeting moiety, and people synthesize and mark Many kinds of 17 beta estradiol derivatives are remembered, as estrogen receptor imaging agent.Nineteen ninety, Katzenellenbogen et al. are closed It is 17 alpha-acetylenes bases-estradiol, 17 β-alkynyl-respectively at a series of C-11 C-17 by the estradiol analog of alkynyl substituted Estradiol, 11 'beta '-methoxies-estradiol and 11 β-ethyl-estradiol.By carrying out rat biodistribution experiments to each probe, and With¹⁸The experimental result of F-FES, which compares, show that the selectivity absorbed in 17 beta estradiol bodies can be enhanced in the introducing of substituent group. 2013, Michel et al. proposition fluoro- -16 α of 11 'beta '-methoxy-of 4- [¹⁸F] estradiol (4FMFES) is fluorinated to estrogen receptor The tumor uptake of positive source of mouse breast cancer (MC7-L1 and MC4-L2) PET tumor imaging is higher than¹⁸F-FES.It is right¹⁸F-FES is carried out The example of retrofit has very much, but these transformations all do not reduce really¹⁸F-FES's is fat-soluble, and some even makes it Fat-soluble increase, these properties limit the development of such probe.

Summary of the invention

The primary purpose of the present invention is that providing a kind of estrogen receptor targeted contrast agents with excellent imaging performance.

It is of the invention another purposefully to be to provide the synthesis and labeling method of above-mentioned receptor target imaging agent.

Another object of the present invention is to provide application of the above-mentioned receptor target imaging agent in tumor imaging.

The specific technical solution of the present invention is as follows:

A kind of receptor target imaging agent, it is characterised in that: the probe has a short chain of polyethylene glycol, anti-by " click " It should be connected with the female alcohol of 17 alpha-acetylenes of targeting estrogen receptor, the imaging agent specific structure general formula is as follows:

The preparation method of a kind of above-mentioned receptor target imaging agent, includes the following steps:

1) by compound 1 and p-methyl benzene sulfonic chloride with molar ratio (1-5): the ratio mixing of (2.5-15), in triethylamine Under effect, compound 2 is obtained；

2) by compound 2 with sodium azide with molar ratio (1-5): the ratio of (5-1) mixes, and obtains compound 3；

3) compound 3 is marked¹⁸F obtains compound 4；

4) " click " is occurred for the female alcohol of the alpha-acetylenes of compound 4 and 17 to react, obtains target label product Compound 5.

It is synthesized and label route are as follows:

In the present invention,¹⁸F is radio-contrast effect,¹⁸F also could alternatively be other radiation conventionally used for photographic developer Property element, this should be regarded as equivalent replacement of the invention.

The present invention marks PEG chain one end¹⁸F, and reacted by " click " and have high-affinity to estrogen receptor Ethinyloestradiol is connected, and obtains a novel estrogen receptor targeting developing immunomodulator compounds 5.Theoretically, this structure is repaired Decorations change less estrogen itself, and it is fat-soluble to reduce its, to reduce hepatic metabolism rate, improves tumour to radiation The intake of property drug.Preliminary Results show that estrogen receptor targeting developing immunomodulator compounds 5 have excellent biological property, When including: to immature rat biodistribution experiments, uterus and ovary to the intake of compound 5 to be apparently higher than its hetero-organization and Intake of the organ to compound 5；When to source of people breast cancer (MCF-7) PET tumor imaging of estrogen receptor positive, tumor uptake It is apparently higher than muscle, and this intake can be suppressed；The source of people breast cancer (MDA-MB-231) of estrogen receptor negative is swollen When tumor PET is imaged, discovery MDA-MB-231 tumor uptake is significantly lower than MCF-7 tumour, these experiments all absolutely prove compound 5 There is very high specificity to estrogen receptor.In addition, the preparation method of compound 5 is simple, at low cost, it is expected to clinically obtain To promote and apply.

The beneficial effects of the present invention are:

(1) estrogen receptor targeted contrast agents of the invention have estradiol skeleton, while having good chemical stabilization Property and bio distribution property, specific activity it is high, targeting is strong, and preparation method is simple and easy to do, can be used for carrying out estrogen receptor positive PET tumor imaging.

(2) receptor target imaging agent of the present invention has excellent biological property.Have in estrogen receptor positive tumors Higher intake, and absorb with specificity, meet the condition for being used as estrogen receptor imaging agent, target to non-target ratio value is high, bright It shows better than existing¹⁸F-FES.In addition, lower non-target internal organs intake can reduce unnecessary radioactive damage.

Detailed description of the invention

Fig. 1 compound 5 (under) and its standard items (on) high performance liquid chromatography (HPLC) analysis chart；

The cell of Fig. 2 compound 5 is saturated binding curve (A)；In 5-90min, MCF-7 cell and MDA-MB-231 cell Intake experiment to compound 5 and intake of the MCF-7 cell to compound 5 in 30-90min, after being suppressed agent inhibition (B)；MCF-7 cell is suppressed front and back and compares (C) to the intake of compound 5；MCF-7 cell and MDA-MB-231 cell are to chemical combination The comparison (D) that object 5 absorbs.

