WO2023130587A1

WO2023130587A1 - Water-soluble methylbenzene ether derivative, positron nuclide probe and nuclide marker, and preparation methods therefor and uses thereof

Info

Publication number: WO2023130587A1
Application number: PCT/CN2022/082160
Authority: WO
Inventors: 李林; 吴小艾; 王玥祺; 潘立立; 李亚伦
Original assignee: 四川大学华西医院
Priority date: 2022-01-05
Filing date: 2022-03-22
Publication date: 2023-07-13
Also published as: CN114573558B; CN114573558A

Abstract

The present invention relates to a water-soluble methylbenzene ether derivative, a positron nuclide probe and a nuclide marker, and preparation methods therefor and the uses thereof. Water-soluble side chains are introduced by means of derivatizing the structure of a methylbenzene ether to prepare a series of probes having a PD-L1 inhibitory activity and a targeting property. A small molecule inhibitor of the water-soluble methylbenzene ether has a higher inhibitory activity, a 68Ga-labeled positron nuclide probe can be used for PET imaging of PD-L1 in vivo, and a 177Lu nucleotide marker can achieve therapeutic targeting of PD-L1. Therefore, the small molecule can achieve the integration of diagnosis and treatment with PD-L1 as a target.

Description

A water-soluble methyl benzyl ether derivative, a positron nuclide probe, a nuclide label, its preparation method and application

technical field

The invention belongs to the field of imaging agents, and in particular relates to a water-soluble methyl benzyl ether derivative, a ⁶⁸ Ga positron nuclide probe and a ¹⁷⁷ Lu nuclide marker, a preparation method and an application thereof.

Background technique

The diagnosis and treatment of malignant tumors are currently difficult problems in the medical field. With the development of molecular biology, multiple potential tumor-related diagnostic/therapeutic targets and mechanisms have been gradually discovered and rapidly applied in the field of tumor diagnosis and treatment. Tumor immune checkpoint inhibition (Immune checkpoint blocking, ICB) therapy is a new therapy developed in recent years that uses the human immune mechanism to treat tumors. It reactivates the immune mechanism of the human body suppressed by the tumor to kill tumor cells, so it has better The advantages of high therapeutic effect and not easy to produce drug resistance are the hotspots in the field of tumor treatment research. So far, a number of drugs based on this mechanism have been officially approved by the FDA/SFDA and play an important role in clinical tumor treatment.

Currently, immune checkpoint inhibitors in clinical use mainly inhibit CTLA-4 (Cytotoxic T lymphocyte associated protein 4, CTLA-4) signaling pathway and programmed death (PD) signaling pathway, preventing CTLA- The mutual recognition and combination of 4/B7 and PD1/PD-L1 (programmed death protein 1/programmed cell death ligand 1) reactivates T cells that have been suppressed by tumor cells, thereby restoring the ability of T cells to kill tumor cells. Taking the PD1/PD-L1 signaling pathway as an example, in order to avoid serious immune reactions from killing normal cells by mistake, the body will have some immunoregulatory proteins on the surface of T cells. PD1 is such a protein. When PD1 on the surface of T cells and the expression of normal cells After the ligand PD-L1 binds, it will transmit immunosuppressive signals and reduce the proliferation of T cells. However, tumor cells can recognize and use this mechanism to express PD-L1 protein on their own surface, so that T cells cannot correctly recognize tumor cells and proliferate, thereby evading this programmed death mechanism, thus blocking the recognition and activation of PD1/PD-L1. The combination can restore the ability of T cells to kill tumors.

Tumor immune checkpoint inhibition therapy has many advantages that traditional therapies (radiotherapy, chemotherapy, targeted therapy) cannot match, such as showing unprecedented clinical activity in certain types of tumors (such as melanoma), and relatively small toxic and side effects ( It mainly causes immune-related side effects), drug resistance is not easy to appear, and some patients can obtain a very long period of remission after using this type of drug. However, in clinical applications, immune checkpoint inhibitor drugs often have a low response rate and are only effective for some patients. The low response rate is a very troublesome problem for scientists and clinicians. How to improve the response rate of patients, or predict and evaluate the treatment effect before using the therapy, and screen patients who can benefit from the therapy in advance is an urgent problem to be solved. scientific issues.

Positron emission tomography (PET) has the advantages of sensitive detection and non-invasiveness. It can not only perform real-time imaging of target organs in vivo to display the distribution of lesions, but also perform quantitative analysis to determine the expression level of biological targets in vivo. . Therefore, if a specific molecular probe targeting the target of tumor immunotherapy is designed and synthesized, an appropriate nuclide (such as ⁶⁸ Ga) is selected for labeling and imaged by PET technology, the immunotherapy in human primary or metastatic tumors can be determined. The content and level of the target can not only perform tumor imaging of immune checkpoint-related targets, but also predict the therapeutic effect of tumor immunotherapy. ^177Lu (half-life 6.7d) is currently the most commonly used radiometal for therapeutic purposes, as it has particulate emission (β- or Auger electrons) for achieving therapy and emits several concomitant signals γ-208keV (11%) and 113 keV (6.4%) photons were used for diagnostic evaluation and dosimetry. Therefore, if ¹⁷⁷ Lu is connected to this kind of radioactive probe at the same time, targeted therapy of tumor can also be realized.

