KR101494429B1 - Glucosamine-containing cyclo RGDfK derivatives conjugated with NODAGA, a process for the preparation thereof, and a nuclear medicine imaging agent and an anticancer agent comprising the same - Google Patents

Abstract

The present invention relates to a novel nuclear medicine imaging contrast agent or radioactive cancer therapeutic compound of formula (2) containing NODAGA as a ligand to which two cyclic RGDfK peptides are introduced in one molecule and which contains glucosamine and lysine and binds radioactive isotopes, (1), a process for their preparation, and a compound of formula (2).

Description

NODAGA-labeled glucosamine-containing cyclo-RGDfK derivatives, a method for producing the same, a nuclear medicine imaging contrast agent containing the same, and a therapeutic agent for cancer, wherein the glucosamine-containing cyclo-RGDfK derivatives are conjugated with NODAGA, a process for the preparation thereof and a nuclear medicine imaging agent and an anticancer agent comprising the same}

The present invention relates to a novel compound which can be used as a nuclear medicine imaging agent for cancer diagnosis or a cancer treatment agent, a preparation intermediate thereof, a method for producing the same, a nuclear medicine imaging contrast agent containing the novel compound, will be.

Positron Emission Tomography (PET) is a system that injects physiologically, chemically, and functionally images into a specific cell or organ by injecting a drug labeled with a radioactive isotope that emits positron into the body, Is a nuclear medicine test. In addition to PET, Single Photon Emission Computed Tomography (SPECT) is also widely known as a useful method of nuclear medicine imaging.

PET and SPECT are now known to be useful for diagnosing various cancers, evaluating recurrence, and evaluating treatment effects. In recent years, the importance of nuclear medicine imaging devices in clinics has been increasing worldwide. In particular, the radiation emitted from specific radioactive isotopes in cells or organ is easily detectable by PET and SPECT imaging devices and is highly sensitive. The high sensitivity of the PET and SPECT imaging devices by these radiopharmaceuticals allows imaging of tumor receptor, metabolism, and various biological functions and information associated therewith (Non-Patent Document 1).

Most of the radiopharmaceuticals used in PET contain radioactive isotopes such as [ ¹⁸ F] fluoride, [ ¹¹ C] carbon (carbon) in the molecules participating in specific metabolism in vivo such as water, oxygen, carbon dioxide, glucose, ), [ ¹⁵ O] oxygen, or [ ¹³ N] nitrogen. For example, [C ¹¹ ] methionine, [ ¹³ N] ammonia, H ₂ ¹⁵ O, [ ¹⁸ F] FDG (Fluoro-deoxy glucose) and the like can be given.

In addition, metallic radioisotopes emitted by radiation are widely used in nuclear medicine. The metallic radioisotopes used for PET diagnosis are Cu-64, Ga-68, Zr-89 and Y-86. The metallic radioactive isotope used is In-111, Tc-99m, and the like. In addition to these nuclear medicine imaging diagnoses, Cu-67, Y-90, Lu-177 and the like are known as therapeutic radioisotopes that can be directly used for the treatment of tumors (Non-Patent Document 2).

Among them, metallic radioisotope [ ⁶⁴ Cu] copper (positron emission rate, about 34%), which has recently been developed in the field of nuclear medicine, is irradiated on a concentrated Ni-64 target by irradiating a cyclotron- (p, n) Cu-64 nuclear reactions, and [ ⁶⁴ Cu] copper with high radiochemical purity is required for clinical use. Radioactive isotope [ ⁶⁴ Cu] copper is usually present as a divalent cation in an aqueous solution and can easily coordinate with a chelate with a negative charge. In addition, the radioactive isotope [ ⁶⁴ Cu] copper has a relatively long half-life of 12.7 hours physically, and the advantage of obtaining a long-lived image after injection that could not be obtained with the conventional half-life radioactive isotopes within 2 hours (Non-Patent Document 3).

Recently, there have been reported many cases in which radioactive isotopes [ ⁶⁴ Cu] copper are labeled as biological molecules ingested into cancer cells and used as radiopharmaceuticals for diagnosing or treating tumors. These radiopharmaceuticals for tumor imaging are important tools for understanding biological phenomena and for accurate diagnosis of various tumors, i) high selectivity of radioactive medicines for the tumor, ii) fast intake and equilibration time after injection, iii) And duration (see Non-Patent Document 4).

On the other hand, there is a receptor on the surface of the cell, which is responsible for informing the cell of the signal transmitted from outside the cell. Cells are exposed to a variety of external signals, and the types of receptors vary. Integrin protein, one of these receptors, is a protein complex composed of α and β subunits involved in cell adhesion or migration, mediating interactions between cells. It is also involved in the early development of the cell tissue. The function of the integrin protein is required for inflammation, blood coagulation, cell movement, etc., and various kinds exist depending on the cell. Among them, α _ν β ₃ integrin, which is called vitronection receptor, is not expressed in normal vascular endothelial cells but is expressed at the time of neovascularization of cancer cells. RGD (R: arginine, G: glycine, D: aspartic acid) sequence ≪ / RTI > RGD, a tumor-selective peptide, has been reported to be applied to tumor treatment because of its selective binding to tumor blood vessels (Non-Patent Document 5).

The Peter S. Conti group of the University of Southern California has labeled the radioactive isotope, Cu-64, for the tumor targeting peptide, RGD, to obtain an effective tumor PET image (non-patent document 6).

