KR102190674B1 - Sodium saccharin conjugated ligand, derivatives thereof and process for the preparetion thereof - Google Patents

Abstract

The present invention provides a compound having a selective target for hypoxic tumors by providing a ligand compound-bound saccharin sodium, a derivative thereof, and a method for preparing the same. Specifically, it can be applied to a contrast medium for tumor diagnosis by providing a compound obtained by binding a ligand to sodium saccharin or a derivative thereof.

Description

Sodium saccharin conjugated to a ligand compound, its derivatives, and its manufacturing method {SODIUM SACCHARIN CONJUGATED LIGAND, DERIVATIVES THEREOF AND PROCESS FOR THE PREPARETION THEREOF}

The present invention relates to a substance having a selective targeting action against hypoxic tumors, and to sodium saccharin to which a ligand compound is bound, a derivative thereof, and a method for preparing the same.

Among the radioactive isotopes, the radioactive isotope [ ⁶⁸ Ga] gallium that emits positrons can be obtained by extraction by decay according to the physical half-life (270.95 days) of the parent nucleus species [ ⁶⁸ Ge] germanium, mainly by a generator. Since it exists as a cation, it can be easily coordinated with a ligand having a negative charge (Non-Patent Document 1). The radioactive isotope [ ⁶⁸ Ga] gallium has long been used for diagnosis and treatment of tumors by labeling biologically active molecules used for targeting cancer cells.

About 90% of all currently known cancers are solid cancers. The concentration of oxygen in solid cancer varies greatly, and when exposed to continuous oxygen deficiency, necrosis occurs, but viable tumor cells surround the necrotic tissue, leading to hypoxia. Hypoxia tumor cells actually change their gene transcription patterns in order to overcome the stress caused by lack of oxygen, so they can resist glycolysis, angiogenesis, proliferation, metastasis, invasion and radiation therapy.

Carbonic anhydrase 9 (CA XI; Carbonic Anhydrase IX) is present on the surface of these hypoxic tumor cells, and after the cancer cells use glucose as an energy source in a state of lack of oxygen, pyruvate is produced as a by-product by glycolysis. Is promoted by anaerobic respiration. The lactic acid (Lactic acid) is the accumulation of tumor Inside the pH is lowered rapidly, the carbon dioxide (CO ₂₎ to neutralize it bicarbonate (HCO ₃ ^-) in the neutralization while the survival of the cancer cells by adjusting the pH in the tumor cells to survive Carbonic anhydrase 9 (CA XI; Carbonic Anhydrase IX) plays a large role in maintaining (Non-Patent Document 2).

In addition, carbonic anhydrase 9 (CA XI) is expressed only in hypoxic tumor cells, not normal cells, and saccharin (SAC) inhibits activity by selectively binding to carbonic anhydrase 9 without affecting surrounding normal cells. Therefore, studies on inhibiting proliferation of tumor cells due to these binding properties have been published. Representative research results are Korea University's SW Kim. These are the results of effective cancer cell proliferation inhibition in the evaluation of antiproliferative properties of saccharin against various cancer cell lines and MSCs in the group (Non-Patent Document 3).

In the present invention, it is intended to develop a molecular imaging agent for tumor diagnosis by using the biological properties of the tumor as described above.

1. Velikyan I. 68Ga-Based Radiopharmaceuticals: Production and Application Relationship. Molecules Review. 2015. 12913-12943. 2. Mahon BP, Hendon AM, Driscoll JM, Rankin GM, Poulsen SA, Supuran CT, McKenna R. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors. 2015. 849-854. 3.JS Choi, Sang Yong Park, Man Gil Dong Beom Lee, Tae Bok Lee, Ji Hye Heo, Min Woo Lee, and Suhng Wook Kim. Estimation of Anti-proliferative Activity of Saccharin against Various Cancer Cell Lines and MSCs. 2016. 169-175.

An object of the present invention is to provide a compound in which sodium saccharin (SAC) and a ligand are combined with sodium saccharin (SAC) having a high binding affinity for carbonic anhydrase 9 (CA IX) in hypoxia tumors, and a derivative thereof.

In addition, an object of the present invention is to provide a method for preparing the compound and its derivatives.

In addition, it is an object of the present invention to provide a contrast agent compound comprising the above compounds and derivatives thereof.

In order to solve the above problems, the present invention

It provides a compound in which a ligand compound is bound to sodium saccharin (SAC) or a derivative thereof.