So far, an antibody targeting CTLA4 (ipilimumab) and multiple antibodies targeting the PD-1/PD-L1 signaling pathway (nivolumab, pembrolizumab and atezolizumab) have been approved by the FDA for clinical use Potential antibodies have entered clinical trials, and these antibodies targeting the immune checkpoint signaling pathway have shown excellent therapeutic effects and response rates in the clinical treatment of melanoma (primary or metastatic), non-small cell lung cancer and renal cell carcinoma , greatly improving the survival of patients who respond to this therapy. At the same time, these targeting antibodies can be used as specific molecular probes to study the expression of target proteins in vivo after being radiolabeled, and the obtained image data can not only be used for tumor distribution imaging, but also for the prediction of tumor immune checkpoint therapy with evaluation. Antibody-based PD-L1 radionuclide imaging has already been carried out by a research group, and important progress has been published in international well-known journals such as Nature and PNAS. Therefore, although antibody-based radionuclide imaging has scientific value, it is not enough. novelty. At the same time, due to the metabolic characteristics of antibody molecules, it is difficult for antibody-based nuclide molecular probes to concentrate in the brain, and it is difficult to detect the expression level of PD-L1 in brain tumors.

So far, although some small molecules with highly active PD-L1 inhibitory activity have been reported, such small molecule PD-L1 inhibitors have not been successfully developed into clinical therapeutic drugs, nor have they been successfully applied to prepare nucleated Tumor imaging and early diagnosis using protein probes. This type of small molecule generally contains a methyl benzyl ether skeleton. Such compounds are generally highly fat-soluble, and will accumulate in the liver after entering the body, or be deeply combined with plasma proteins, making it difficult to penetrate multiple layers of physiological barriers to reach the tumor site. Therefore, by introducing a water-soluble fragment DOPA into the molecule, adjusting its lipophilicity, and realizing the isotope 68Ga labeling for positron radioactivity diagnosis, a series of nuclide probes that maintain the affinity of PD-L1 will be obtained, enabling tumor imaging in vivo and The ability to evaluate the expression and distribution of PD-L1 in vivo. At the same time, when DOTA is used as a chelate to label 68Ga to prepare positron imaging drugs, ¹⁷⁷ Lu can be conveniently labeled to "convert" the imaging drugs into targeted therapeutic drugs, realizing the integration of "diagnosis-treatment" of tumor targeting .

In summary, the present invention provides a series of highly active PD-L1-targeted positron nuclide probes, which can be used for the target content and level of immunotherapy in human primary or metastatic tumors. Tumor imaging at the spot can predict the therapeutic effect of tumor immunotherapy; it can also be labeled with therapeutic nuclides such as ¹⁷⁷ Lu for tumor treatment.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a water-soluble methyl benzyl ether derivative, a positron nuclide probe, a preparation method and an application.

The purpose of the present invention is achieved by the following technical solutions: a water-soluble methyl benzyl ether derivative, structural formula is as follows:

In the formula, n is an integer greater than or equal to 0, _R is a hydroxyl group or a halogen atom, and Linker is a linear unsubstituted alkane of different lengths or an ethylene glycol side chain (the carbon atom is directly connected to the nitrogen atom of piperazine).

Preferably, said n is 0, 1, 2, 3, 4... etc., constituting cyclic aliphatic amine substituents of different sizes.

Preferably, the R ₁ is an aliphatic cyclic amine substituted by a carboxyl group in the adjacent position, and the configuration of the chiral atom is S configuration, and the chiral atom is the carbon atom of the carboxyl group.

Preferably, the R ₂ is a hydroxyl group, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

Preferably, the Linker is

Wherein, m=1, 2, 3, 4; p=1, 2, 3, 4. The Linker directly connects the parent ring of methyl benzyl ether and piperazine, and inserts the DOTA fragment capable of coupling metal radionuclides into the whole molecule.

The present invention also provides an application of the above-mentioned water-soluble methyl benzyl ether derivative in tumor imaging.

The present invention also provides a kind of preparation method of above-mentioned water-soluble methylbenzyl ether derivative, comprising the following steps:

1) Compound 1 is provided, and compound 1 and methyl benzyl ether intermediate 2I or 2II generate compound 3I or compound 3II under the action of a base, wherein R ₃ is an amino protecting group, and R ₄ is a halogen;

2) The compound 3I or compound 3II is condensed with an amine under acidic conditions and then reduced to generate compound 4I or compound 4II;

3) The compound 4I or compound 4II is under the action of an acid, and the _R3 amino protecting group is removed to generate compound 5I or compound 5II;

4) Compound 5I or Compound 5II undergoes a condensation reaction to generate Compound 6I or Compound 6II (the water-soluble methyl benzyl ether derivative);

Further, in step 1), the alkali is one or more of potassium carbonate, sodium carbonate, sodium hydride, DBU, triethylamine; the solvent used is acetonitrile, tetrahydrofuran, ethanol, dimethylformamide, One or more of dimethyl sulfoxide;

And/or, in step 2), the acid is glacial acetic acid; the reagent used for the reduction is sodium borohydride or sodium cyanoborohydride; the solvent used is methanol;

And/or, in step 3), the acid is trifluoroacetic acid or hydrochloric acid; the solvent used is one or more of methanol, ethanol, tetrahydrofuran, and methylene chloride;

And/or, in step 4), the condensation reaction is catalyzed by EDCI-HOBT or HATU-HOBT; the solvent used is one or more of dimethylformamide, dimethylsulfoxide, dimethylacetamide kind.

The present invention also provides a positron nuclide probe or a nuclide label prepared from the water-soluble methylbenzyl ether derivative described above.