In addition to the RGD, studies on tumor selective peptides binding to the? _V ? ₃ integrin have been actively conducted. cyclo-RGD, RGDyK, RGDfK, RGDfV, and the like, or a combination of two RGDs or a plurality of RGDs (Non-Patent Document 7). Studies have been conducted on the synthesis of gluco-RGD derivatives by introducing monosaccharides such as glucose into the carbon chains linked to RGD in an effort to prevent the accumulation of certain organs (e.g., liver) in the body of radioactive pharmaceuticals injected into the body Patent Document 8).

In order to efficiently and stably label metallic radioactive isotopes such as Cu-64, various chelators in the form of NOTA, DOTA and NODAGA have been studied (Non-Patent Document 9).

1. Richard J. Kowalsky, Steven W. Falen, Radiopharmaceuticals in Nuclear Medicine. American Pharmacists Association. 2rd, 2004 2. Ji-Ae Park, Jung Young Kim, Recent Advances in Radiopharmaceutical Applications of Matched-Pair Radiometals. Current Topcs in Medicinal Chemistry. 2013; 13 (4): 458-469 3. Thaddeus J. Wadas, Edward H. Wong, Gary R. Weisman, Carolyn J. Anderson, Coordinating Radiometals of Copper, Gallium, Indium, Yttrium, and Zirconium for PET and SPECT Imaging of Disease. Chemical Review. 2010, 110, 2858-2902 4. Carolyn J. Anderson, Riccardo Ferdani, Copper-64 Radiopharmaceuticals for PET Imaging of Cancer: Advances in Preclinical and Clinical Resaerch. Cancer Biotherapy and Radiopharmaceuticals. 2009, 24, 379-393 5. Arab W, Pasqualine R, Ruoslahti E, Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 1998 Jan 16; 279 (5349): 377-80 6. Chen X, Park R, Tohme M, Shahinian AH, Bading JR, Conti PS. MicroPET and autoradiographic imaging of breast cancer alpha v-integrin expression using 18F- and 64Cu-labeled RGD peptides. Bioconjugate Chem. 2004, 15, 41-49 7. Cai W, Sam Gambhir S, Chen X. Multimodality tumor imaging targeting integrin. Biotechniques. 2005 Dec; 39 (6 Suppl): S14-25 8. Kyung-Ho Jung, Kyung-Han Lee, Jin-Young Paik, Bong-Ho Ko, Jun-Sang Bae, Byung Chul Lee, Hyun Ju Sung, Dong Hyun Kim, Yearn Seong Choe and Dae Yoon Chi. Favorable Biokinetic and Tumor-Targeting Properties of 99mTc-Labeled Glucosamine RGD and Effect of Paclitaxel Therapy. J Nucl Med 2006; 47: 2000-2007 9. Dumont RA, Deininger F, Haubner R, Maecke HR, Weber WA, Fani M. Novel (64) Cu- and (68) Ga-labeled RGD conjugates and facile radiosynthesis. J Nucl Med. 2011 Aug; 52 (8): 1276-84

The present inventors have studied for the development of an effective nuclear medicine imaging contrast agent and a therapeutic agent for nuclear medicine and as a result have found that not only tumor selectivity is high but also the ability to bind to radioactive isotopes sufficiently to be stable in the blood for the time required for nuclear medicine imaging and cancer treatment And to develop new RGD peptide derivatives that can act as an antioxidant.

Therefore, an object of the present invention is to provide a method for producing a nuclear medicine image contrast agent for cancer diagnosis, which is capable of stably binding with radioactive isotopes in blood, Or RGD peptide derivatives which are intermediates for the production of cancer therapeutic agents.

Another object of the present invention is to provide a novel compound which can be used as a nuclear medicine imaging contrast agent or cancer treatment agent in which the RGD peptide derivative is bound to a radioactive isotope.

It is another object of the present invention to provide a process for preparing the above compounds.

It is another object of the present invention to provide a PET contrast agent comprising the novel compound.

It is still another object of the present invention to provide a SPECT contrast agent comprising the novel compound.

It is still another object of the present invention to provide a therapeutic agent for cancer comprising the novel compound.

In order to achieve the above object, one aspect of the present invention provides a compound of formula 1:

[Chemical Formula 1]

In Formula 1,

는

를 의미하고,

The

Lt; / RTI >

n is an integer from 3 to 6;

Another aspect of the present invention provides a compound of formula 2:

(2)

및 n은 상기 화학식 1에서 정의된 바와 같으며, In the formula (2)

And n is as defined in Formula 1,

The M ^* may be selected from the group consisting of Cu-64 (II), Cu-67 (II), Ga-68 (III), Zr-89 (IV), Y- III), Tc-99m (I-VII) and In-111 (III), which are coordinately bound to NODAGA in the above formula.

Another aspect of the present invention provides a process for preparing a compound of formula (1), comprising subjecting a compound of formula (3) to a peptide coupling reaction with a NODAGA-NHS ester:

(3)

In Formula 3, n is an integer of 3 to 6.

In another aspect of the present invention, there is provided a process for preparing a compound represented by the formula (I), which comprises reacting a compound represented by the above formula (1) with at least one compound selected from the group consisting of Cu-64 (II), Cu- (M ^* ) selected from -90 (III), Lu-177 (III), Tc-99m In the presence of a base.

