CN114853827A

CN114853827A - Glucose derived ligand compound and preparation method and application thereof

Info

Publication number: CN114853827A
Application number: CN202210624052.0A
Authority: CN
Inventors: 杨大参; 杨凌云; 戴娟; 罗志刚
Original assignee: Nanjing Pet Tracer Co ltd
Current assignee: Nanjing Pet Tracer Co ltd
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-08-05
Anticipated expiration: 2042-06-02
Also published as: CN114853827B

Abstract

The invention provides a glucose derivative ligand compound and a preparation method and application thereof, and relates to the technical field of radiopharmaceutical chemistry. The compound has a structure shown in a formula (II), and the preparation method is simple. The longer linking agent introduced between glucosamine and DOTA makes the complex after radionuclide labeling more stable and can also improve the pharmacokinetic property at the same time. The radionuclide labeled glucose derivative formula (I) provided by the invention has the advantages of high labeling rate, high in vivo and in vitro stability, good targeting property and the like, and can be further used for preparing nuclide diagnosis or therapeutic drugs for tumors.

Description

Glucose derived ligand compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of radiopharmaceutical chemistry, and particularly relates to a glucose-derived ligand compound and a preparation method and application thereof.

Background

Glucose is an important substance required for energy metabolism, and after entering blood circulation, the glucose enters cells in vivo depending on a glucose transporter, and the energy metabolism of tumor cells is abnormal, and the glucose uptake is higher than that of normal cells. Therefore, the glucose and the analogues thereof can be taken up by tumor tissues to carry out radionuclide labeling modification on glucose molecules, and a radioactive glucose metabolism molecular probe is prepared, so that the radioactive glucose metabolism molecular probe is used for targeted diagnosis and treatment of in vivo tumors.

At present, the number of the current day, ¹⁸ F-FDG is the most commonly used glucose-based radiopharmaceutical and is known as a century molecule in the field of molecular imaging. ¹⁸ F-FDG combined with PET/CT has important values in diagnosis, staging, monitoring treatment response and evaluating prognosis. ¹⁸ The strong impact of F-FDG has prompted intensive research into other glucose-based radiopharmaceuticals for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging.

Of these, SPECT imaging studies have been mainly conducted ^99m Tc-labeled glucose and derivatives thereof, currently studied ^99m Tc-labeled substances are ^99m Tc-EC-DG、 ^99m Tc-DTPA-DG、 ^99m TcN-DGDTC and ^99m Tc-CN5DG, and the like. 2003, Yang et al ^99m Tc-EC-DG tumor imaging findings, although ^99m Tumor uptake ratio of Tc-EC-DG ¹⁸ F-FDG is low, however ^99m The ratio of Tc-EC-DG tumor/brain tissue and tumor/muscle tissue are superior to each other ¹⁸ F-FDG (radio, 2003,226 (2)), 465 and 473. At present, the number of the current day, ^99m Tc-EC-DG has entered phase II/III clinical trials. 2012, Yang et al use ¹¹¹ In radiolabelling DOTA-DG, which is slowly taken up In tumors, the metabolic mechanism still needs to be further evaluated (J radio nucleic Ch,2013,295(2), 1371-1375).

PET imaging is also ⁶⁸ Ga and ⁶⁴ studies on Cu-labeled glucose and derivatives thereof. In 2008, Simon R et al ⁶⁴ Cu-labelled glucose derivatives ⁶⁴ Cu-ATSE/A-G studies found that the marker had some tumor uptake, but no uptake was observed in brain and heart, and was not considered a replacement for glucose metabolism (J Nucl Med,2008,49(11), 1862-. 2012, Yang et al will ⁶⁸ The Ga-labeled DOTA-DG research shows that the uptake in tumors is far lower than that in the tumor ¹⁸ F-FDG(AJNucl Med Mol I,2012,2,499–507)。

Therefore, the development of a radiolabeled glucose derivative with good stability, high tumor uptake and good imaging effect is the focus of current research.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a glucose derivative ligand compound and a preparation method and application thereof.

The second objective of the invention is to provide a radionuclide-labeled glucose derivative and application thereof.