Further, the preparation method of the positron nuclide probe is as follows: take the water-soluble methyl benzyl ether derivative, carry out positron nuclide ⁶⁸ Ga labeling, or nuclide ¹⁷⁷ Lu labeling, respectively obtain ⁶⁸ Ga positron Nuclide probe, ¹⁷⁷ Lu nuclide label; the structural formula of the ⁶⁸ Ga positron nuclide probe and ¹⁷⁷ Lu nuclide label is as follows:

Further, when the positron nuclide ⁶⁸ Ga is labeled, the following labeling reaction can be easily used to complete the labeling of the metal nuclide:

The present invention also provides an application of the above-mentioned ⁶⁸ Ga positron nuclide probe in tumor targeting imaging and/or tumor radionuclide therapy.

The present invention also provides an application of the above-mentioned ¹⁷⁷ Lu nuclide marker in tumor targeting therapy.

The present invention derivates the reported methyl benzyl ether structure, introduces DOTA groups through Linkers of different lengths, and prepares a series of new compounds that can label ⁶⁸ Ga and ¹⁷⁷ Lu and have PD-L1 targeting. The ⁶⁸ Ga and ¹⁷⁷ Lu labels of this series of compounds were successfully prepared in the experiment of prime labeling. The targeting of the probe was verified by cell experiments, and the pharmacological and drug metabolism properties of the positron label in the body, such as absorption, distribution, metabolism and excretion, were studied through normal animals, and the tumor imaging experiments were verified. Targeting of a series of compounds and their ability to detect tumor PD-L1 receptor expression.

The present invention uses substituted benzaldehyde as a starting material to synthesize a series of water-soluble methyl benzyl ether derivatives containing DOTA groups. Compared with the reported molecules, the series of compounds have higher activity. On this basis, the present invention prepared its ⁶⁸ Ga positron nuclide-labeled compound, and used the small molecule for PD-L1 expression tumor imaging for the first time, and verified the tumor targeting and detection of tumors of the probe The ability to express PD-L1 has clinical translational value. At the same time, the present invention also prepares ¹⁷⁷ Lu nuclide markers, which can be used for tumor nuclide therapy targeting PD-L1.

The beneficial effects of the present invention are: the new ⁶⁸ Ga-labeled water-soluble methylbenzyl ether derivatives of the present invention have better PD-L1 targeting, and can be used as tumor imaging agents, and the mechanism is that it interacts with PD-L1 expressed by tumor cells. -L1 receptor binding. At the same time, compared with the reported PD-L1 probes based on antibodies and polypeptides, the positron nuclide probes involved in the present invention have the characteristics of easy operation, high "target/non-target" ratio, and better tumor imaging The image can better show the distribution of PD-L1 receptors in the whole body. Therefore, the positron nuclide markers involved in the present invention can be used as tumor-targeted imaging probes for clinical application, which can reflect the expression level of tumor PD-L1, diagnose tumors, and formulate treatment plans. At the same time, the efficacy of tumor immunotherapy can also be evaluated, that is, imaging is performed before and after the initiation of targeted or immunotherapy. If the expression of PD-L1 is significantly reduced, it can prove that the therapy is effective. The probe is easy to synthesize, the raw materials are readily available, and the imaging effect is good. At present, there are no reports of small-molecule nuclide probes targeting PD-L1 in this field, and it has clinical transformation value.

Description of drawings

Fig. 1 is the release inhibition experiment of IFN-Gamma;

Fig. 2 is the cellular uptake experiment of radioactive probe;

Figure 3 is the PET imaging experiment and 'Time-SUV' curve of B16F10 tumor model compound 18;

Figure 4 shows the PET imaging experiment and 'Time-SUV' curve of B16F10 tumor model compound 29.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following description.

Embodiment 1 The preparation method of water-soluble methyl benzyl ether derivative

Take n as 2, _R2 as Br, and Linker as

m=3, R ₃ is Boc, and R ₄ is Br as an example, the preparation of the water-soluble methyl benzyl ether derivative on the right side includes the following steps:

DMF (23.7mL, 0.31mol) was dissolved in acetonitrile (70mL), POCl ₃ (24.3mL, 0.26mol) was slowly added dropwise at room temperature, and the reaction temperature was controlled below 30°C, then stirred at room temperature for 1h, and then dropped to - 17°C, compound 7 (32 g, 0.22 mol) dissolved in acetonitrile (70 mL) was added dropwise to the reaction liquid, and reacted at this temperature for 2 h. The reaction solution was added dropwise to water (500 mL) at 40°C, and the temperature was raised to 52°C for 1 h to react, and the acetonitrile was spun off, and the aqueous layer was extracted three times with EA, dried over anhydrous sodium sulfate, spun to dry the solvent, and purified by column (PE:EA =5:1) 15.3 g of light yellow solid was obtained, yield 40%. ¹ H NMR (400MHz,DMSO-d ₆ )δ11.14(s,1H),10.69(s,1H),10.00(s,1H),7.37(s,1H),6.55(s,1H).

Compound 9 (396mg, 2.3mmol), compound 10 (657mg, 2.8mmol) and triphenylphosphine (926mg, 3.2mmol) were dissolved in dry THF (10mL), and DIAD (0.7ml, 1.4eq ) in THF solution (10 mL) was added dropwise to the reaction, and after the reaction was completed overnight at room temperature, TLC showed (PE:EA=2:1) that the reaction of the raw materials was complete. The solvent was spin-dried and purified by column (PE:EA=10:1) to obtain 581 mg of milky white solid compound 11 with a yield of 72%. ¹ H NMR (400MHz, Chloroform-d) δ11.43(s, 1H), 9.69(s, 1H), 7.71(s, 1H), 7.49(dd, J=6.9, 2.1Hz, 1H), 7.42(dd ,J＝8.0,6.5Hz,2H),7.33–7.39(m,1H),7.24–7.33(m,4H),6.61(s,1H),5.21(s,2H),2.25(s,3H).

