WO2018079882A1 - Compound for positron emission tomography image of atherosclerotic arterial plaque, and method for producing same - Google Patents

Abstract

The present invention relates to a compound marked with a radioactive isotope used for positron emission tomography (PET) for imaging atherosclerotic arterial plaque, which is a cause of cardio-cerebrovascular diseases; and a production method and an application thereof. More specifically, the present invention relates to a compound, a method for producing the same, and a method for imaging atherosclerotic arterial plaque by using the same, wherein the compound is marked by a radioactive isotope and coupled with 2 to 4 peptides capable of selectively targeting atherosclerotic arterial plaque, and thus an excellent PET image of atherosclerotic plaque in an artery that has a fast flow of blood can be obtained in a short amount of time using a small amount of the compound.

Description

Compound for positron emission tomography imaging of atherosclerotic plaques and preparation method thereof

The present invention relates to a radioisotope-labeled compound for use in positron emission tomography (PET) for imaging atherosclerotic plaques, which is the cause of cardiovascular disease, and to a method and application thereof. Preferably, two to four peptides labeled by F-18 and capable of selectively targeting atherosclerotic plaques combine to provide a good positron emission monolayer for atherosclerotic plaques in arteries with fast blood flow in small amounts and in a short time. The present invention relates to a compound capable of obtaining imaging images, a method of manufacturing the same, and a method of imaging atherosclerotic plaque using the same.

Cardiovascular diseases such as stroke and myocardial infarction are the world's No. 1 mortality rate and the second largest in Korea after cancer, mainly due to atherosclerotic plaques. Atherosclerotic plaques continue to form and develop with age, especially due to poor eating habits, overweight, and lack of exercise. When the atherosclerotic plaque deteriorates, blood is ejected from the thinned vascular membrane and blood clots in the surrounding blood vessels. If this process is repeated, eventually the coagulated blood blocks the blood vessels and obstructs blood flow, and the coagulated blood bursts due to the elevated blood pressure, thereby causing a blood clot in the blood vessels. The thrombus flows along the blood vessels and blocks the thin blood vessels. The blood clots cause myocardial infarction in the coronary arteries and stroke in the brain. Atherosclerotic plaques usually form in the coronary, carotid and cerebral arteries. The reason why these cardiovascular diseases are fatal is that they occur suddenly. Especially, cerebrovascular diseases such as cerebral infarction and cerebral hemorrhage are characterized by no prior symptoms before onset. Radiotracers targeting atherosclerotic plaques can be used as a drug for early diagnosis of cerebrovascular disease, but remain at the basic research stage using animals.

Therefore, the present inventors have developed a compound capable of selectively binding to atherosclerotic plaques and labeled with radioactive isotopes for positron emission, thereby obtaining Positron Emission Tomography (PET) images of atherosclerotic plaques. Confirmed and completed the present invention

[Patent Document] US Patent No. 8,436,140, Japanese Patent Application Publication No. 2012-82166

It is an object of the present invention to provide a compound for positron emission tomography imaging of atherosclerotic plaques.

Another object of the present invention is to provide a method for preparing a compound for positron emission tomography imaging of atherosclerotic plaque.

Still another object of the present invention is to provide a radiopharmaceutical comprising a compound for positron emission tomography imaging of atherosclerosis as an active ingredient and a method for obtaining a positron emission tomography image for atherosclerosis using the same.

In order to achieve the above object, the present invention provides a compound for positron emission tomography imaging of atherosclerotic plaque represented by the following formula (1).

[Formula 1]

In Chemical Formula 1,

Peptides are peptides that specifically bind to atherosclerotic plaques,

n is 2 to 4,

L _1, L ₂ and L ₃ are each independently a hydrocarbon group of C _1-50 containing at least one element selected from the group consisting of nitrogen, hydrogen, halogen and sulfur;

X is a radioisotope.

Preferably, in Formula 1, n peptides may be the same or different from each other, HO ₂ C-Arg-Pro-Pro-Arg-Gln-Cys-NH-, HO ₂ C-Cys-Arg-Pro-Pro Cyclic both ends of -Arg-Gln-Cys-NH- or HO ₂ C-Cys-Arg-Pro-Pro-Arg-Gln-Cys-NH- are bound by disulfide bonds (-SS-) It may be a peptide.