The purpose of the invention is realized by the following technical scheme:

a glucose-derived ligand compound having the structure shown in formula II:

wherein m and n are integers of 0 to 10, but not 0 at the same time.

Preferably, m is an integer of 2 to 5, and n is 0 to 5. More preferably, n is 3 and n is 0.

Specifically, the glucose-derived ligand compound has the structure:

the invention also provides a preparation method of the glucose-derived ligand compound II, and the synthetic route is as follows:

wherein R is ₁ Is composed of

Any one of the above; r ₂ Any one selected from Boc, Bn, Cbz, Fmoc and Tos; m and n are as defined for compound I.

The specific reaction steps of the route are as follows:

firstly, protecting the amino group of the compound a by using an amino protecting reagent to obtain a compound b.

In the first step of reaction, the compound a reacts with a protective reagent under alkaline conditions; the protective reagent is selected from any one of Boc anhydride, Bn-Br, Cbz-Cl, Fmoc-Cl and Tos-Cl. And the pH value of the alkaline condition is controlled to be between 8 and 9 by sodium hydroxide or sodium bicarbonate. The reaction temperature is-10 to 30 ℃.

Preferably, the amino protecting reagent is Boc anhydride.

In the second step, the carboxyl group of the compound b is activated with an ester group R ₁ Modification to obtain the compound c.

In the second reaction step, the ester group R is activated ₁ With R ₁ the-OH form participates in the reaction. R ₁ The compound c is generated by the action of-OH and the compound b under the action of DCC; the R is ₁ -OH is selected from any one of N-hydroxysuccinimide (NHS), Tetrafluorophenol (TFP) or pentafluorophenol (PFP). The reaction temperature is-10 to 30 ℃.

Preferably, said R is ₁ -OH is tetrafluorophenol.

And thirdly, condensing the compound c and glucosamine under the condition of organic base to obtain a compound d.

In the third step of the reaction, the organic base is selected from triethylamine or DIPEA; preferably the organic base is triethylamine. The reaction solvent is selected from CH ₃ Cl、CH ₂ Cl ₂ One or more than two of DMF, DMSO, THF or 1, 4-dioxane; preferably, the reaction solvent is CH ₂ Cl ₂ Or DMF. The reaction temperature is 0-30 ℃, and the preferable temperature is 25 ℃.

And fourthly, removing the amino protecting group on the compound d to obtain a compound e.

In the fourth step of reaction, the amino protecting group on the compound d is removed under the acidic condition; the acid is hydrochloric acid or trifluoroacetic acid.

And fifthly, reacting and condensing the compound e and DOTA or DOTA derivative to obtain a compound II.

In the fifth reaction step, DOTA or a DOTA derivative is reacted with compound e directly or in the form of an active ester. Preferably, the DOTA active ester form participates in the reaction; the DOTA active ester is selected from any one of DOTA-TFP, DOTA-PFP or DOTA-NHS; more preferably, the active ester is DOTA-TFP.

The invention also provides a glucose derivative marked by the radioactive nuclide, which is prepared by marking the glucose derivative ligand compound by the radioactive nuclide M and has the structure shown as the formula I:

wherein M is a radionuclide, and M and n are as defined in Compound II.

M is selected from ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁹⁰ Y、 ¹¹¹ In、 ¹³³ La、 ¹³⁵ La、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁶⁶ Ho、 ¹⁷⁷ Lu、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ²⁰³ Pb、 ²¹² Pb、 ²¹³ Bi、 ²²⁵ Ac、 ²²⁷ Th.

Preferably, M is ⁶⁸ Ga、 ¹⁷⁷ Lu or ^133/135 La。

Specifically, the radionuclide-labeled glucose derivative structure is selected from:

the reaction of labeling the glucose-derived ligand compound with the radionuclide M is carried out under acidic and heating conditions.

The acidic condition is pH 4-7; preferably pH 4-5; the acidic reagent is selected from hydrochloric acid or nitric acid. The heating temperature is 50-100 ℃; the heating temperature is preferably 90 ℃.

The invention also provides the application of the radionuclide-labeled glucose derivative in preparing a tumor diagnosis or treatment medicament.

Further, the tumor diagnosis medicament is a PET or SPECT molecular diagnosis imaging agent.