Compound 12 (5.0 g, 26.8 mmol) was dissolved in dry DMF (100 mL), 1,4-dibromopropane (4.1 mL, 40.1 mmol) was added, and then potassium carbonate (6.0 g, 43.4 mmol) was added to the reaction After completing the reaction at 60°C for 2 hours, TLC monitored the complete reaction (PE:EA=1:1), added water (200mL) to the reaction system to quench the reaction, extracted with ethyl acetate (200mL×3), and combined The organic layer was washed with water (400mL×1) and saturated brine (400mL×1), dried over anhydrous sodium sulfate, spin-dried the solvent under reduced pressure, separated and purified by silica gel column chromatography (PE:EA=3:1) to obtain Milky white solid (2.8g), yield 34%. ¹ H NMR (400MHz, Chloroform-d) δ3.47(t, J=6.6Hz, 2H), 3.42(t, J=5.1Hz, 4H), 2.49(t, J=7.0Hz, 2H), 2.39( t,J=5.1Hz,4H),2.03(p,J=6.8Hz,2H),1.46(s,9H).

Dissolve compound 11 (300mg, 0.75mmol) and compound 13 (270mg, 0.88mmol) in dry DMF (5ml), then add potassium hydroxide (63mg, 1.1mmol) into the reaction, and react overnight at room temperature , TLC (PE:EA=1:1) raw materials have a small amount remaining. Water was added, EA was extracted three times, the organic layer was washed once with water and saturated brine, dried, spin-dried, and purified by column (PE:EA=1:1.5) to obtain 420 mg of white solid compound 14 with a yield of 89%. ¹ H NMR ( 400MHz, Chloroform-d)δ10.26(s,1H),8.04(s,1H),7.50(dd,J=6.6,2.5Hz,1H),7.43(t,J=7.4Hz,2H),7.36( t,J=7.2Hz,1H),7.33–7.27(m,4H),6.58(s,1H),5.24(s,2H),4.15(t,J=6.1Hz,2H),3.45(t,J =5.1Hz, 4H), 2.58(t, J=7.2Hz, 2H), 2.44(t, J=5.1Hz, 4H), 2.29(s, 3H), 2.07(q, J=6.6Hz, 2H), 1.46(s,9H).

Compound 14 (90mg, 0.14mmol) and D-pipercolic acid (74mg, 0.57mmol) were dissolved in 2ml DMF, then acetic acid (8ul, 0.14mmol) was added, and then sodium cyanoborohydride (45mg, 0.71mmol) Add it, complete the reaction at room temperature for 1 hour, raise the temperature to 47 degrees and heat the reaction overnight. TLC shows (PE:EA=1:1) that the raw materials are basically reacted completely, and DCM:MeOH=10:1 shows that there are new spots with high polarity. . Water was added, EA was extracted three times, the organic layer was washed once with water and saturated brine, dried, spin-dried, and purified by column (DCM:MeOH=6:1) to obtain 46 mg of compound 15 as a white solid with a yield of 45%. ¹ H NMR ( 600MHz,Chloroform-d)δ8.06(s,1H),7.52(dd,J=6.8,2.3Hz,1H),7.43(t,J=7.8Hz,2H),7.33(t,J=7.2Hz, 1H),7.33–7.25(m,4H),6.95(s,1H),5.07(s,2H),4.42–4.40(m,1H),4.14–4.12(m,1H),4.05–4.00(m, 2H),3.45(s,3H),3.45–3.38(m,8H),2.73–2.70(m,1H),2.62–2.57(m,2H),2.47–2.37(m,3H),2.21–2.17( m,1H),2.06–1.87(m,4H),1.80–1.68(m,2H),1.44(s,9H).

Compound 15 (35mg, 0.05mmol) was dissolved in dry DCM (1ml), then trifluoroacetic acid (105ul, 1.4mmol) was added to the reaction solution, and the reaction was completed overnight at room temperature, TLC (DCM:MeOH=10: 1) It shows that the reaction of raw materials is complete, and new points with higher polarity are formed. The solvent was spin-dried to obtain 28 mg of compound 16 with a yield of 82.6%.

Compound 16 (11mg, 0.017mmol) was dissolved in dry DMF (1ml), then DOTA-GA-Anhydride (9.5mg, 0.02mmol) and triethylamine (2.6ul, 0.018mmol) were added respectively, and completed at room temperature After reacting overnight, the reaction liquid entered the mass spectrometer, which showed that a product was formed. After the reaction solution is diluted with water, it is separated and purified by HPLC. The preparative column is (A8), and the mobile phase is acetonitrile and 0.1% aqueous formic acid. , 14-25min was 5%, and finally 10 mg of product was obtained, with a yield of 55%. ¹ H NMR (400MHz, Methanol-d ₄ )δ7.69(d, J=8.3Hz, 1H), 7.54(d, J=7.8Hz, 1H), 7.45(t, J=7.4Hz, 2H), 7.32 (t,J=7.5Hz,1H),7.25–7.15(m,4H),6.95(s,1H),5.42(s,2H),4.50–4.42(m,1H),4.36–4.11(m,5H ),3.96–3.33(m,21H),3.26–2.63(m,13H),2.42–2.19(m,8H),2.02–1.60(m,6H),1.57–1.53(m,1H).