In Formula 1, L ₁ may be the same as or different from each other, and may include one or more triazole groups. Preferably L ₁ is

Can be.

In Formula 1, L ₂ may include one or more triazine groups, preferably L ₂

Can be.

L ₃ may comprise one or more ether groups, preferably L ₃ is

Can be.

The radioisotope X is F-18, Cu-64, Ga-68, Br-77, Zr-89, Y-90, Tc-99m, In-111, I-123, I-124, I-125, It may be selected from the group consisting of I-131 and Lu-177, most preferably using F-18.

Method for producing a compound for positron emission tomography imaging of atherosclerotic plaque according to the present invention,

Binding a linker material (L ₁ ) precursor to a peptide that specifically binds to atherosclerotic plaque (S1);

Preparing a linker material (L ₂ ) precursor (S2);

Binding a radioisotope anion to a linker precursor (L ₃ ) to prepare a compound labeled with a radioisotope (S3);

Bonding the peptide to which the linker material (L ₁ ) precursor is bound and the linker material (L ₂ ) precursor (S4); And

Comprising the step (S5) of combining the compound prepared in the step (S3) with the radioisotope labeled compound prepared in the step (S4).

The L ₁ , L ₂ , L ₃ and radioisotopes mentioned above are as defined above.

In the method for preparing a compound for positron emission tomography imaging of atherosclerotic plaque according to the present invention, the step (S1) of binding the linker material (L ₁ ) precursor to the peptide comprises: (1-a) Stirring the compound having an azido group dissolved in a solvent; (1-b) mixing acetic acid, trifluoroethanol, and dichloromethane with the material prepared in step (1-a), concentrating the filtrate and separating by column chromatography to obtain a compound from which resin has been removed; And (1-c) separating the peptide bound to the linker material (L ₁ ) precursor from the material obtained through the step (1-b). 4-azido butanoic acid etc. can be used as a compound which has an azido group.

In preparing the linker material (L ₂ ) precursor (S2), the (2-a) cyanuric chloride is dissolved in a solvent, alcohol and triethylamine are mixed and stirred, and then the solvent is removed and separated by column chromatography. And (2-b) dissolving the compound obtained through the step (2-a) in a solvent, adding triethylamine and propanediamine to react, removing the solvent, and separating by column chromatography.

In the linker (L ₂ )

Compounds such as may also be combined.

In the step (S3) of preparing a compound labeled with a radioisotope by binding the radioisotope anion to a linker material (L ₃ ) precursor, (3-a) the aqueous solution of radioisotope anion is passed through the Cryptofix-Potassium carbonate solution. To elute the radioisotope anion, and (3-b) mixing and stirring the linker material (L ₃ ) precursor to the solution prepared from (3-a).

The compound for positron emission tomography imaging of atherosclerotic plaques according to the present invention has two to four peptides capable of selectively targeting atherosclerotic plaques, thereby better binding to atherosclerotic plaques in the arteries with rapid blood flow. The radioactive isotopes that emit positrons are labeled to obtain excellent positron emission tomography images of atherosclerotic plaques.

Figure 1 is a comparison of the conventional micro PET / CT image when applying the compound for positron emission tomography imaging according to the present invention.

Figure 2 is a comparison of the conventional technique with the autoradiography image in the arterial vessel 2 hours after the injection of the compound for positron emission tomography imaging according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited thereto.

Preparation Example 1 Preparation of a Linker Compound (L ₂ )

Step 1:

Cyanuric chloride (2.0 g, 10.8 mmol) was dissolved in dichloromethane (20 mL), cooled to 0 ° C., and then propazyl alcohol (0.94 mL, 16.2 mmol) and triethylamine (3.0 mL, 21.6 mmol) were added. After 5 minutes, the temperature was raised to room temperature, the mixture was stirred for 3 hours, and then dichloromethane was removed by concentration under reduced pressure, followed by column chromatography (10% ethyl acetate / hexane) to obtain target compound 2 (0.6 g, 25%). .