According to an embodiment, the ligand compound is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) Can include.

According to an embodiment, the compound may have a structure such as Formula 1.

[Formula 1]

According to an embodiment, the compound may have a structure such as Formula 2.

[Formula 2]

In Formula 2,

Sodium saccharin (SAC) is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) and ions Bonded, and M ^* is a radioactive isotope, which is coordinated to the NOTA.

According to one embodiment, the radioactive isotope may be ⁶⁸ Ga.

According to another embodiment of the present invention, preparing a sodium saccharin-ligand-binding compound by ion-binding reaction of sodium saccharin (SAC) with a ligand compound, and

It provides a method for preparing a compound or a derivative thereof, comprising the step of reacting the sodium saccharin-ligand-binding compound with a radioactive isotope.

According to an embodiment, the ligand compound is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) Can include.

According to one embodiment, the radioactive isotope may be ⁶⁸ Ga.

According to another embodiment, it is possible to provide a contrast medium including the above compound or a derivative thereof.

In addition, the contrast medium can be used for positron emission tomography (PET) for cancer diagnosis.

Other details of the embodiments according to the present invention are included in the detailed description below.

The present invention relates to sodium saccharin to which a ligand compound is bound, a derivative thereof, and a method for preparing the same, and a compound or a derivative thereof that specifically binds to hypoxic tumors can be provided. In addition, by applying the above compound or a derivative thereof to a contrast medium for cancer diagnosis, it is possible to effectively diagnose tumors by improving discrimination.

1 is a graph showing the result of thin film chromatography (TLC) for the compound according to the present invention.
2 is a positron emission computed tomography (PET/CT) image using the compound according to the present invention.
3 is a PET/CT image for confirming tumor selectivity.
4 is a graph showing the results of distribution in tissues of the compound according to the present invention.

Since the present invention can apply various transformations and have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. The contents of all publications referred to herein by reference are incorporated herein by reference in their entirety. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Hereinafter, a compound or a derivative thereof according to an embodiment of the present invention will be described in more detail.

The present invention provides a compound or a derivative thereof in which a ligand compound is bound to sodium saccharin (SAC).

According to an embodiment, the ligand compound is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) Can include. These metal-affinity ligand compounds can act as a protective agent by reducing cytotoxicity since they can act to release radioactive substances out of the body by preventing the release of radioactive isotopes in the body (Marouan Rami et al., Carbonic). anhydrase inhibitors: Gd(III) complexes of DOTA- and TETA-sulfonamide conjugates targeting the tumor associated carbonic anhydrase isozymes IX and XII, New J. Chem., 2010, 34, 2139-2144; Silvio Aime et al., NMR relaxometric studies of Gd(III) complexes with heptadentate macrocyclic ligands, Magnetic Resonance in Chemistry (1998) Volume: 36, Issue: S1, Pages: S200-S208).

Specifically, the compound according to the present invention may include the following formula (1) compound.

In Formula 1, sodium of saccharin and 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA ) Of the carboxyl group is bonded.

According to one embodiment, the present invention may include a structure such as the following formula (2).

In Formula 2,

Sodium saccharin (SAC) is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) and ions Are combined,

The M ^* is a radioactive isotope and is coordinated to the NOTA.

According to one embodiment, the radioactive isotope may include ⁶⁸ Ga.

The NOTA can easily form a complex by coordinating with a radioactive isotope.

That is, as described above, the present invention can provide a compound in which a ligand is bound to stably bind a radioactive isotope to sodium saccharin (SAC).

According to another embodiment of the present invention,

Preparing a sodium saccharin-ligand-binding compound by ionic bonding reaction of sodium saccharin (SAC) with a ligand compound, and

It provides a method for preparing a compound or a derivative thereof, comprising the step of reacting the sodium saccharin-ligand-binding compound with a radioactive isotope.

According to an embodiment of the present invention, for example, a ⁶⁸ Ga solution is mixed with a buffer solution having a pH of 5 to 6 and a compound of Formula 1, and reacted at, for example, 50 to 100°C, for example, 80°C. It may include forming a compound. The buffer solution may include, for example, sodium acetate, but is not limited thereto.

According to an embodiment, the ligand compound is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) Can include.

Specifically, the manufacturing process according to an embodiment of the present invention is shown in Scheme 1.