Further, the therapeutic agent is a radionuclide therapeutic agent.

The PET or SPECT molecular diagnostic imaging agent refers to the use of ⁶⁸ Ga、 ¹³³ La or ¹⁷⁷ A Lu-labelled compound of formula (II); the therapeutic drug is prepared by utilizing alpha ray or beta ray released by radionuclide in decay process to closely and accurately kill diseased cell nucleus tissue, such as malignant tumor, and so on ¹³⁵ La or ¹⁷⁷ Lu-labelled compounds of formula (II).

In particular, the amount of the solvent to be used, ⁶⁸ ga or ¹³³ The La marked compound of formula (II) is a PET molecular diagnosis developer; ¹⁷⁷ the Lu-labeled compound shown as the formula (II) is a SPECT molecular diagnostic imaging agent, ¹³⁵ la and ¹⁷⁷ the Lu-labeled compound of formula (II) is a nuclide therapeutic drug.

The invention designs and synthesizes a novel glucose derivative ligand compound, the compound connects glucose derivatives and bifunctional chelating agent DOTA together through a longer linking agent, the raw materials are easy to obtain, and the preparation is simple. The introduction of the linking agent can reduce the steric hindrance of a chelating group directly coupled with a glucose group, and the complex labeled by the metal nuclide is more stable and can improve the pharmacokinetic characteristic. The radionuclide-labeled glucose derivative provided by the invention has the advantages of high labeling rate, high in-vivo and in-vitro stability and good targeting property. Can be used for preparing nuclide diagnosis or treatment medicines for tumors.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 Compounds provided in example 2 ⁶⁸ HPLC charts of Ga-I and compound Ga-I standard products.

FIG. 2 Compounds provided in example 4 ¹³³ HPLC chart of La-I and compound La-I standard substance.

FIG. 3 Compounds provided in example 6 ⁶⁸ Ga-I and ¹⁸ F-FDG in tumor bearing mice in microPET contrast map.

Detailed Description

The features and properties of the present invention are described in further detail below with reference to examples. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a glucose-derived ligand compound II-1, having the formula:

this example also provides a method for preparing the above compound II-1, the synthetic route is as follows:

the specific operation is as follows:

step 1: to a 250ml round-bottomed flask, 10ml of NaOH (1N) and 20ml of 1.4-dioxane were added compound a' (1mmol), and the mixture was cooled to 0 ℃. Di-tert-butyl dicarbonate (Boc anhydride) (1.1mmol) was added to the reaction mixture. Stirred at room temperature for 8 hours. The 1, 4-dioxane was removed under reduced pressure. Followed by saturationKHSO of ₄ The solution was acidified and the resulting solution was extracted with ethyl acetate (3 × 40 mL). The organic layer was collected and rotary evaporated to give material b' in 85% yield.

Step 2: substance b' (1mmol) and 2,3,5, 6-tetrafluorophenol (1.2mmol) were mixed in 10mL of DMF and the mixture was reacted in an ice bath for 0.5 h. Then, DCC (1.5mmol) was added to the mixture and stirred at room temperature overnight. At the end of the reaction, the white precipitate in the reaction mixture was filtered and the filtrate was extracted twice with dichloromethane (50mL) and water (50 mL). The organic layer was collected and rotary evaporated to give crude product. The crude product was purified by silica gel column chromatography to give product c' in 65% yield.

And step 3: compound c' (1mmol) and glucosamine (1.1mmol) were dissolved in 50ml of anhydrous CH ₂ Cl ₂ Then triethylamine (4.8mmol) was added. Stirred at room temperature for 3 h. The solvent was dried by spinning and the crude product was recrystallized from acetone to give the product d' with a yield of 58%.

And 4, step 4: the compound d' is CH ₂ Cl ₂ Dissolving, adding trifluoroacetic acid, and reacting at room temperature for 2 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the solid matter was washed 3 times with ethyl acetate. The crude product obtained was purified by silica gel column chromatography to give the product e' in 87% yield.