Step 2:

Compound 2 (0.6 g, 2.68 mmol) obtained in step 1 was dissolved in dichloromethane (5 mL), cooled to −10 ° C., triethylamine (0.45 mL, 3.21 mmol) was added thereto, and 1,3-propanediamine ( 0.23 mL, 2.68 mmol) was added slowly. After 30 minutes, the reaction was terminated, and dichloromethane was removed by concentration under reduced pressure, followed by column chromatography (8% methanol / dichloromethane) using DIOL silica gel (FUJI SILYSIA Chemical Ltd) to obtain target compound 3 (0.16 g, 23 %) Was obtained.

¹ H NMR (400 MHz, CD ₃ OD) δ 1.87-1.95 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.96 (t, J = 2.4 Hz, 1H), 2.99 (t, J = 2.4 Hz, 1H), 3.50 (t, J = 6.8 Hz, 2H), 4.97 (d, J = 2.4 Hz, 2H), 5.01 (d, J = 2.4 Hz, 2H).

¹³ C NMR (100 MHz, CD ₃ OD) δ 29.9, 38.8, 38.9, 55.8, 56.0, 76.6, 76.7, 78.9, 79.0, 169.3, 172.1, 172.6.

MS (ESI) m / z 261.7 [M + H] ⁺ .

Preparation Example 2 Preparation of Peptide-L ₁ Compound

Step 1:

Dichloromethane was added to the reaction vessel containing Merrifield resin (4, 2.0 g, 0.46 mmol) to which the peptide was bound and swelled for 30 minutes. 4-azido butanoic acid (5, 178 mg, 1.38 mmol) HOBt (1-hydroxybenzotriazole, 186 mg, 1.38 mmol) and TBTU (o- (bentotriazol-1-yl) ) -N, N, N ', N'-tetramethyluronium detrafluoroborate, 443 mg, 1.38 mmol) was dissolved in dimethylformamide (5.0 mL) and stirred for 10 minutes and added to the reaction vessel containing the resin. Next, diisopropylethylamine (0.24 mL, 1.38 mmol) was added thereto, followed by stirring for 3 hours using vortex at room temperature. The resin was transferred to a syringe with polyethylene frit and washed with dimethylformamide, methanol and dichloromethane to obtain resin 6.

Step 2:

Acetic acid (1 mL), trifluoroethanol (1 mL) and dichloromethane (8 mL) were added to the reaction vessel containing the resin 6 obtained in step 1, followed by stirring for 1 hour using vortex at room temperature. The filtrate obtained by filtration was concentrated under reduced pressure, and column chromatography (6% methanol / dichloromethane) was performed using COOH silica gel to give a white solid product 7 (540 mg, 63%).

MS (ESI) m / z 1857 [M + H] ⁺ , 1855 [MH] ⁻ ,

Step 3;

Compound 7 (1.40 g, 0.754 mmol) obtained in step 2 was added to the reaction vessel and trifluoroacetic acid (12 mL), water (1 mL), ethanedithiol (1 mL), and triisopropylsilane (1 mL) were added to the reaction vessel. It was added sequentially and stirred at room temperature for 4 hours. The solvent was removed under reduced pressure, ethyl ether (200 mL) was added to the reaction mixture, followed by centrifugation to remove the white precipitate, and the remaining solution was obtained using compound 8 (320 mg, 49%) using a high performance liquid chromatography.

Preparation Example 3 Preparation of Compound 9

Compound 3 (15 mg, 0.058 mmol) obtained in Preparation Example 1 was dissolved in ethanol (2.0 mL), and then Compound 8 (150 mg, 0.173 mmol) dissolved in water (2.0 mL) was added thereto. A mixed aqueous solution of 0.2 M copper sulfate (0.058 mL) and 0.2 M sodium ascorbate (0.087 mL) was added thereto, followed by stirring at room temperature for 3 hours. Separation by high performance liquid chromatography gave a white solid compound 9 (45 mg, 39%).

MS (ESI) m / z 997 [M + 2H] ²⁺ , 665 [M + 3H] ³⁺

Preparation Example 4 Preparation of Compound 11

Compound 9 (35 mg, 0.018 mmol) obtained in Preparation Example 3 was dissolved in 0.1 M aqueous sodium bicarbonate solution (2.0 mL), and compound 10 (25 mg, 0.053 mmol) dissolved in acetonitrile (1.0 mL) was added thereto. The reaction mixture was stirred overnight and then separated by high performance chromatography to give white solid compound 11 (10 mg, 24%).