Example 2 Preparation of ⁶⁸ Ga positron nuclide probe

Rinse the 68Ge/68Ga generator with 0.1M HCl solution, and cut off the 3-5mL eluent (the eluent at this stage has the highest radioactive specific activity) for later use. Add 400uL 68Ga eluent to the 4mL vial containing compound 17 (50uG), and add 40uL 1M sodium acetate solution to the reaction bottle to adjust the pH value to about 3.5, and seal the reaction at 90 degrees for 20 minutes. The reaction solution was identified by HPLC with a radioactive probe (Agilent ZORBAX SB-C18 5um), the conditions were 25% A (MeCN): 75% B (0.1% TFA aqueous solution), and the flow rate was 1 mL/Min. The retention time of unmarked 68Ga is about 3 minutes, and the peak time of compound 18 is about 8 minutes. Under this condition, the labeling rate is about 98%, and the in vivo and in vitro evaluation or live imaging can be directly performed without separation.

Example 3 Preparation of ¹⁷⁷ Lu nuclide marker

Add 400uL ¹⁷⁷ LuCl3 solution to the 4mL vial containing compound 17 (50uG), and add 40uL 1M sodium acetate solution to the reaction bottle to adjust the pH value to about 3.5, and seal the reaction at 90°C for 20 minutes. The reaction solution was identified by HPLC with a radioactive probe (Agilent ZORBAX SB-C18 5um), the conditions were 25% A (MeCN): 75% B (0.1% TFA aqueous solution), and the flow rate was 1 mL/Min. The retention time of unlabeled ¹⁷⁷ Lu is about 3.5 minutes, and the peak time of compound 19 is about 8 minutes. Under this condition, the labeling rate is about 98%, and there is no need for separation, and the treatment experiment can be carried out directly.

Embodiment 4 The preparation method of water-soluble methyl benzyl ether derivative

Take n as 1, _R2 as cl, and Linker as

p=2, R ₃ is Boc, and R ₄ is Br as an example, preparing the water-soluble methyl benzyl ether derivative on the left side above, comprising the following steps:

Compound 20 (2.0g, 9.9mmol), compound 21 (1.1g, 11.9mmol) and Pd(dppf)Cl2 (55mg, 0.01mmol) were dissolved in toluene (16mL) and ethanol (8mL), nitrogen protection, and then added Aqueous sodium bicarbonate solution (14.9mL, 2M, 29.7mmol) was heated to 80°C for 3 hours, TLC showed (PE:EA=4:1), the reaction of raw materials was complete. Ethyl acetate (30 mL) and water (10 mL) were added, the aqueous layer was extracted three times with EA, the organic layers were combined, dried over anhydrous sodium sulfate, spin-dried, purified by column (PE:EA=10:1), and the Yellow solid 1.89g, yield 74%. ¹ H NMR (400MHz, Chloroform-d) δ: 7.35(dd, J=7.3, 1.6Hz, 1H), 7.22(t, J=7.5Hz, 1H), 7.19–7.14(m, 1H), 6.89(d ,J=8.2Hz,1H),6.81(d,J=2.1Hz,1H),6.75(dd,J=8.2,2.1Hz,1H),4.75(s,2H),4.29(s,4H),2.25 (s,3H).

Compound 22 (1.1 g, 4.3 mmol), 5-chloro-2,4-dihydroxybenzaldehyde (1.2 g, 5.2 mmol) and triphenylphosphine (1.6 g, 6.1 mmol) were dissolved in THF (30 mL), DIAD (1.2mL, 6.1mmol) in THF solution (30mL) was added dropwise to the reaction in an ice-water bath, and after completion of the reaction at room temperature for 2 hours, TLC showed (PE:EA=2:1) that the reaction of the starting material was complete. The solvent was spin-dried and purified by column (PE:EA=10:1) to obtain 1.6 g of white solid with a yield of 82%. ¹ H NMR (400MHz, Chloroform-d) δ: 11.38(s, 1H), 9.67(s, 1H), 7.38(q, J=4.1Hz, 1H), 7.30–7.16(m, 3H), 6.91(d ,J=8.3Hz,1H),6.82(d,J=2.1Hz,1H),6.77(dd,J=8.2,2.1Hz,1H),6.64(d,J=6.7Hz,1H),5.18(s ,2H),4.30(s,4H),2.26(s,3H).

Compound 12 (5.0g, 26.8mmol) was dissolved in dry DMF (100mL), 1,2-bis(2-bromoethoxy)ethane (7.6g, 40.1mmol) was added, and potassium carbonate ( 5.5g, 40.2mmol) was added to the reaction, and after completion of the reaction at 60°C for 2 hours, the reaction was monitored by TLC (PE:EA=1:2), and water (200mL) was added to the reaction system to quench the reaction, and ethyl acetate was used to Extraction (200mL×3), combined organic layers, washed with water (400mL×1) and saturated brine (400mL×1), dried over anhydrous sodium sulfate, spin-dried the solvent under reduced pressure, separated and purified by silica gel column chromatography (PE: EA=1:1), a milky white solid (4.3g) was obtained with a yield of 42%. ¹ H NMR (400MHz, Chloroform-d) δ3.87(t, J=6.6Hz, 2H), 3.62–3.52(m, 8H), 3.22(t, J=5.1Hz, 4H), 2.52(t, J =7.0Hz, 4H), 2.48(t, J=5.1Hz, 2H), 1.45(s, 9H).

Compound 23 (500mg, 1.22mmol) and compound 24 (558mg, 1.46mmol) were dissolved in dry DMF (5ml), then potassium hydroxide (103mg, 1.83mmol) was added to the reaction, and reacted at room temperature overnight , TLC (DCM:MeOH=20:1) showed that the starting material was completely reacted. Water was added, EA was extracted three times, the organic layer was washed once with water and saturated brine, dried, spin-dried, and purified by column (DCM:MeOH=20:1) to obtain 659 mg of white solid with a yield of 76%. ¹ H NMR (600MHz, Chloroform-d) δ10.29(s, 1H), 7.82(d, J=1.9Hz, 1H), 7.46(dt, J=6.7, 3.3Hz, 1H), 7.22(q, J =3.6,3.0Hz,2H),6.96(d,J=8.5Hz,1H),6.83(d,J=2.6Hz,1H),6.78(dd,J=8.24,2.3Hz,1H),6.56(s ,1H),5.16(s,2H),4.30(s,4H),4.28(t,J＝5.0Hz,2H),3.66–3.50(m,8H),2.57–2.55(m,2H),2.48– 2.39(m,6H),2.28(s,3H),2.05–2.03(m,2H),1.44(s,9H).