And 3, step 3: compound e' (1mmol) and DOTA-TFP (1.1mmol) were dissolved in anhydrous CH ₂ Cl ₂ Then triethylamine (4.8mmol) was added. Stirring at room temperature for 3h, spin-drying the solvent, recrystallizing the crude product with diethyl ether, and purifying by silica gel column chromatography to obtain product II-1 with yield of 60%.

LC-MS：[M/2+H] ⁺ ＝378.18。

¹ HNMR(500MHz,Chloroform-d)δ7.49–7.43(m,1H),6.48(t,J＝5.7Hz,1H),5.22(t,J＝5.9Hz,1H),5.10(d,J＝5.7Hz,1H),5.01(t,J＝7.3Hz,1H),4.95(dd,J＝8.0,0.9Hz,1H),4.49–4.39(m,2H),4.39–4.32(m,1H),3.90–3.81(m,2H),3.84–3.76(m,2H),3.79–3.65(m,6H),3.65–3.53(m,6H),3.47(d,J＝0.7Hz,6H),3.22(s,2H),2.56(d,J＝3.0Hz,16H),2.50(t,J＝7.7Hz,2H)。

Example 2

The present embodiment provides a radionuclideLabeled glucose derivative obtained by Using Compound II-1 prepared in example 1 ⁶⁸ Ga is marked to obtain a complex ⁶⁸ Ga-I, the structure of which is shown as follows:

using 5mL of 0.1M HCl solution ⁶⁸ Ga]GaCl ₂ (300. mu. Ci, 100. mu.L) to a reaction flask, add 350. mu.L of 2M NaOAc buffer solution to adjust pH to 4; was added to an aqueous solution (20. mu.L, 1mg/mL) of the compound II-1 prepared in example 1 and reacted at 90 ℃ for 15 min. Diluting 10mL of the reaction crude product, adsorbing by using a C18 column, and washing 10mL of sterilized water for injection; eluting the C18 column with 0.5mL of 80% ethanol to obtain the product ⁶⁸ Ga-I, radiochemical purity 97%.

And (3) identification:

preparing a standard substance: an aqueous solution (5ml,10mg/ml) of the compound II-1 prepared in example 1 was taken in with GaCl ₃ Mixed with the sodium acetate buffer solution (5ml,20mg/ml, pH 4-4.5), and reacted at 90 ℃ for 24 hours. And separating and purifying by preparative HPLC to obtain the stable metal Ga-marked metal complex standard Ga-I.

LC-MS：[M/2+H] ⁺ ＝411.13。

HPLC: the standard UV peak Rt is 6.128min, and the radioactivity peak Rt is 6.308min, which confirm that the peak positions of the two are consistent. The HPLC spectrum is shown in FIG. 1.

Example 3

Labeling the product ⁶⁸ After Ga-I is placed at room temperature for 4 hours, the radioactive chemical purity is 97 percent; after the strain is respectively placed in mouse serum and physiological saline and incubated in a 37 ℃ water bath for 4 hours, the radioactive chemical purities are respectively 95% and 97%, which shows that the strain has good in vitro stability.

Example 4

This example provides a radionuclide-labeled glucose derivative, and the compound II-1 prepared in example 1 was used ¹³³ Labeling La to obtain complex ¹³³ La-I, the structure of which is shown as follows:

using 1mL of 0.05M HCl solution ¹³³ La]LaCl ₃ (200 μ Ci, 100 μ L) into a reaction flask and the pH of the solution was adjusted to 4.5 with 50 μ L NaOAc buffer (pH 9.0). Was added to an aqueous solution (50. mu.L, 0.4mg/mL) of the compound II-1 prepared in example 1, and reacted at 90 ℃ for 30 min. The reaction solution is passed through a C18 column, and then the C18 column is washed by 10mL of water to obtain a product ¹³³ La-I, radiochemical purity 99%.

And (3) identification:

preparing a standard substance: an aqueous solution (2ml,5mg/ml) of the compound II-1 prepared in example 1 was taken in with LaCl ₃ Mixing the sodium acetate buffer solution (2ml,10mg/ml, pH 4.5), heating to 90 ℃ for reaction for 15h, and separating and purifying by preparative HPLC to obtain the stable metal La marked metal complex standard La-I.