Another aspect of the present invention provides a PET contrast agent comprising the compound of formula 2:

(2)

In the formula (2)

는

를 의미하고,

The

Lt; / RTI >

n is an integer from 3 to 6,

The M ^* is a metallic radioisotope selected from Cu-64 (II), Ga-68 (III), Zr-89 (IV), and Y-86 (III) and coordinated to NODAGA.

Another aspect of the present invention provides a SPECT contrast agent comprising a compound of formula 2:

(2)

In the formula (2)

는

를 의미하고,

The

Lt; / RTI >

n is an integer from 3 to 6,

The M ^* is a metallic radioactive isotope selected from In-111 (III) and Tc-99m (I-VII) and coordinated to NODAGA.

Another aspect of the present invention provides a cancer therapeutic agent comprising a compound of the following formula:

(2)

In the formula (2)

는

를 의미하고,

The

Lt; / RTI >

n is an integer from 3 to 6,

The M ^* is a metallic radioisotope selected from Cu-67 (II), Y-90 (III), and Lu-177 (III) and is coordinated to NODAGA.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to in this specification are incorporated herein by reference in their entirety.

The present inventors have developed an RGD peptide derivative of the above formula (1) containing NODAGA as a ligand that incorporates two cyclic RGDfK peptides in one molecule and contains glucosamine and lysine and binds radioactive isotopes. The RGD peptide derivative of Formula 1 may be stably present in the blood for about 24 hours without being rapidly metabolized in the body due to the glucosamine structure when bound to a radioactive isotope for nuclear medicine imaging (Experimental Example 1) (Experimental Example 2). In addition, the cRGDyK (cyclo-Arg-1) -dependent cytotoxicity was observed in the tumor region after the intravenous injection of the substance conjugated with the radioactive isotope to the tumor, Gly-Asp-Tyr-Lys) was first administered to block the vitronectin receptor, and then the nuclear magnetic resonance imaging was taken after the administration of the above substance. As a result, the tumor signal was lower (Experimental Example 3). Therefore, it has been confirmed that when the RGD peptide derivative of Formula 1 is combined with a radioactive isotope for nuclear medicine image, it can be used as a nuclear medicine imaging contrast agent effective for cancer diagnosis. In addition, when the RGD peptide derivative of Formula 1 is bound to a radioactive isotope for nuclear medicine treatment, it can be used as an effective cancer treatment agent.

Thus, in one aspect, the present invention provides a compound of formula 1:

[Chemical Formula 1]

In Formula 1,

는

를 의미하고,

The

Lt; / RTI >

n is an integer from 3 to 6;

Specifically, n is 3 or 4, and more specifically, 3.

In the present specification, the compound of Chemical Formula 1 is abbreviated as cRGDfK ₂ -NODAGA, and cRGDfK ₂ -NODAGA of Chemical Formula 1 is significantly higher in tumor selectivity than conventional tumor selective RGD peptide derivatives.

Since the compound of Formula 1 can be efficiently and stably bound to a radioactive isotope for nuclear medicine imaging and can be used as a PET or SPECT contrast agent compound, the compound of Formula 1 is an intermediate for preparing PET contrast agent or SPCT contrast agent compound have.

Accordingly, in another aspect, the invention provides compounds of formula 2:

(2)

In Formula 2,

및 n은 상기 화학식 1에서 정의한 바와 같고,

And n are the same as defined in Formula 1,

The M ^* may be selected from the group consisting of Cu-64 (II), Cu-67 (II), Ga-68 (III), Zr-89 (IV), Y- (III), Tc-99m (I-VII) and In-111 (III), which are coordinately bound to NODAGA in the structure of Formula 2 above.

In the compound of Formula 2, n is specifically 3 or 4, and more specifically, 3. The M ^* is more specifically Cu-64.

In the formulas 1 and 2, cRGDfK is a cyclic compound having an amino acid sequence of Arg-Gly-Asp-D-Phe-Lys formed by a peptide bond and has a property of binding to a bitonectin receptor found in tumor cells.

The RGD peptide derivative of Formula 2 is not rapidly metabolized in the body due to the glucosamine structure and can stably exist in the blood for about 24 hours (Experimental Example 1). After injection of the substance of Formula 2 into the tumor-bearing mouse, (Experimental Example 2). Further, cRGDyK (cyclo-Arg-Gly-Asp-Tyr-Lys) was first administered to block the vitronectin receptor, 2, the signal of the tumor site was lower than that of the tumor not blocking the vitronectin receptor (Experimental Example 3). Accordingly, it has been confirmed that the substance of the above formula (1) can be used as an effective nuclear medicine imaging contrast agent and an anticancer drug for nuclear medicine treatment which are stable in the body and highly selective for tumor.

As used herein, the term "complex" refers to a group of atoms consisting of several different atoms, ions, molecules or atoms centered on one or more atoms or ions in a directionally coordinated manner . Here, atomic ion molecules (chelator) or atomic group coordinated to a central atom or ion are called a ligand.

As used herein, the term NODAGA is an abbreviation for 1,4,7-triazacyclononane-1-glutaric acid-4,7-triacetic acid and is a metal-compatible ligand compound. The ligand acts as a ligand of a metal affinity, and thus the radioactive metals Cu-64, Cu-67, Ga-68, Zr-89, Y-86, Y-90, Lu-177 and In- (Marouan Rami et al., Carbonic anhydrase inhibitors: Gd (R)), and can act as a chemical protective agent against radiation damage because it can reduce the cytotoxicity of radioactive materials due to the action of removing radioactive materials from the body 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).