Compound 25 (300mg, 0.42mmol) and L-proline (194mg, 1.68mmol) were dissolved in 8ml DMF, then acetic acid (24ul, 0.42mmol) was added, then sodium cyanoborohydride (133mg, 2.1mmol) Add it, complete the reaction at room temperature for 1 hour, raise the temperature to 47 degrees and heat the reaction overnight. TLC shows (PE:EA=1:1) that the raw materials are basically reacted completely, and DCM:MeOH=10:1 shows that there are new spots with high polarity. . Water was added, EA was extracted three times, the organic layer was washed once with water and saturated brine, dried, spin-dried, and purified by column (DCM:MeOH=6:1) to obtain 154 mg of compound 15 as a white solid, with a yield of 45%. ¹ H NMR (600MHz, Chloroform-d) δ7.62(s, 1H), 7.47(d, J=4.8Hz, 1H), 7.19(dd, J=4.1, 2.3Hz, 2H), 6.89–6.72(m ,3H),6.55(s,1H),5.16(s,2H),4.42–4.40(m,1H),4.28(s,4H),4.14–4.12(m,1H),4.05–4.00(m,2H ),3.66–3.50(m,8H),2.57–2.55(m,2H),2.48–2.39(m,6H),2.21–2.17(m,1H),2.28(s,3H),2.06–1.87(m ,6H),1.80–1.68(m,2H),1.46(s,9H).

Compound 26 (120mg, 0.15mmol) was dissolved in dry DCM (5ml), then trifluoroacetic acid (1.15ml, 15mmol) was added to the reaction solution, and the reaction was completed overnight at room temperature, TLC (DCM:MeOH=8: 1) It shows that the reaction of raw materials is complete, and new points with higher polarity are formed. The solvent was spin-dried to obtain 85 mg of compound 16 with a yield of 81%.

Compound 27 (50mg, 0.07mmol) was dissolved in dry DMF (1ml), then DOTA-GA-Anhydride (40mg, 0.084mmol) and triethylamine (10ul, 0.07mmol) were added respectively, and the reaction was completed overnight at room temperature , the reaction liquid into the mass spectrometer, showing the formation of products. After the reaction solution is diluted with water, it is separated and purified by HPLC. The preparative column is (A8), and the mobile phase is acetonitrile and 0.1% aqueous formic acid. , 14-25min was 5%, and the final product was 41 mg, and the yield was 59%. ¹ H NMR (400MHz, Methanol-d ₄ ) δ7.52–7.38(m,2H),7.25–7.10(m,2H),6.95(d,J=8.3Hz,1H),6.80(d,J=8.6 Hz,1H),6.72–6.54(m,2H),5.18(s,2H),5.54–5.42(m,1H),4.26(s,4H),4.25–4.06(m,4H),4.02–3.31( m,24H),3.30–2.58(m,18H),2.52–1.12(m,16H).

Example 5 Preparation of ⁶⁸ Ga positron nuclide probe

Add 400uL 68Ga eluent to the 4mL vial containing compound 28 (50uG), and add 40uL 1M sodium acetate solution to the reaction bottle to adjust the pH value to about 3.5, and seal the reaction at 90 degrees for 20 minutes. The reaction solution was identified by HPLC with a radioactive probe (Agilent ZORBAX SB-C18 5um), the conditions were 25% A (MeCN): 75% B (0.1% TFA aqueous solution), and the flow rate was 1 mL/Min. The retention time of unmarked 68Ga is about 3 minutes, and the peak time of compound 18 is about 10 minutes. Under this condition, the labeling rate is about 98%, and the in vivo and in vitro evaluation or live imaging can be directly performed without separation.

Example 6 Preparation of ¹⁷⁷ Lu nuclide marker

Add 400uL ¹⁷⁷ LuCl3 solution to the 4mL vial containing compound 28 (50uG), and add 40uL 1M sodium acetate solution to the reaction bottle to adjust the pH value to about 3.5, and seal the reaction at 90°C for 20 minutes. The reaction solution was identified by HPLC with a radioactive probe (Agilent ZORBAX SB-C18 5um), the conditions were 25% A (MeCN): 75% B (0.1% TFA aqueous solution), and the flow rate was 1 mL/Min. The retention time of unlabeled ¹⁷⁷ Lu is about 3.5 minutes, and the peak time of compound 30 is about 10 minutes. Under this condition, the labeling rate is about 98%, and there is no need for separation, and the treatment experiment can be carried out directly.

Example 7 In vitro enzyme activity test

Using homogeneous time-resolved fluorescence (HTRF) technology, since small molecule inhibitors can block the binding of PD-L1 and PD-1, inhibit the approach of the two fluorescent labels on PD-L1 and PD-1, thereby inhibiting the excitation of fluorescence, Therefore, it can be indirectly used to determine the ability of small molecule compounds to inhibit the combination of the two.

Purchase PD-1/PD-L1 Binding Assay Kit (CISBIO, Part#64ICP01PEG & 64ICP01PEH), operate according to the instructions of the kit, prepare the compounds to be tested into solutions of different concentrations, and use the kit to determine the half inhibitory concentration (IC50 )value. The activity data are shown below (with BMS202 reported by BMS company as a positive control substance).