LC-MS：[M/2+H] ⁺ ＝446.12。

HPLC: the standard UV peak Rt is 6.002min, and the radioactivity peak Rt is 6.215min, which prove that the peak positions of the two correspond. The HPLC spectrum is shown in FIG. 2.

Example 5

Labeling the product ¹³³ After the La-I is placed at room temperature for 24 hours, the radioactive chemical purity is 96 percent; after the strain is respectively placed in mouse serum and physiological saline and incubated in a 37 ℃ water bath for 24 hours, the radioactive chemical purities are respectively 95% and 96%, which shows that the strain has good in vitro stability.

Example 6

For example 2 ⁶⁸ Ga-I (150 mu L,200 mu Ci) is subjected to MicroPET imaging research of A549 tumor-bearing mouse model, and the dosage is equal ¹⁸ F-FDG was used as a control and dynamic scan was performed for 60min (5X 1 min-2X 2.5 min-2X 5 min-4X 10min) simultaneously with administration. The impact data was reconstructed using a 3D MAP algorithm, and the region of interest (ROI) was manually drawn on the major organs and tumor. The results of the experiment are shown in FIG. 3, the tumor tissue pairs ⁶⁸ The uptake of Ga-I is higher than ¹⁸ F-FDG, and the analysis result shows that in 5-20min, ⁶⁸ tumor/muscle ratio of Ga-I(TBR) significantly higher than ¹⁸ F-FDG， ⁶⁸ The TBR value of Ga-I is 1.89 +/-0.2, ¹⁸ F-FDG is 1.34 +/-0.16 (n is 7, p)<0.002)， ⁶⁸ Ga-I has a higher TBR value. In addition, with ¹⁸ Liver vs. F-FDG ⁶⁸ The uptake of Ga-I is low.

What has been described above is a specific embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.

Claims

1. A glucose-derived ligand compound having the structure shown in formula II:

wherein m and n are integers of 0 to 10, but not 0 at the same time.

2. The glucose-derived ligand compound according to claim 1, wherein m is an integer of 2 to 5, and n is an integer of 0 to 5.

3. The process for preparing a glucose-derived ligand compound as set forth in claim 1, wherein the synthesis route is as follows:

wherein R is ₁ Is composed of

Any one of the above; r ₂ Any one selected from Boc (tert-butyloxycarbonyl), Bn (benzyl), Cbz (benzyloxycarbonyl), Fmoc (fluorenyl methoxycarbonyl) and Tos (p-toluenesulfonyl); m and n are the same as in claim 1.

4. The process of claim 3, comprising the reaction steps of:

the first step is as follows: using protecting groups R ₂ Protecting the amino group of the compound a to obtain a compound b;

the second step: compounds b using an activated ester group R for the carboxyl group ₁ Modifying to obtain a compound c;

the third step: condensing the compound c and glucosamine under the condition of organic base to obtain a compound d;

the fourth step: removing the amino protecting group on the compound d to obtain a compound e;

the fifth step: and (3) reacting and condensing the compound e with DOTA or DOTA derivative to obtain a compound II.

5. A radionuclide-labeled glucose derivative prepared by labeling the glucose-derived ligand compound of claim 1 with a radionuclide M, having a structure represented by formula I:

wherein M is a radionuclide, and M and n are the same as in claim 1.

6. The radionuclide-labeled glucose derivative according to claim 5, wherein M is selected from the group consisting of ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁹⁰ Y、 ¹¹¹ In、 ¹³³ La、 ¹³⁵ La、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁶⁶ Ho、 ¹⁷⁷ Lu、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ²⁰³ Pb、 ²¹² Pb、 ²¹³ Bi、 ²²⁵ Ac、 ²²⁷ Th is any one.

7. The radionuclide-labeled glucose derivative according to claim 5, wherein the labeling reaction is carried out under acidic and heating conditions.

8. Use of the glucose-derived ligand compound of claim 1 or the radionuclide-labeled glucose derivative of claim 5 for the preparation of a tumor diagnostic or therapeutic drug.

9. The use of claim 8, wherein the diagnostic agent is a PET or SPECT molecular diagnostic imaging agent; the therapeutic agent is a radionuclide therapeutic agent.