As used herein, the term "glyco-dimeric cyclo-RGDfK" refers to a compound containing two cyclic compounds, lysine and glucosamine, in which the amino acid sequence of Arg-Gly-Asp-D-Phe- 1 is a compound before the lysine structure is bound to NODAGA, i.e., a compound of the following formula (3).

(3)

In Formula 3, n is an integer of 3 to 6.

The glyco-dimeric cyclo-RGDfK compound of formula (3) can be coupled with the NODAGA to form a compound of formula (1) via lysine. The compound of formula (1) is coordinately bound to radioactive isotopes by NODAGA, A compound of formula (2) that can be used as a therapeutic agent for radioactive cancer can be formed.

Α _ν β ₃ integrin, which is not expressed in normal vascular endothelial cells and expressed during neovascularization of cancer cells, is characterized by binding to a cyclopeptide having an RGD (Arg-Gly-Asp) sequence. The glyco-dimeric cyclo-RGDfK compound of Formula 3 has a significantly higher binding affinity for cancer cells than conventional RGD derivatives, and thus can form a nuclear medicine imaging contrast agent and an anticancer agent with significantly increased targeting effect on cancer tissues.

In another aspect, the present invention provides a process for preparing a compound of formula (1), which comprises reacting a compound of formula (3) with a NODAGA-NHS ester in a peptide coupling reaction:

(3)

In Formula 3, n is an integer of 3 to 6.

The NODAGA-NHS ester refers to NODAGA- (N-hydroxysuccinimide) ester. In the above process, the amine group at the lysine structural end of the formula 3 reacts with NODAGA-NHS ester to form a peptide bond, ≪ / RTI > can be formed by any method capable of forming a < RTI ID = 0.0 > Also, the reaction solvent can be any solvent that does not inhibit the peptide-binding reaction of the compound of formula (III) with NODAGA-NHS ester. For example, DMF, DMSO, CH ₃ CN, water, methanol, ethanol and the like can be used.

(III), Zr-89 (IV), Y-86 (III), Cu-67 (II) (M ^* ) selected from the group consisting of Y-90 (III), Lu-177 (III), Tc-99m A method for producing the compound is provided.

In the method of preparing the compound of Formula 2, the radioactive isotope bonds with the ligand NODAGA present in the compound of Formula 1 to form the compound of Formula 2. [ In the step of preparing the compound of Formula 2, any solvent which does not inhibit the reaction may be used as the reaction solvent.

In the method for preparing the compound of Formula 2, the compound of Formula 2 can be produced by mixing a solution (pH 5 to 6) of the radioactive isotope in a buffer solution with a solution of the compound of Formula 1 at about 40 to 60 ° C have. As the buffer solution, for example, sodium acetate buffer solution and the like may be used, but the present invention is not limited thereto. The product thus obtained can be removed using any method known in the art, and preferably the impurities can be removed by HPLC.

An embodiment of the process for preparing the compound of formula (2) according to the present invention is shown in the following reaction formula (1).

[Reaction Scheme 1]

The compound of Formula 2 may be coordinately bound to a radioactive isotope via NOTA and may be conjugated to a PET contrast agent, a SPECT contrast agent, or the like depending on the kind of a radioisotope according to a radioactive isotope for nuclear medicine imaging or a radioactive isotope for cancer treatment. Can be effectively used as a cancer therapeutic agent.

Accordingly, in another aspect, the present invention provides a PET contrast agent comprising the compound of Formula 2:

(2)

In the formula (2)

는 상기 화학식 1에서의 정의한 바와 같고,

Is as defined in Formula 1,

n is an integer from 3 to 6,

The M ^* is a metallic radioisotope selected from Cu-64 (II), Ga-68 (III), Zr-89 (IV), and Y-86 (III) and coordinated to NODAGA.

In another aspect, the present invention provides a SPECT contrast agent comprising a compound of formula 2:

(2)

In the formula (2)

는 상기 화학식 1에서의 정의한 바와 같고,

Is as defined in Formula 1,

n is an integer from 3 to 6,

The M ^* is a metallic radioactive isotope selected from In-111 (III) and Tc-99m (I-VII) and coordinated to NODAGA.

In another aspect, the present invention provides a cancer therapeutic agent comprising a compound represented by the following formula (2): < EMI ID =

(2)

In the formula (2)

는 상기 화학식 1에서의 정의한 바와 같고,

Is as defined in Formula 1,

n is an integer from 3 to 6,

The M ^* is a metallic radioisotope selected from Cu-67 (II), Y-90 (III) and Lu-177 (III) and is coordinately bound to NODAGA.