[Scheme 1]

As shown in Scheme 1, in the present invention, a ligand was used to coordinate sodium saccharin with a metal radioactive isotope, and 1,4,7-triazacyclononene-1,4,7 as the ligand compound -A compound in which sodium of saccharin is bound to the carboxy group of triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) can be provided.

delete

Sodium saccharin (SAC) has the property of specifically binding to carbonic anhydrase 9 (CA IX), which is not expressed in normal cells and is expressed only in hypoxic tumors. Sodium saccharin (SAC) is labeled as a radioactive isotope through a ligand compound, and the substance according to the present invention can be applied to a contrast agent by targeting to such hypoxic tumors.

Specifically, the contrast agent including the compound according to the present invention or a derivative thereof may be used as a positron emission tomography (PET) contrast agent for hypoxic tumors.

According to one embodiment, the PET contrast agent according to the present invention may be formulated as an injection.

According to an embodiment, when the compound according to the present invention is used as a contrast medium, a non-toxic solution, which is an isotonic component with blood, may be included as a diluent. The diluent may include, for example, sodium chloride solution, potassium chloride solution, sodium hydrogen carbonate solution, Hartmann's solution, glucose solution, glucose physiological saline solution, and the like, and as a specific example, a phosphate buffer solution of pH 7.4, etc. may be included as a diluent. I can.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, these Examples and Experimental Examples are merely illustrative to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Example 1: Preparation of Compound 1 (SAC-NOTA)

In order to prepare the compound of Formula 1 (SAC-NOTA), a K ₂ CO ₃ solution and 1,4,7-triazacyclononene-1,4,7-triacetic acid (1,4,7-triazacyclononane- 1,4,7-triacetic acid, NOTA) 1 mg of a ligand compound was added to dissolve it. In another container, sodium saccharin (SAC) 1.83 mg (5 equivalents) was dissolved in sterile distilled water (DW), and then added to the reaction container. After stirring at room temperature for 24 hours, separation and purification were performed using a column (Pharmacia Fine Chemicals, Sephadex G-25).

Example 2: Compound 2 (SAC-NOTA- ⁶⁸ Ga) Manufacturing

To prepare the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga), gallium was used as a radioactive isotope. Extracted from the ⁶⁸ Ga generator ⁽⁶⁸ Ga-Generator) ⁶⁸ Ga was obtained as a colorless transparent solution was dissolved in dilute hydrochloric acid solution, and then appropriately obtained in accordance with the radiation dose required for the experiment (at least 0.1mCi), suitable for Ga solution ⁶⁸ It was put in a glass container and dried at 100°C while blowing nitrogen. The dried ⁶⁸ Ga was applied in the form of a transparent film on a glass container. Here, 0.5 mL of the compound of Formula 1 (SAC-NOTA) separated and extracted in Example 1 was obtained and added to the reaction vessel coated with the ⁶⁸ Ga, followed by labeling reaction at pH 5 to 6 and 80°C for 10 minutes Made it. Reaction formula according to Example 2 is shown in Reaction Formula 2.

[Scheme 2]

The final material, the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga), was adsorbed on thin layer chromatography (TLC) and sufficiently developed with a 0.1M citrate buffer solution developing solution, and then the radiochemical yield and purity were confirmed. , The results are shown in FIG. 1. 1 is a graph showing the radio-TLC (AR-2000, Eckert & Ziegler) results of the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga).

As shown in FIG. 1, it can be seen that the radiochemical yield and purity are 98% or more on average in the TLC image.

Experimental Example 1: Stability evaluation

In order to evaluate the stability of the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga) prepared in Example 2, stability in human serum and mouse serum was measured. Formula 2 compound (SAC-NOTA- ⁶⁸ Ga) 0.4mCi, 100 μL were mixed with 0.5 mL of human serum and mouse serum, and then cultured at 37°C. Thin layer chromatography (TLC) was performed using a radioactive thin film chromatography (AR-2000, Eckert & Ziegler) equipment at the time period to be measured (1 hour, 3 hours, 6 hours). Table 1 shows the stability measurement results for the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga) in serum by time.

division 1 hours 3 hours 6 hours Formula 2 compound Human serum 100% 100% 98% Mouse serum 100% 98.26% 98.50%

As shown in Table 1, by maintaining the purity of the target compound, the compound of Formula 2, it can be confirmed that the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga) is stable for more than 6 hours in human and mouse serum. .