Tissue distribution patterns when Fig. 3 compound 5 is 1 hour in normal immature rat body, different tissues organ are taken the photograph Value is indicated in the form of per gram of tissue percentage injection dose rate (%ID/g)；

Fig. 4¹⁸Tissue distribution patterns when F-FES is 1 hour in normal immature rat body, different tissues organ are taken the photograph Value is indicated in the form of per gram of tissue percentage injection dose rate (%ID/g)；

Fig. 5 compound 5 absorbs compound 5 in the intracorporal PET imaging (A) of lotus MCF-7 tumor nude mice and tumour and muscle Comparison (B)；The ratio (C) of tumour and muscle intake.

Fig. 6 compound 5 by the intracorporal PET imaging (A) of estrogen-receptor inhibitor treated MCF-7 tumor nude mice and The comparison (B) of tumor uptake value in inhibited dose of processing and unprocessed experiment；

Fig. 7 compound 5 is swollen in the intracorporal PET imaging (A) of lotus MDA-MB-231 tumor nude mice and the experimental result and MCF-7 Comparison (B) of the tumor to 5 uptake values of compound.

Fig. 8¹⁸F-FES is in the intracorporal PET imaging (A) of lotus MCF-7 tumor nude mice and the experimental result and MCF-7 tumour pair The comparison (B) of 5 uptake values of compound.

Specific embodiment

Specific embodiment combination attached drawing below by way of compound 5 is that technical solution of the present invention carries out further Bright and description.

1. the preparation of compound 5

1) synthesis of compound 2:

At 0 DEG C, 1.5g compound 1 and 1.7mL triethylamine are dissolved in 10mL methylene chloride, 4.77g is added to methyl Benzene sulfonyl chloride reacts at room temperature 5 hours.After reaction, reaction solution is extracted with dichloromethane three times, collects organic phase and rotates and remove Remove solvent.Product is crossed into silicagel column, mobile phase ratio is petroleum ether: ethyl acetate=2:1.Obtaining pure white crystal is chemical combination Object 2.

2) synthesis of compound 3:

0.65g sodium azide is dissolved completely in 10mL n,N-Dimethylformamide, 4.59g compound 2, room is added Temperature stirring 5 hours.After the reaction was completed, it is dissolved with 50mL ethyl acetate, is washed with 40mL deionized water, collect organic phase and rotated Remove solvent.Product silica gel column purification, mobile phase ratio is petroleum ether: ethyl acetate=2:1.Obtain pale yellowish oil substance As compound 3.

3) label of compound 4:

It will¹⁸F^-It is enriched on QMA column, then by 4,7,13,16,21,24- six oxygen -1,10- phenodiazine of 4mg potassium carbonate and 13mg Bicyclic [8.8.8] hexacosane is dissolved in 1mL solvent (acetonitrile: water=9；1) QMA column will, be eluted with the solution.At 100 DEG C, Solvent is dried up with nitrogen evaporator, and separately plus three times anhydrous acetonitrile dries up, guarantee system is anhydrous.It is anhydrous that compound 3 is dissolved in 0.5mL Acetonitrile is added to drying¹⁸F^-In, 20 DEG C of reaction 20min.Reaction product is separated with C18 column, is eluted with 1mL tetrahydrofuran. Obtain light yellow liquid.

4) label of compound 5:

By the female alcohol of 17 alpha-acetylenes of 2mg, 2mg diisopropyl ethyl amine and 1mg cuprous iodide are added separately to the four of compound 4 In hydrogen tetrahydrofuran solution, 60 DEG C of reaction 20min.It is purified by radioactivity-HPLC, radiochemical purity is greater than 99%.Efficient liquid phase Chromatograph condition are as follows: C18 column (250 × 4.6mm, 5 μm), mobile phase A are mutually water, 35% acetonitrile of B phase, flow velocity 4mL/min.

By taking 5 imaging agent of above compound as an example, the experimental results showed that, basic performance is as follows:

1. the cell experiment of compound 5

The saturation experiment of A.MCF-7 cell

1) orifice plate (24 orifice plate) for forming monolayer adherence cell is taken out, all culture mediums is carefully sucked out, then to each 0.5mL culture medium is added in orifice plate, then is sucked out to wash away the cell that surface dies or falls off.At this moment orifice plate is lifted, through light Line can be seen that one layer of uniform cell membrane is arranged at orifice plate bottom from bottom, preferably the region not fallen off uniformly and obviously.

2) 5 solution of compound of appropriate HPLC after purification is taken, is diluted with culture medium, the compound 5 of 1-100nM is obtained, it will The compound of various concentration, which is added in cell, to be cultivated, and capping is placed at room temperature for 1h.

3) it is washed three times with 1mL PBS (pH=7.4), is being incubated for 5-10min with 1mL sodium hydroxide.Then it is inhaled with pipettor The light scraper plate hole bottom of head, completely disengages cell, and all solution and cell are in small test tube in absorption orifice plate.It is examined with gamma counter Survey the radioactivity of every pipe.

It is calculated by Fig. 2A and through software it is found that the dissociation constant Kd of compound 5 is 26.54nM, Bmax Bmax is 1.12×10⁴/ cell.