According to the following experimental example, the stability of the compound of formula (2) in serum was measured and it was found that the compound was stable without decomposition for more than one day (Experimental Example 1). In addition, the compound of formula (2) was administered to a tumor-bearing mouse and the PET / CT imaging system for small animals was observed to show an increased signal at the tumor site (Experimental Example 2). Thus, the compound of formula 2 can be used as a nuclear medicine imaging contrast agent or cancer treatment agent that is specifically selective for cancer. In addition, mice with tumors were first administered cRGDyK (cyclo-Arg-Gly-Asp-Tyr-Lys) to block the vitronectin receptor and then injected with the compound of formula 2, The signal of the tumor site was lower than in the case of not blocking (Experimental Example 3). Thus, it was found that the compound of formula (2) has specific selectivity for the vitronectin receptor expressed in cancer cells. Actually, the amount of radioactivity present in the tumor was 3.9% ID / g or more after 1 hour and 1.12 (after 12 hours) after 40 hours. % ID / g. Significantly lower levels of radiation were observed in most of the organs, and the distribution of the compound of Formula 2 was found to be highly selective for the tumor in the tissues, and the imaging effect Was confirmed to be continuous. Therefore, it has been confirmed that it can be used as a nuclear medicine imaging contrast agent which is very useful for cancer diagnosis, and it has been confirmed that the side effect is remarkably low and can be used as an effective cancer treatment agent.

The dose of the PET contrast agent may vary depending on the kind of the radioactive isotope of the compound of formula (2) as the active ingredient, but may be 0.1 to 30 mCi to the adult on the basis of Curie (Ci, 1Ci = 1,000 mCi) can do.

The dosage of the SPECT contrast agent may vary depending on the kind of the radioactive isotope of the compound of formula (2) as the active ingredient, but may be 0.1 to 30 mCi to the adult on the basis of Curie (Ci, 1Ci = 1,000 mCi) can do.

The dose of the cancer therapeutic agent may vary depending on the kind of the radioactive isotope of the compound of formula (2) as the active ingredient, but may be 0.1 to 100 mCi to the adult on the basis of Curie (Ci, 1Ci = 1,000 mCi) can do.

The PET contrast agent, SPECT contrast agent, and cancer treatment agent can be used for the diagnosis or treatment of any cancer in which cancer cells are known to express a vitronectin receptor, and all cancers in tissues are capable of expressing a bitonectin receptor, But is not limited thereto.

The PET contrast agent, SPECT contrast agent, and cancer treatment agent according to the present invention can be formulated as an injectable agent. When formulated into an injectable preparation, a non-toxic buffer solution that appears as blood can be contained as a diluent. For example, Solution. The PET contrast agent, the SPECT contrast agent, and the cancer treatment agent may contain other diluents or additives in addition to the buffer solution. Excipients and additives that may be added to such injections are well known to those skilled in the art and can be found, for example, in the following references: Dr. HP Fiedler "Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete "[Encyclopedia of auxiliaries for pharmacy, cosmetics and related fields]).

As described above, the compound of formula (2) according to the present invention contains two cyclic RGDfKs having high binding affinity for bitonectin, which not only has a high tumor selectivity but also contains glucosamine and is highly stable in the body, Can be used as a more specific, safe and effective nuclear medicine imaging contrast agent and cancer treatment agent for cancer cells as compared with the conventional nuclear medicine imaging contrast agent and cancer treatment agent. Therefore, the compound of formula (2) according to the present invention can be usefully used as a more effective and safe nuclear medicine imaging contrast agent and cancer treatment agent. Further, the compound of formula (I) according to the present invention can form a complex of formula (II) through coordination with the radioactive isotope for PET, and thus can function as a ligand capable of imparting safety to radioactivity, stability in the body, and tumor selectivity .

Figure 1 is an HLPC image reflecting the chemical purity of cRGDfK ₂ -NODAGA of Formula 1 prepared according to one embodiment of the present invention.
2 is a MALDI-TOF-MS image of cRGDfK ₂ -NODAGA of formula (1) prepared according to one embodiment of the present invention.
3 is a TLC image of cRGDfK ₂ -NODAGA- ⁶⁴ Cu of Formula 2 prepared according to one embodiment of the present invention.
Figure 4 is a PET / CT image of a mouse with U87MG tumor injected with 0.2 mCi of cRGDfK ₂ -NODAGA- ⁶⁴ Cu.
FIG. 5 shows the results of PET / CT imaging (right), in which cRGDfK ₂ -NODAGA- ⁶⁴ Cu was injected and shot without blocking the vitronectin receptor present in the mouse tumor, and cRGDyK peptide, (Left) after cRGDfK ₂ -NODAGA- ⁶⁴ Cu injection after blocking the receptor.
FIG. 6 is a graph showing the time-based comparison of the tissue radioactivity ratio (% ID / g) measured in various organs by injection of cRGDfK ₂ -NODAGA- ⁶⁴ Cu into U87MG tumor-bearing mice.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Abbreviation definitions

DCM: Dichloromethane

TFA: Trifluoroacetic acid

DMF: N, N-dimethylformamide

ACN: Acetonitrile

AcOH: Acetic acid

TFE: Tetrafluoroethylene

DIPEA: diisopropylethylamine

 NODAGA: 1,4,7-triazacyclononane-1-glutaric acid-4,7-triacetic acid

HOAt: 1-hydroxy-7-azabenzotriazole

HOBt: N-hydroxybenzotriazole

HATU: 2- (1H-7-Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium.

