KR20130128285A - Aminocyclohexane-containing cyclo rgd derivatives, a process for the preparation thereof and a pet contrast agent comprising the same - Google Patents

Abstract

The present invention provides cyclo ACH-RGDK which is prepared by inserting aminocyclohexanecarboxylic acid (ACH) between lysine and aspartate of cyclo Arg-Gly-Asp-Lys (cyclo RGDK peptide) which is known for having selectivity to cancer cells by peptide bonds; novel PET contrast compounds in which cyclo ACH-RGDK is conjugated with radioisotope, 64Cu2+, through a ligand; an intermediate of the novel compounds; a method for preparing the same; and a PET contrast agent containing the novel compounds.

Description

Aminocyclohexane-containing cyclo RGD derivatives, a method for preparing the same, and a PET contrast agent comprising the same {aminocyclohexane-containing cyclo RGD derivatives, a process for the preparation etc and a PET contrast agent comprising the same}

The present invention relates to a novel compound that can be used as a novel positron emission tomography (PET) contrast agent, a method for preparing the same, and a PET contrast agent containing the same.

Positron emission tomography (PET) is a device for imaging the distribution of radiopharmaceuticals injected into a living body, and by imaging the biological changes of the human body due to the disease, it provides accurate information for the early diagnosis of the disease and the determination of treatment for the disease. Can provide. Therefore, the importance of nuclear medical imaging equipment is increasing.

Radiopharmaceuticals used in PET include radioisotopes [ ¹⁸ F] fluoride, [ ¹¹ C] carbon, [ ¹⁵ ] in molecules that participate in certain metabolisms in vivo, such as water, oxygen, carbon dioxide, and glucose. Synthetic positrons such as C ¹⁵ O, [ ¹³ N] ammonia, H ₂ ¹⁵ O, [ ¹⁸ F] FDG (F-Deoxy Glucose), which label O] oxygen or [ ¹³ N] nitrogen Emitters. These radiopharmaceuticals and highly sensitive PET can be used to image data on tumor receptors, metabolism, and various biological functions and information.

Among the radioisotopes capable of positron emission, relatively recently developed metallic radioisotopes [ ⁶⁴ Cu] copper (positive electron emission rate, about 34%) in the field of nuclear medicine are those in the form of beams of protons accelerated by cyclotron in concentrated Ni-64. Irradiation can be achieved by Ni-64 (p, n) Cu-64 nuclear reactions, and for clinical use requires Cu-64 with high radiochemical purity. High-purity Cu-64 can be obtained by separating Cu, Ni and other metal impurities through a column using electrostatic properties. Thus purified Cu-64 is present as a divalent cation in dilute hydrochloric acid solution, and has the characteristic of easily coordinating with a negatively charged chelate. In addition, Cu-64 has a relatively long half-life of 12.7 hours due to the physical properties of Cu-64, and thus has an advantage of obtaining images after a long time after injection, which cannot be obtained with a conventional short-lived radioisotope labeled radiopharmaceutical within 2 hours.

Radiopharmaceuticals for tumor imaging are important tools for understanding biological phenomena or for the accurate diagnosis of a variety of tumors, and therefore: 1) high selectivity of radiopharmaceuticals for the tumor, 2) fast intake equilibrium time after injection, and 3) suitable for imaging. It should satisfy characteristics such as duration.

Receptors are present on the surface of the cell, which serves as a pretext for signaling signals from outside the cell into the cell. Cells are exposed to various external signals, and the types of receptors vary. Integrin protein, one of these receptor types, is a protein complex composed of α and β subunits involved in cell adhesion and migration, and mediates interactions between cells. It is also involved in the early development of cell tissue. In addition, inflammation, blood clotting, cell movement, etc. require the function of integrin protein. Among them, α _ν β ₃ integrins, called vitronection receptors, are not expressed in normal vascular endothelial cells but are expressed during neovascularization of cancer cells and bind to peptides having an arg-gly-asp (RGD) sequence. There is this. RGD, a tumor-selective peptide, has been reported to be applied to tumor treatment due to its characteristic of selectively binding tumor vessels (Non-Patent Document 1).