Example 1

Aqueous [ ¹⁸ F] fluoride anion aqueous solution (103 mCi) produced from cyclotron was captured by passing through chromafix ^® , washed with ethanol (1.0 mL), and then purified by Cryptofix-Potassium (Kryptofix [2.2.2]. ] -K ₂ CO ₃ ) was passed through ethanol (1.0 mL) in which [ ¹⁸ F] fluoride anion was eluted into a vial vessel. The solution was stirred well at 100 ° C. and blown with nitrogen to remove the ethanol solvent. Then compound 12 (3 mg, 0.010 mmol) was added, t-amyl alcohol (0.5 mL) was added, the lid was well covered, and the mixture was stirred at 120 ° C. for 10 minutes. I was. After blowing nitrogen at 100 ° C. to remove t-amyl alcohol, the remaining material was dissolved in acetonitrile (1.0 mL) and separated by high performance liquid chromatography. Distilled water (10 mL) was added to a solution containing F-18-labeled compound [ ¹⁸ F] 13 (8 mL) and passed through a C-18 SePak ^® cartridge to capture compound [ ¹⁸ F] 13, followed by distilled water ( 5.0 mL). Nitrogen was blown to remove water from inside the C-18 SePak ^® cartridge and then slowly passed through acetonitrile (0.5 mL) to elute Compound [ ¹⁸ F] 13 (65 mCi).

Example 2

The acetonitrile solution containing Compound [ ¹⁸ F] 13 obtained in Example 1 was placed in a vial vessel containing Compound 11 (1.0 mg) and stirred at 40 ° C. for 30 minutes. Distilled water (1 mL) was added to the reaction mixture, followed by injection into a high performance liquid chromatography to separate [ ¹⁸ F] 1a. [ ¹⁸ F] 1a of distilled water (10 mL) was added to the solution, which was then passed through a C-18 SePak ^® cartridge, captured, washed with distilled water (3.0 mL) and blown with nitrogen into the C-18 SePak ^® cartridge. Drained off. Ethanol (1.0 mL) was passed through a C-18 SePak ^® cartridge to elute compound [ ¹⁸ F] 1a and blow nitrogen to remove ethanol as much as possible. The final compound [ ¹⁸ F] 1a obtained was 1.45 mCi (2.6%), and diluted with an appropriate amount of saline solution for animal experiments using mice.

Example 3: Compound [ ¹⁸ F] 1a Atherosclerotic Plaque Micro PET / CT Imaging

The F-18 labeling compound [ ¹⁸ F] 1a (0.300-500 mCi) prepared in Example 2 was intravenously injected into ApoE (− / −) mice, and the image was then obtained through a micro PET / CT scanner for 90 minutes.

Comparative Example 1: Atherosclerotic plaque micro PET / CT image of [ ¹⁸ F] FDG (2- [ ¹⁸ F] fluoro-2-deoxy-glucose)

[ ¹⁸ F] FDG (0.500mCi) was intravenously injected into ApoE (-/-) mice and images were obtained for 90 minutes using a micro PET / CT scanner.

1 is a micro PET / CT image of an ApoE (− / −) mouse injected with [ ¹⁸ F] FDG and [ ¹⁸ F] 1a of the present invention, respectively, with arrows showing atherosclerotic plaques. In the case of [ ¹⁸ F] FDG, the intake of the heart and other organs is high, whereas in the case of [ ¹⁸ F] 1a of the present invention, the intake of organs and tissues other than the atherosclerotic plaque is almost absent.

FIG. 2 is an autoradiography image obtained by injecting [ ¹⁸ F] FDG and [ ¹⁸ F] 1a of the present invention into ApoE (− / −) mice, respectively, and detaching the arterial tube 2 hours later. It can be seen that a lot of intake occurred in the portion of the aortic arch (aortic arch) is formed a lot.