HBTU: 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate

HPLC: High-performance liquid chromatograph

Example 1: cRGDfK ₂ Preparation of -NODAGA (Formula 1)

Tert-Butyl-3-aminopropylcarbamate (5 g) was dissolved in acetonitrile under argon gas, followed by K ₂ CO ₃ . To this, propargyl bromide (8.1 mL, 80% toluene) was slowly dropped. After stirring at room temperature for 5 hours, filtering, the mother liquor was removed under reduced pressure. The reaction mixture was dissolved in methylene chloride, and water was added to extract the organic layer. The obtained organic layer was washed with brine again. The washed organic layer was passed through anhydrous NaSO ₄ layer to remove residual water and the residual solvent was removed under reduced pressure. Liquid chromatography (30% EA / Hx) Colorless In oil form Compound 1 (6.88 g, 96%) was obtained.

_{^{1 H NMR (CDCl 3, 400}} MHz); 4.89 (br s, 1 H) , 3.42 (d, J = 2.0 Hz, 4H), 3.22-3.16 (m, 2H), 2.60 (t, 2H, J = 6.8 Hz), 2.22 (t, 2H, J = 2.4 Hz), 1.70-1.60 (m, 2H), 1.43 (s, 9H) 13 C NMR (100 MHz, CDCl 3) δ 156.14,79.15, 73.19, 50.50, 42.30, 39.05, 28.56, 27.41 MS (ESI) m / z 251.2 [M ⁺ H] < ^{+ &} gt ;.

The compound 1 was dissolved in 30% TFA / DCM, stirred at room temperature for 2 hours, and then concentrated under reduced pressure. After re-dissolved in water, NaHCO ₃ Solution and concentrated. The residue was dissolved in DCM and the salt was filtered off. Subsequently, the residue was dissolved in acetonitrile, succinic anhydride (3.03 g) was added thereto, and the mixture was stirred at room temperature for 1 hour and 30 minutes. Purification by liquid chromatography (5% methanol / DCM) Yellow Compound 2 (4.26 g, 64%) was obtained in the form of an oil.

_{^{1 H NMR (CDCl 3, 400}} MHz) δ 6.96 (br s, 1 H), 3.46 (d, J = 2.8 Hz, 4H), 3.38-3.32 (m, 2H), 2.62-2.48 (m, 8H), 2.28 (t, J = 2.4 Hz , 2H), 1.69 (m, 2H) 13 C NMR (100 MHz, CDCl 3) δ 175.60, 172.81, 73.99, 51.03, 42.03, 38.82, 31.04, 30.39, 25.72 MS (ESI) m / z 251.2 [M ⁺ H] < ^{+ &} gt ;.

The reaction schemes for preparing the compounds 1 and 2 are shown in the following Reaction Scheme 2

[Reaction Scheme 2]

The compound 2 Compound 3 was sequentially subjected to Solid-Phase Peptide Synthesis according to the following reaction scheme to obtain Compound 4 (505 mg, 43%) in the form of a yellow oil.

¹ H NMR (MeOH-d 4, 400 MHz) ? 8.20-8.12 (m, 1.5H), 7.98-7.92 (m, 0.5H), 7.38-7.26 (m, 5H), 7.04-6.98 2H), 4.26-4.18 (m, 1H), 3.45 (d, J = 2.4 Hz, 4H), 3.26-3.08 (m, 6H), 2.68-2.40 J = 7.4 Hz, 2H), 1.90-1.30 (m, 12H) ¹³ C NMR (100 MHz, MeOH-d ₄ ) ? 176.96,176.22,75.28,174.59,174.48,158.95,138.46,129.46,128.95,128.77 (ESI) m / z 598.4 [M + H] < ⁺ >. [ .

The compound 2 and The reaction scheme of the solid phase peptide synthesis method in which compound 3 is reacted to produce compound 4 is as follows.

[Reaction Scheme 3]

* Synthesis of Solid-Phase Peptide Synthesis :

Peptides were synthesized sequentially using 2-chlorotrityl chloride resin and all couplings were performed using 3 equivalents of N-Fmoc-protected amino acids and HBTU, HOBt, and DIPEA. Each coupling reaction was monitored by Kaiser test and N-Fmoc protection was deprotected with 20% (v / v) piperidine / DMF. After each coupling and deprotection reaction, the resin was thoroughly washed with DMF and DCM. The separation in the resin was carried out with AcOH / TFE / DCM = 3/1/6 (v / v / v). The separated peptides were purified by Prep-HPLC.

The compound 4 (920 mg), HATU (702 mg), HOAt (251 mg) and DIPEA (1.19 g) were dissolved in DMF, stirred under argon gas, and then added with glucosamine HCl (365 mg) and stirred at room temperature. After 6 hours, D- (+) - glucosamine HCl (0.5 eq), HATU (0.6 eq) and DIPEA (1 eq) were added and stirred. Distilled water was added and the mixture was concentrated under reduced pressure. Separation and purification by Prep-HPLC gave Compound 5 (750 mg, 49%) as a colorless oil.

HPLC conditions; Tilt: 20-60% ACN linear ramp for 30 minutes, flow rate = 12 mL / min, Rt = 14 min; MS (ESI) m / z 763.6 [M + H] < ⁺ >.

The compound 5 (569 mg), azido cRGDfK (1.29 g), CuSO ₄ (270 ml, 1M aqueous solution) and Na-ascorbate (270 ml, 1M aqueous solution) were dissolved in DMF and distilled water (2: 1) and stirred at room temperature. After concentration under reduced pressure, it was separated and purified by Prep-HPLC to obtain Compound 6 (1.1 g, 59%) as a white solid.