Peter S. Conti Group of the University of Southern caifornia labeled the radioisotope Cu-64 on RGD, a tumor target peptide, to obtain an effective tumor PET image (Non-Patent Document 2).

In order to label Cu-64 efficiently and stably, studies on various chelators in the form of DOTA and NODAGA have been conducted (Non-Patent Document 3).

In an effort to increase the ability to bind to tumors, studies have been made on various amino acid sequences such as cyclo RGD, RGDyK, RGDfK, RGDfV, or a combination of two RGDs or multiple RGDs. Patent Document 4)

1. 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 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 peptide. Bioconjugate Chem. 2004, 15, 41-49 3.Dumont RA, Deininger F, Haubner R, Maecke HR, Weber WA, Fani M. Novel (64) Cu- and (68) Ga-labeled RGD conjugates show improved PET imaging of α (ν) β (3) integrin expression and facile radiosynthesis. J Nucl Med. 2011 Aug; 52 (8): 1276-84 4. Cai W, Sam Gambhir S, Chen X. Multimodality tumor imaging targeting integrin alphavbeta3. Biotechniques. 2005 Dec; 39 (6 Suppl): S14-25

It is an object of the present invention to provide an RGD peptide derivative having high binding affinity for tumors.

It is another object of the present invention to provide a PET contrast agent compound to which an RGD peptide derivative having a high binding affinity for a tumor is bound.

Still another object of the present invention is to provide an intermediate for preparing the PET contrast agent compound.

Another object of the present invention is to provide a method for preparing the compounds.

Still another object of the present invention is to provide a PET contrast agent including the PET contrast agent compound.

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

[Formula 1]

Another aspect of the invention provides a compound of formula

(2)

Wherein the ligand is DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA,

The structure of cyclic aminocyclohexanecarboxylic acid -Arg-Gly-Asp-Lys (cyclo-ACH-RGDK) corresponding to the compound of Formula 1 is amide bonded to the ligand via lysine.

Another aspect of the present invention provides a compound of formula 3:

(3)

Wherein the ligand is as defined in Formula 2,

The ⁶⁴ Cu ²⁺ is coordinated to the ligand,

The structure of the cyclo-ACH-RGDK is amidated with the ligand via lysine.

In another aspect of the present invention, lysine and (1S, 3R) -3-aminocyclohexane-1-carboxylic acid are subjected to peptide coupling reaction by the Fmoc solid phase synthesis method and then reacted with Fmoc solid phase A method for producing the compound of formula (1), which comprises peptide bonding by a synthetic method.

Yet another aspect of the present invention provides a method for preparing a compound of formula 1, comprising subjecting a compound of formula 1 to a peptide coupling reaction with a ligand selected from DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA, 2. ≪ / RTI >

Another aspect of the present invention provides a method of preparing the compound of Formula 3, including reacting the compound of Formula 2 with a ⁶⁴ Cu solution.

Another aspect of the invention provides a PET contrast agent comprising the compound of formula (3).

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 found that when cyclo-ACH-RGDK is formed by inserting aminocyclohexanecarboxylic acid (ACH) between a lysine and an aspartic acid of a conventional cyclo-RGDK peptide (cyclo Arg-Gly-Asp-Lys) (Α _ν β ₃ integrin) expressed in cancer cells significantly increases the binding affinity to cancer tissues (Experimental Example 1). In addition, the substance in which the cyclo ACH-RGDK is bound to the radioisotope ⁶⁴ Cu ²⁺ via a ligand is stable in the blood for about 24 hours (Experimental Example 2), and in fact, PET of a mouse having a tumor using the substance As a result of the imaging, an increased signal at the tumor site could be observed (Experimental Example 3). In addition, when cRGDyK (cyclo Arg-Gly-Asp-Tyr-Lys) was administered first to block the Vitronectin receptor, and then PET image was taken, the signal of the tumor area was higher than that of the case where the Vitronectin receptor was not blocked. It was found to be low (Experimental Example 4). Therefore, it was found that the radioisotope ⁶⁴ Cu ²⁺ conjugated with cyclo ACH-RGDK has specific selectivity for the vitronectin receptor expressed in cancer cells.