Claims (15)

1. Compound for positron emission tomography imaging of atherosclerotic plaques represented by Formula 1 below.

   [Formula 1]

   

   In Chemical Formula 1,

   Peptides are peptides that specifically bind to atherosclerotic plaques,

   n is 2 to 4,

   L _1, L ₂ and L ₃ are each independently a hydrocarbon group of C _1-50 containing at least one element selected from the group consisting of nitrogen, hydrogen, halogen and sulfur;

   X is a radioisotope.
2. The method according to claim 1,

   N peptides may be the same or different from each other, HO ₂ C-Arg-Pro-Pro-Arg-Gln-Cys-NH-, HO ₂ C-Cys-Arg-Pro-Pro-Arg-Gln-Cys-NH Atherosclerosis characterized in that both ends of cysteine (Cys) of HO ₂ C-Cys-Arg-Pro-Pro-Arg-Gln-Cys-NH- are cyclic peptides bound by disulfide bonds (-SS-) Compound for positron emission tomography imaging of cure plaques.
3. The method according to claim 1,

   Wherein L ₁ may be the same or different from each other, the compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it comprises one or more triazole groups.
4. The method according to claim 1,

   L ₂ is a compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it comprises at least one triazine group.
5. The method according to claim 1,

   L ₃ is a compound for positron emission tomography imaging of atherosclerotic plaques, characterized in that it comprises one or more ether groups.
6. The method according to claim 1,

   X is F-18, Cu-64, Ga-68, Br-77, Zr-89, Y-90, Tc-99m, In-111, I-123, I-124, I-125, I-131 And Lu-177 compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it is selected from the group consisting of.
7. The method according to claim 6,

   X is F-18 compound for positron emission tomography imaging of atherosclerotic plaque.
8. Binding a linker material (L ₁ ) precursor to a peptide that specifically binds to atherosclerotic plaque (S1);

   Preparing a linker material (L ₂ ) precursor (S2);

   Binding a radioisotope anion to a linker precursor (L ₃ ) to prepare a compound labeled with a radioisotope (S3);

   Bonding the peptide to which the linker material (L ₁ ) precursor is bound and the linker material (L ₂ ) precursor (S4); And

   Positron emission tomography images of atherosclerotic plaques represented by Formula 1 below, including the step (S5) of binding the compound labeled with the radioisotope prepared in step (S3) to the compound prepared in step (S4). Method for preparing the compound.

   [Formula 1]

   

   In Chemical Formula 1,

   Peptides are peptides that specifically bind to atherosclerotic plaques,

   n is 2 to 4,

   L _1, L ₂ and L ₃ are each independently a hydrocarbon group of C _1-50 containing at least one element selected from the group consisting of nitrogen, hydrogen, halogen and sulfur;

   X is a radioisotope.
9. The method according to claim 8,

   The peptide is HO ₂ C-Arg-Pro-Pro-Arg-Gln-Cys-NH-, HO ₂ C-Cys-Arg-Pro-Pro-Arg-Gln-Cys-NH- or HO ₂ C-Cys-Arg Compounds for positron emission tomography imaging of atherosclerotic plaques characterized in that both cysteines (Cys) of -Pro-Pro-Arg-Gln-Cys-NH- are cyclic peptides bound by disulfide bonds (-SS-) Manufacturing method.
10. The method according to claim 8,

    The linker material (L ₁ ) precursor is 4-azido butanoic acid production method of the compound for positron emission tomography imaging of atherosclerotic plaque.
11. The method according to claim 8,

    The linker material (L ₂ ) precursor is a method for producing a compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it comprises at least one triazine group.
12. The method according to claim 8,

    The linker material (L ₃ ) precursor is a method for producing a compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it comprises at least one ether group.
13. The method according to claim 8,

    The radioisotope is F-18, Cu-64, Ga-68, Br-77, Zr-89, Y-90, Tc-99m, In-111, I-123, I-124, I-125, I A method for preparing a compound for positron emission tomography imaging of atherosclerotic plaque characterized in that it is selected from the group consisting of -131 and Lu-177.
14. Radiopharmaceuticals for imaging positron emission tomography of atherosclerotic plaques comprising the compound for positron emission tomography imaging of claims 1 to 7 as an active ingredient.
15. A method of obtaining positron emission tomography images of atherosclerotic plaques using the radiopharmaceuticals for imaging positron emission tomography.

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