HPLC condition; water (0.1% TFA) / ACN = 70/30, flow rate = 12 mL / min, Rt = 10.8 min; MS (MALDI-TOF) m / z 2284.35 [M + H] < ⁺ >, 2285.34 [M + 2H] < ⁺ >.

Compound 6 (1.1 g) was dissolved in DMF and MeOH under an argon atmosphere, Pd / C (283 mg) was added thereto, and the solution was substituted with hydrogen gas. After stirring at room temperature for 18 hours, the solution was filtered. The reaction mixture was concentrated under reduced pressure, dissolved in a minimum amount of methanol, and then excess diethyl ether was added to precipitate. The precipitated solid was filtered to give compound 7 (1.085 g) as a white solid and used in the next step without further purification.

MS (MALDI-TOF) m / z 2150.18 [M + H] < ⁺ >, 2151.19 [M + 2H] < ⁺ >.

Compound 7 was obtained from Compound 4 The reaction scheme of the synthetic method to be produced is as follows.

[Reaction Scheme 4]

Compound 7 (200 mg) and DIPEA (77 mg) were dissolved in DMF under an argon atmosphere, and a solution of 125 mg of NODAGA-NHS ester dissolved in DMF was slowly added thereto. After stirring at room temperature, distilled water (0.1% TFA) was added to terminate the reaction, followed by concentration under reduced pressure. Separation and purification by Prep-HPLC were performed to obtain Compound 8 as a white solid. HPLC condition (Figure 1); gradient: 10 to 45% ACN linear gradient for 30 min, flow rate = 12 mL / min, Rt = 20.5 min; MS (FIG. 2, MALDI-TOF) m / z 2506.53 [M] ⁺ , 2507.53 [M + H] ⁺ , 2508.55 [M + 2H] ⁺ .

From the compound 7 corresponding to the formula 3, the compound 8 corresponding to the formula 1 The reaction scheme of the synthetic method to be produced is shown in the following reaction formula (5).

[Reaction Scheme 5]

Example 2: cRGDfK ₂ -NODAGA- ⁶⁴ Preparation of Cu (Formula 2)

⁶⁴ Cu produced from a cyclotron is obtained as a colorless transparent solution dissolved in a dilute hydrochloric acid solution. After appropriately obtaining (according to the amount of radioactivity required for the experiment), ⁶⁴ Cu solution is put into a suitable glass container and nitrogen is blown And dried at 100 ° C. The dried ⁶⁴ Cu was attached to the glass vessel in the form of a transparent membrane, and 0.2-0.3 mL of 1 M sodium acetate buffer solution (pH 5-6) was added and dissolved. Subsequently, cRGDfK ₂ -NODAGA prepared in Example 1 was dissolved in ethanol to obtain 250 μg / 0.05 mL of the solution. The solution was mixed with the ⁶⁴ Cu solution and the labeling reaction was allowed to proceed at 50 ° C. for 30 minutes. The final product cRGDfK ₂ -NODAGA- ⁶⁴ Cu (Compound 9 ) was adsorbed on the thin layer chromatography and sufficiently developed with a developing solution (0.1 M citrate buffer solution) to confirm the radiochemical yield and purity.

From the compound 8 corresponding to the formula 1, the compound 9 corresponding to the formula 2 The reaction scheme of the synthetic method to be produced is shown in the following reaction formula (6).

[Reaction Scheme 6]

Figure 3 is a TLC image of cRGDfK ₂ -NODAGA- ⁶⁴ Cu prepared according to one embodiment of the present invention. From the TLC image of Figure 3, it can be seen that the radiochemical yield and purity are above 98% on average.

Experimental Example 1: cRGDfK ₂ -NODAGA- ⁶⁴ Measurement of in-vitro serum stability of Cu

The stability of the compound of formula (2) (cRGDfK ₂ -NODAGA- ⁶⁴ Cu) prepared in Example 2 in human serum and mouse serum was measured. cRGDfK ₂ -NODAGA- ⁶⁴ Cu (0.38 mCi, 100 mL) was mixed with human serum and mouse serum (0.5 mL) and cultured at 37 ° C. TLC was performed in the time zone (30 minutes, 1 hour, 2 hours, 24 hours) to be measured. The stability results of cRGDfK ₂ -NODAGA- ⁶⁴ Cu in the serum by time are shown in Table 1 below.

division 30 min 1 h 2 h 24 h cRGDfK ₂ -NODAGA- ⁶⁴ Cu Human serum 96.45% 91.44% 92.99% 97.76% Mouse serum 94.7% 94.93% 99.94% 94.43%

According to the above results, cRGDfK ₂ -NODAGA- ⁶⁴ Cu is stable in human and mouse serum for 24 hours.

Experimental Example 2: cRGDfK ₂ -NODAGA- ⁶⁴ Acquisition of PET image of Cu

PET images were taken with multiple PET / SPECT / CT systems (INVEON, Simens Medical Solutions). U87MG cells, a malignant glioma cell line, were injected subcutaneously into the nude mouse shoulder to create a tumor. PET / CT images were obtained by injecting 0.2 mCi of cRGDfK ₂ -NODAGA- ⁶⁴ Cu prepared in Example 2 through the tail vein of a tumor-bearing mouse (nude, 20 g). The results are shown in FIG. CT images were taken at the same location and matched to PET images to facilitate anatomic location.