Accordingly, in one aspect of the present invention,

There is provided a compound of formula 1:

[Formula 1]

In the present specification, the compound of Formula 1 is abbreviated as cyclo ACH-RGDK, and the cyclo ACH-RGDK of Formula 1 is a structure in which cyclohexane is inserted into a conventional cyclo RGDK tumor selective peptide, and compared to a conventional cyclo RGDK derivative in tumors. Selectivity is significantly higher.

In order to more efficiently and stably bind the compound of Formula 1 with ⁶⁴ Cu ²⁺ , a ligand may be bound to Formula 1. Thus, in another aspect, the present invention provides a compound of Formula 2:

(2)

Wherein the ligand is DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA,

The structure of the cyclo-ACH-RGDK corresponding to the compound of the formula (1) is amide-bonded to the ligand via lysine.

According to one embodiment, the compound of Formula 2 may have the structure of Formula 2a:

(2a)

By coordination to the ⁶⁴ Cu ^{2 +} binding to the ligand of the compound of Formula 2 to form a complex, can be manufactured in a compound of formula (3). Thus, in another aspect, the present invention provides a compound of Formula 3:

(3)

Wherein the ligand is as defined in Formula 2,

The ⁶⁴ Cu ²⁺ is coordinated to the ligand,

The structure of the cyclo-ACH-RGDK is amidated with the ligand via lysine.

As used herein, the term "complex" refers to the formation of one atomic group by directing three or more different atoms, ions, molecules, or groups of atoms centered on one or more atoms or ions in a three-dimensional manner. . Here, a chelator or group of atoms coordinated with a central atom or ion is called a ligand.

As used herein, the term DTPA is Diethylene triamine penta aeetic acid, DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DO3A is 1,4,7,10-tetraazacyclododecane-1, 4,7-triacetic acid, NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid, NODAGA is 1,4,7-Triazacyclononane, 1-glutaric acid-4,7-acetic acid, and TETA is 1,4,8,11-tetraazacyclotetradecane-N, N ', N'',N'''-tetraacetic acid, TE3A is 1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic acid, TE2A is 1,4,8,11-Tetraazabicyclohexadecane-4,11-diacetic acid, PCTA is 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1,11,13-triene-3,6,9,- Triacetic acid is an abbreviation for metal affinity ligand compound. The ligand acts as a metal affinity ligand to prevent the release of radioactive metal ⁶⁴ Cu in the body and to remove the radioactive material outside the body can reduce the cytotoxicity caused by the radioactive material chemical protection against radiation interference (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).

As used herein, the term "cyclo-ACH-RGDK" means a compound of cyclic structure in which the amino acid sequence of Arg-Gly-Asp-Lys is formed by peptide bonds via aminocyclohexanecarboxylic acid and aspartic acid and lysine, 1 < / RTI > In the above formula (3), the cyclo-ACH-RGDK is amidated with the ligand via lysine. Α _ν β ₃ integrin, which is not expressed in normal vascular endothelial cells and is expressed during neovascularization of cancer cells, is characterized by binding to a cyclo peptide having an RGD (Arg-Gly-Asp) sequence. The cyclo ACH-RGDK has a significantly higher binding affinity for cancer cells than RGD cyclopeptides, thereby forming a PET contrast agent with a markedly increased targeting effect on cancer tissues.

In the present specification, "ligand → ⁶⁴ Cu ²⁺ " means a complex formed by coordinating radioactive copper element ion ⁶⁴ Cu ²⁺ to the ligand.

The compound of formula 3 may, according to one embodiment, be a compound of formula 3a:

[Chemical Formula 3]

According to another aspect of the present invention, there is provided a method for producing a lysine compound, which comprises subjecting lysine and (1S, 3R) -3-aminocyclohexane-1-carboxylic acid to a peptide coupling reaction by a Fmoc solid phase synthesis method and then reacting the aspartic acid, glycine and arginine The present invention provides a method for producing a compound represented by the above formula (1), which comprises peptide bonding by Fmoc solid phase synthesis.