Experimental Example 2: SAC-NOTA- ⁶⁸ PET image acquisition of Ga

Positron emission tomography (PET) images were taken with small animal pet/city (PET/CT) equipment (INVEON, Simens Medical Solutions). The malignant glioma cell line U87MG cells were implanted subcutaneously into the left shoulder of nude mice to form a tumor model. In the tail vein of the tumor-transplanted mouse (nu/nu nude, 20 g), 0.23mCi of the formula 2 compound (SAC-NOTA- ⁶⁸ Ga) prepared in Example 2 was injected into the tail vein for 30 minutes, 60 minutes and 90 minutes. PET/CT images were acquired by time, and the results are shown in FIG. 2. As shown in Figure 2, it can be observed that the signal is increased at the location of the tumor after administration of the compound of Formula 2 (SAC-NOTA- ⁶⁸ Ga).

Experimental Example 3: SAC-NOTA- ⁶⁸ Confirmation of Ga's tumor selectivity

An experiment was conducted to confirm the selective specificity of the SAC-NOTA- ⁶⁸ Ga prepared in Example 2 for a receptor expressed in tumor cells. Tumors were made by subcutaneous injection of U87MG cells, a malignant glioma cell line, into the shoulder of nude mice. First, sodium saccharin (10 mg/kg) was injected through the tail vein of anesthetized mice for receptor blocking. After 30 minutes, the SAC-NOTA- ⁶⁸ Ga 0.2 mCi was injected to obtain PET/CT images in the same manner as in Experimental Example 2. The results are shown in FIG. 3.

Figure 3 is a PET/CT image taken by injecting SAC-NOTA- ⁶⁸ Ga without blocking receptors present in the tumor (middle) and after blocking receptors present in the tumor by injecting sodium saccharin, SAC-NOTA- ⁶⁸ This is a PET/CT image taken by scanning Ga (right).

According to FIG. 3, it can be seen that the signal at the tumor site where the receptor is blocked is lower than that at the tumor site where the receptor is not blocked. From these results, it can be seen that SAC-NOTA- ⁶⁸ Ga has a selective specificity for a receptor expressed in tumor cells.

Experimental Example 4: SAC-NOTA- ⁶⁸ Check the distribution of Ga within the organization

The distribution of mouse tissues of SAC-NOTA- ⁶⁸ Ga prepared in Example 2 was confirmed. U87MG cells, a malignant glioma cell line, were injected subcutaneously into the shoulder of a nude mouse (weight 20 g) to create a tumor. After injecting 0.01 mCi of SAC-NOTA- ⁶⁸ Ga through the tail vein of a tumor-bearing mouse (n=4), organs (blood, muscle, heart, lung, liver, spleen, stomach) were injected at 30 and 60 minutes, respectively. , Intestines, kidneys, bones, tumors) were excised and the radioactivity of the tissue was measured with a gamma counter, as shown in FIG.

FIG. 4 is a graph showing a comparison of tissue radioactivity ratio (% ID/g) to the injection amount measured in various organs by injecting SAC-NOTA- ⁶⁸ Ga prepared according to an embodiment of the present invention by time.

According to FIG. 4, it can be seen that the level of radioactivity present in the tumor is maintained at 1.52% or more at 30 minutes after injection and 0.69% or more at 60 minutes after injection. In addition, it can be seen that the level of radioactivity in liver tissue is maintained low at 0.99% at 30 minutes and 0.63% at 60 minutes after injection.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the technical field to which the present invention pertains can make various modifications and variations without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

Claims (10)

A compound in which a ligand is bound to sodium saccharin (SAC),
The compound, wherein the compound is the formula 2
[Formula 2]

In Formula 2,
Sodium saccharin (SAC) is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) and ions Are combined,
The M ^* is a radioactive isotope and is coordinated to the NOTA. delete delete delete The method of claim 1,
The compound, wherein the radioactive isotope is ⁶⁸ Ga. 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) ligand compound with sodium saccharin (SAC) Preparing a sodium saccharin-ligand-binding compound of Formula 1 by ionic bonding reaction with; And
A method for preparing a compound of Formula 2, comprising reacting the sodium saccharin-ligand-binding compound with a radioactive isotope:
[Formula 1]

[Formula 2]

In Formula 2,
Sodium saccharin (SAC) is 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) and ions Are combined,
The M ^* is a radioactive isotope and is coordinated to the NOTA. delete The method of claim 6,
The method for producing a compound, wherein the radioactive isotope is ⁶⁸ Ga. A contrast agent comprising the compound according to claim 1 or 5. The method of claim 9,
The contrast agent is to be used in positron emission tomography (PET) for cancer diagnosis.

Images