Note: For the preparation methods of Compound 31 and Compound 32, please refer to Examples 1-6.

The test results show that the compound of the example can significantly inhibit the binding of PD-1 and PD-L1 at nanomolar concentration, and the activity is higher than that of the positive control substance.

Example 8 In vitro cell experiment

The proliferative activity of T lymphocytes can be indirectly reflected by IFN-Gamma. Using extracted human mononuclear cells (PBMC), use anti-CD3/anti-CD28 antibody to activate T lymphocytes, add PD-L1 antibody to inhibit T lymphocytes, and add PD-L1 small molecule inhibitor to detect IFN-Gamma The expression of , can reflect the ability of small molecule inhibitors to release PD-L1 antibody from inhibiting T lymphocyte activation. The activity data is shown in 1 (with BMS1166 reported by BMS company as a positive control substance).

It can be seen from Figure 1 that compounds 17, 28, 31 and 32 exhibited obvious dose-dependent effects when releasing PD-L1 antibody from inhibiting IFN-Gamma release, and the positive control substance BMS202 could obviously release the inhibitory effect of PD-L1 at a concentration of 100nM ; Among the other three compounds, compound 17 has the strongest ability to release inhibition, and at 10nM, it can obviously release the inhibitory ability of PD-L1 and reactivate the release of IFN-Gamma.

Example 9 Cell uptake experiment

Probe uptake experiments by tumor cells can be used to verify the binding of compounds to tumor cells. In this study, B16F10 melanoma cells were used for evaluating the uptake of radioactive probes. The uptake experiment is briefly described as follows: the cells were cultured to the plateau stage, transferred to a 6-well plate and cultured overnight to allow them to adhere to the wall, and the cells were counted to ensure that about 500,000-1 million cells per well could be used for further experiments. Dilute the prepared radioactive compound 18 ( ⁶⁸ Ga-18) into 1mCi/mL of 20% ethanol-water solution, and transfer 5uL (about 5uCi) to each culture well with a pipette, and after adding the probe At different time points (such as 5min, 15min, 30min, 60min), the cells were separated by centrifugation, and the radioactive counts of the cells and the medium were measured using a gamma counter to obtain the ratio of the radioactive marker uptake by the cells, and the "time-radioactive uptake ratio" was drawn. curve. In the inhibition experiment (Blocking), 1 hour before adding the radioactive probe, a non-radioactive standard (compound 16) was added to the cells in each well to reach a concentration of 1 nM to inhibit probe uptake. The result is shown in Figure 2.

It can be seen from the figure that the uptake of radioactive probes by cells shows a significant time-dependent effect in PD-L1 positive cells, and as time goes on, the uptake gradually reaches saturation. At the same time, the uptake can be inhibited by the added non-radioactive standard, indicating that This intake is optional. However, in cells negative for PD-L1 expression, the uptake rate of radioactivity was low, about 10% of positive cells. In B16F10 cells, the uptake rate was 1.62% at 5 minutes, 4.06% at 15 minutes, 5.33% at 30 minutes, and 6.08% at 60 minutes; The rate was 0.58%, 2.12% in 15 minutes, 2.84% in 30 minutes, and 3.40% in 60 minutes. In U87MG cells, the uptake rate was 1.37% at 5 minutes, 4.20% at 15 minutes, 5.59% at 30 minutes, and 5.86% at 60 minutes; The rate is 0.55%, 2.52% in 15 minutes, 2.83% in 30 minutes, and 3.10% in 60 minutes. In MCF7 cells, the uptake rate was 0.13% at 5 minutes, 0.28% at 15 minutes, 0.43% at 30 minutes, and 0.51% at 60 minutes.

The experimental results showed that in cells positively expressing PD-L1, compound 18 (that is, ⁶⁸ Ga-18) had a higher cell concentration and could be inhibited by non-radioactive compound 17. In cells with low expression of PD-L1, the concentration of radioactivity was lower.

Example 10 PET imaging experiment of tumor model ( ⁶⁸ Ga radioactive markers 18 and 29)

To verify the targeting distribution of radiolabeled probes in living tumor models, a tumor model with high PD-L1 expression (B16F10) was used for PET imaging studies. The experiment is briefly described as follows: a tumor model with high expression (Bl6F10) was used and inoculated in the armpit of nude mice. When the tumor grew to a size of 0.5 cm ³ -1 cm ³ , PET scanning of radioactive markers could be performed. Tumor-nude mice were anesthetized with isoflurane-oxygen mixed gas using a small animal anesthesia machine, and injected into the tail vein at a dose of 0.16mCi/Kg (the maximum injection volume did not exceed 1ml), and Micro PET/CT (IRIS Micro-PET /CT, INVISCAN) static scanning, static acquisition of PET signals at different time points after injection and reconstruction of PET images. In order to quantify the uptake of radiopharmaceuticals in the body, the SUV (Standard Uptake Value) is used to evaluate the uptake of the drug. SUV=radioactive concentration of lesion (kBq/ml)/injected dose (MBq, calculated decay)/body weight (kg), the higher the value, the higher the concentration of radioactive probe at this site. In this experiment, the brain, lung, thigh muscle, tumor, liver and kidney were drawn as the regions of interest, the SUV was calculated by software, and the graph was drawn according to "SUV-injection time".