B. cellular uptake and Inhibition test

Saturation experiment step 1) of the step 1) with appeal MCF-7 cell

2) 5 solution of compound of appropriate HPLC after purification is taken, is diluted with culture medium, radioactivity amount is diluted to 3 μ Ci/mL.

3) 10 μ L estradiol solution (1mg/mL) are added into inhibition group orifice plate first, then add respectively into institute's abacus Enter the compound 5 that 100 μ L have diluted, capping is placed at room temperature for 5-90min.

Saturation experiment step 3) of the step 4) with appeal MCF-7 cell.

As shown in Fig. 2 B, 2C and 2D, the intake of the MCF-7 of estrogen receptor positive will be significantly larger than estrogen receptor negative MDA-MB-231 cell, and the intake of MCF-7 cell can be suppressed agent inhibition.Illustrate the level pair in vitro of compound 5 Estrogen receptor has high specific.

2. compound 5 and¹⁸The normal rat biodistribution experiments of F-FES

The female sd inbred rats of normal rat biodistribution experiments selection 3-4 week old.Rat tail vein injects 1.85MBq chemical combination Object 5 or¹⁸F-FES, 1 hour progress biodistribution experiments after injection.Intake situation such as Fig. 3 A of each organ to compound 5 It is shown, wherein the intake in organ uterus and ovary rich in estrogen receptor will be apparently higher than its hetero-organization and organ, and liver Intake it is very low.Illustrate that level has very strong targeting to estrogen receptor to compound 5 in vivo.For inhibition group, first Experimental rat is handled with fulvestrant, then tail vein injection compound 5, as can be seen from Figure 3B, the absorption in inhibition group uterus Significantly lower than normal group, illustrate that compound 5 is high to the specificity of estrogen receptor.

Each organ pair¹⁸The intake situation of F-FES as shown in figure 4, wherein the intake of uterus and ovary it is slightly higher, liver has very Height intake, i.e. non-specific uptake are high.In contrast,¹⁸F-FES will be lower than change to the compatibility of the organ rich in estrogen receptor Close object 5.

3. the PET in breast cancer model Mice Body of compound 5 is imaged

8 weeks big Balb/c Female nude mices are selected, in the MCF-7 tumour and estrogen of right shoulder plantation estrogen receptor positive The MDA-MB-231 tumour of receptor negative.PET imaging is carried out when tumour length to diameter is 0.5-1cm, every nude mice passes through tail It is injected intravenously about 5.92MBq compound 5,0.5h, 1h and 2h carry out suction-type anesthesia with isoflurane respectively after injection, and prostrate is solid Static scanning imaging is carried out after fixed.The PET image results of MCF-7 model nude mice are as shown in Figure 5.By image results it is found that female swash The breast cancer tumour of plain receptor positive has a very high intake to compound, this intake change with time it is unobvious, and also There is very high target to non-target ratio, illustrates that the tumour of compounds on estrogen receptor positive has targeting.

Be illustrated in figure 6 the PET image results with the pretreated MCF-7 model nude mice of fulvestrant, from image it is found that After the estrogen receptor of tumour is sufficiently destroyed by fulvestrant, the tumour of MCF-7 model nude mice hardly absorbs compound 5, by This, which demonstrates compound 5, has specificity to estrogen receptor.

The PET image results of MDA-MB-231 model nude mice are as shown in fig. 7, it can be seen from the figure that MDA-MB-231 mould The tumour of type nude mice hardly absorbs compound 5, and compound 5 only has a high intake in the tumour of estrogen receptor positive, and Intake in estrogen receptor negative tumour is close with non-target tissue's intake, it was demonstrated that compound 5 is to estrogen receptor Specific target tropism.

Every nude mice passes through tail vein injection about 5.92MBq¹⁸F-FES, 0.5h, 1h and 2h use isoflurane respectively after injection Suction-type anesthesia is carried out, carries out static scanning imaging after prostrate is fixed.PET image results such as Fig. 8 institute of MCF-7 model nude mice Show.MCF-7 tumour pair¹⁸The intake of F-FES illustrates that compound 5 swells to estrogen positive well below the intake to compound 5 The targeting of tumor is better than¹⁸F-FES。

The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e., Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.

Claims (2)

1. the molecular targeted imaging agent of receptor class, it is characterised in that: the imaging agent has estrogen targeting group and polyethylene glycol short Chain, specific structure general formula are as follows:

。

2. a kind of synthesis and labeling method of the molecular targeted imaging agent of receptor class as described in claim 1, include the following steps:

1) by compound 1 and p-methyl benzene sulfonic chloride with molar ratio (1-5): the ratio mixing of (2.5-15) is acted in triethylamine Under, obtain compound 2；

2) by compound 2 with sodium azide with molar ratio (1-5): the ratio of (5-1) mixes, and obtains compound 3；

3) compound 3 is marked¹⁸F obtains compound 4；

4) " click " is occurred for the female alcohol of the alpha-acetylenes of compound 4 and 17 to react, obtains target label product Compound 5；

Wherein, 1 to 5 structure of compound is as follows:

。