Figure 4 is a PET / CT image of a mouse with tumor injected with 0.2 mCi of cRGDfK ₂ -NODAGA- ⁶⁴ Cu.

According to Fig. 4, an increased signal (bright color) can be observed in the tumor site after administration of cRGDfK ₂ -NODAGA- ⁶⁴ Cu.

Experimental Example 3: cRGDfK ₂ -NODAGA- ⁶⁴ Determination of tumor selectivity of Cu

Experiments were carried out to confirm the selective specificity for the receptor expressed in tumor cells of cRGDfK ₂ -NODAGA- ⁶⁴ Cu prepared in Example 2. U87MG cells, a malignant glioma cell line, were injected subcutaneously into the nude mouse shoulder to create tumors. First, cRGDyK peptide (10 mg / kg) was injected through the tail vein of anesthetized mice to block the receptor. After 30 minutes, 0.2 mCi of cRGDfK ₂ -NODAGA- ⁶⁴ Cu was injected, and PET / CT images were obtained in the same manner as in Experimental Example 3. The results are shown in Fig.

FIG. 5 shows the results of PET / CT imaging (right), in which cRGDfK ₂ -NODAGA- ⁶⁴ Cu was injected and shot without blocking the vitronectin receptor present in the mouse tumor, and cRGDyK peptide, (Left) after cRGDfK ₂ -NODAGA- ⁶⁴ Cu injection after blocking the receptor.

According to Fig. 5, the signal of the tumor site where the receptor is blocked is lower than the signal of the tumor site where the receptor is not blocked. These results suggest that cRGDfK ₂ -NODAGA- ⁶⁴ Cu has selective specificity for receptors expressed in tumor cells.

Experimental Example 4: cRGDfK ₂ -NODAGA- ⁶⁴ Confirming the distribution of Cu in tissues

The intra-tissue distribution of cRGDfK ₂ -NODAGA- ⁶⁴ Cu prepared in Example 2 was confirmed by intravenous injection. U87MG cells, a malignant glioma cell line, were injected subcutaneously into the shoulder of nude mice (BW 20 g) to create tumors. The mice were injected with 0.01 mCi of cRGDfK ₂ -NODAGA- ⁶⁴ Cu through the tail vein of the tumor-bearing mouse (n = 4), and the organs (blood, muscle, heart, lung , Liver, spleen, stomach, intestine, kidney, bone, tumor) were measured and the radioactivity of tissue was measured with a gamma counter.

FIG. 6 is a graph showing a comparison of the tissue radioactivity ratio (% ID / g) measured in various organs by time of injection with cRGDfK ₂ -NODAGA- ⁶⁴ Cu prepared according to an embodiment of the present invention.

According to FIG. 6, the radioactivity present in the tumor was distributed over 3.9% ID / g at 1 hour after injection, and the radioactivity was the highest when compared with other tissues. Therefore, it was confirmed that the selectivity to the tumor was high. It was confirmed that it remained above 1.12% ID / g at 40 hours after the injection.

In addition, it was confirmed that the radioactivity level of liver tissue was maintained at 1.5% ID / g at 4 hours after injection and 0.8% ID / g at 40 hours after injection.

Claims (12)

A compound of formula
[Chemical Formula 1]

In Formula 1,

The

Lt; / RTI >
n is an integer from 3 to 6; 2. The compound according to claim 1, wherein n is 3. A compound of formula 2:
(2)

In the formula (2)

The

Lt; / RTI >
n is an integer from 3 to 6,
The M ^* may be selected from the group consisting of Cu-64 (II), Cu-67 (II), Ga-68 (III), Zr-89 (IV), Y- III), Tc-99m (I-VII), and In-111 (III), which are coordinately bound to NODAGA. 4. The compound according to claim 3, wherein n is 3. The compound according to claim 3 or 4, wherein M ^* is Cu-64. A process for the preparation of a compound of formula (1) according to claim 1, comprising subjecting a compound of formula (3) to a peptide coupling reaction with a NODAGA-NHS ester:
(3)

In Formula 3, n is an integer of 3 to 6. A compound of formula 1 according to claim 1 is reacted with a radioactive isotope M ^* solution selected from Cu-64, Cu-67, Ga-68, Zr-89, Y-86, Y-90, Lu-177 and In- Wherein R < 1 > and R < 2 > are as defined above. A PET contrast agent comprising a compound of Formula 2:
(2)

In the formula (2)

The

Lt; / RTI >
n is an integer from 3 to 6,
The M ^* is a metallic radioisotope selected from Cu-64 (II), Ga-68 (III), Zr-89 (IV), and Y-86 (III) and coordinated to NODAGA. The PET contrast agent according to claim 8, which is used for cancer diagnosis. A SPECT contrast agent comprising a compound of formula 2:
(2)

In the formula (2)

The

Lt; / RTI >
n is an integer from 3 to 6,
The M ^* is a metallic radioactive isotope selected from In-111 (III) and Tc-99m (I-VII) and coordinated to NODAGA. 11. The SPECT contrast agent according to claim 10, which is used for diagnosis of cancer. A cancer therapeutic agent comprising a compound of formula (2):
(2)

In the formula (2)

The

Lt; / RTI >
n is an integer from 3 to 6,
The M ^* is a metallic radioisotope selected from Cu-67 (II), Y-90 (III) and Lu-177 (III) and is coordinately bound to NODAGA.

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