The PET imaging of Bl6F10 tumor model compound 18 ( ⁶⁸ Ga-18) is shown in Figure 3: 30 minutes after tail vein injection, the tumor imaging was obvious, and some drugs were accumulated in the liver, while the kidney and bladder had strong radioactive concentrations. The heart and lungs are also partially concentrated, and the muscle uptake is low. At 60 minutes, the radioactivity of the tumor was further concentrated, the radioactivity of the liver decreased, the radioactivity concentration of the bladder increased to a certain extent, and the radioactivity of the gastrointestinal tract was distributed to a certain extent. In the time window of ⁶⁸ Ga-18 imaging, there was no obvious radioactivity concentration in the brain, bone, bone joints and muscles. The "Time-SUV" curve of the radiolabel is shown in Figure 3.

The PET imaging of Bl6F10 tumor model compound 29 ( ⁶⁸ Ga-29) is shown in Figure 4. The tumor was clearly visualized 15 minutes after tail vein injection, and a large amount of drug was accumulated in the liver, and radioactivity was concentrated in the kidney and bladder. At the same time, part of the chest area such as the lungs and heart has concentrated radioactivity. At 60 minutes, the drug concentration concentrated in the liver gradually decreased, and the radioactive residue in the urinary system also gradually decreased, and the radioactive concentration in the tumor gradually increased. After 90 minutes of drug injection, the radioactivity concentration in the liver and urinary system further decreased, and the radioactivity in the liver was transferred to the intestinal tract. At the same time, the background radioactivity in the whole body was reduced, and the radioactivity concentration in the tumor remained basically unchanged. During the entire ⁶⁸ Ga-29 imaging time window, there was no obvious radioactivity concentration in the brain and bone; no abnormal concentration of radioactivity was found in other major organs.

It can be seen from Figure 4 that the radioactive markers with this skeleton are mainly metabolized by the kidney and liver, and both organs have high radioactive concentrations; the radioactive uptake in the brain is low; the radioactive uptake in muscles is low; there is some radioactivity in the lungs Uptake (higher than muscle and brain); tumor radioactivity concentration is higher, and the radioactivity concentration can be stably retained (about 6 times that of muscle uptake), which can be used for the detection of PD-L1 positive tumors.

The above descriptions are only preferred embodiments of the present invention, and it should be understood that the present invention is not limited to the forms disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and Modifications can be made within the scope of the ideas described herein, by virtue of the above teachings or skill or knowledge in the relevant art. However, changes and changes made by those skilled in the art do not depart from the spirit and scope of the present invention, and should all be within the protection scope of the appended claims of the present invention.

Claims

A water-soluble methyl benzyl ether derivative is characterized in that the structural formula is as follows:

In the formula, n is an integer of 0 or greater than 0, R is a hydroxyl or a halogen atom, and Linker is a straight-chain unsubstituted alkane or an ethylene glycol side chain.
The water-soluble methyl benzyl ether derivative according to claim 1, wherein said n is 0, 1, 2, 3, 4.
The water-soluble methyl benzyl ether derivative according to claim 1, wherein said R is an aliphatic cyclic amine substituted by a carboxyl group in the adjacent position, and the configuration of the chiral atom is an S configuration.
The water-soluble methyl benzyl ether derivative according to claim 1, wherein said R 2 is a hydroxyl group, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
The water-soluble methylbenzyl ether derivative according to claim 1, wherein the Linker is
Wherein, m=1, 2, 3, 4; p=1, 2, 3, 4.
The application of the water-soluble methylbenzyl ether derivative described in any one of claims 1-5 in tumor imaging.
The preparation method of the water-soluble methyl benzyl ether derivative described in any one of claims 1-5, is characterized in that, comprises the following steps:

1) Compound 1 is provided, and compound 1 and methyl benzyl ether intermediate 2I or 2II generate compound 3I or compound 3II under the action of a base, wherein R 3 is an amino protecting group, and R 4 is a halogen;

2) The compound 3I or compound 3II is condensed with an amine under acidic conditions and then reduced to generate compound 4I or compound 4II;

3) The compound 4I or compound 4II is under the action of an acid, and the R3 amino protecting group is removed to generate compound 5I or compound 5II;

4) Compound 5I or Compound 5II undergoes a condensation reaction to generate Compound 6I or Compound 6II (the water-soluble methyl benzyl ether derivative);
The preparation method according to claim 7, wherein, in step 1), the alkali is one or more of potassium carbonate, sodium carbonate, sodium hydride, DBU, triethylamine; the solvent used is acetonitrile , tetrahydrofuran, ethanol, dimethylformamide, dimethyl sulfoxide in one or more;

And/or, in step 2), the acid is glacial acetic acid; the reagent used for the reduction is sodium borohydride or sodium cyanoborohydride; the solvent used is methanol;

And/or, in step 3), the acid is trifluoroacetic acid or hydrochloric acid; the solvent used is one or more of methanol, ethanol, tetrahydrofuran, and methylene chloride;

And/or, in step 4), the condensation reaction is catalyzed by EDCI-HOBT or HATU-HOBT; the solvent used is one or more of dimethylformamide, dimethylsulfoxide, dimethylacetamide kind.
A positron nuclide probe or a nuclide label prepared from the water-soluble methyl benzyl ether derivative according to any one of claims 1-5.
According to the described positron nuclide probe of claim 9, it is characterized in that, the preparation method of described positron nuclide probe is: take described water-soluble methyl benzyl ether derivative, carry out positron nuclide 68 Ga labeling, or nuclide 177 Lu labeling, to obtain 68 Ga positron nuclide probes and 177 Lu nuclide markers respectively; the structural formulas of the 68 Ga positron nuclide probes and 177 Lu nuclide markers are as follows:
The application of the 68 Ga positron nuclide probe in claim 10 in tumor targeting imaging and/or tumor radionuclide therapy.
The application of the 177 Lu radionuclide label in claim 10 in tumor targeting therapy.