The preparation of peptides by the Fmoc solid phase synthesis method is well known in the art, and one skilled in the art can select the appropriate reaction conditions to prepare the compound of formula 1 above.

In another aspect, the present invention provides a method for preparing a compound of formula (I), which comprises reacting a compound of formula (I) with a ligand selected from DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA, To provide a 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, and a small amount of DMSO may be preferably used. In addition, a catalyst may be added during the reaction to promote the reaction. These catalysts can be appropriately selected by those skilled in the art depending on the reactants, for example, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide or sulfo-NHS, 1-hydroxybenzotriazole DCC (Dicyclohexylcarbodiimide ), DIC (diisopropylcarbodiimide), HBTu (O- (Benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate) or a combination thereof. According to one embodiment, a compound of formula (2) can be obtained by adding a mixture of a ligand and a compound of formula (1) to a solvent, adding a catalyst, reacting for about one day and then extracting. The reaction can be carried out at room temperature and the reaction can be completed by reacting overnight. The product thus obtained can be purified by any method known in the art to obtain the compound of formula 2, preferably by HPLC, to remove impurities. According to one embodiment, the ligand may be DOTA.

In still another aspect, the present invention provides a method of preparing the compound of Formula 3, including reacting the compound of Formula 2 with a ⁶⁴ Cu solution.

In this method, a compound of Formula 3 may be produced by mixing a solution of ⁶⁴ Cu (pH 5-6) in a buffer solution with a solution of the compound of Formula 2 at about 40 to 60 ° C. As the buffer solution, for example, sodium acetate buffer solution may be used, but 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. According to one embodiment, the ligand may be DOTA.

An embodiment of the process for preparing the compound of formula (3) according to the present invention is shown in the following reaction formula (1).

[Reaction Scheme 1]

The compound of formula 3 was confirmed that can be effectively used as a PET contrast agent.

Therefore, in another aspect, the present invention provides a PET contrast agent comprising the compound of Formula 3 or Formula 3a.

According to the following experimental example, when the stability of the mouse in the serum of the compound of Formula 3 or Formula 3a was measured, it was found that the compound is stable without degradation for more than one day (Experimental Example 2). In addition, the compound of Formula 3 or Formula 3a was administered to mice with tumors and photographed on a small animal PET imaging system, PET images were obtained. As a result, an increased signal was observed at the tumor site (Experimental Example 3). Thus, the compounds of Formula 3 or Formula 3a may be used as PET contrast agents that are specifically selective for cancer. In addition, cRGDyK (cyclo Arg-Gly-Asp-Tyr-Lys) was first administered to mice with tumors to block the vitronectin receptors, and then PET images were taken. Tumor sites were compared with those without blocking the vitronectin receptors. Was found to be lower (Experimental Example 4). Thus, it has been found that the compound of formula (3) or (3a) has a specific selectivity for the vitronectin receptor expressed in cancer cells. Therefore, it has been found that it can be used as a PET contrast agent which is very useful for diagnosing cancer.

The PET contrast agent may be administered 0.1 to 30 mCi to adults based on the compound of Formula 3 or 3a as the active ingredient.

The PET contrast agent according to the present invention may be formulated as an injection, and when formulated as an injection may include a blood and isotonic non-toxic buffer as a diluent, for example, a phosphate buffer solution of pH 7.4. The PET contrast agent may include other diluents or additives in addition to the buffer solution. Excipients and excipients that can be added to such injectables are well known to those of ordinary skill in the art and can be found, for example, by reference to Dr. HP Fiedler "Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete "[Encyclopaedia of auxiliaries for pharmacy, cosmetics and related fields]).

As described above, the compound of formula 3 or 3a according to the present invention contains a cyclo RGD peptide derivative having a high binding affinity for Vitronectin, and thus can be used as a PET specific agent which is more specific and safe for cancer cells than conventional PET contrast agent. Can be. Therefore, it can be usefully used as a more effective and safe cancer diagnostic contrast agent.

1 is an HPLC image of ACH-RGDK prepared according to an embodiment of the present invention, and FIG. 2 is a MALDI-TOF-MS image of ACH-RGDK prepared according to an embodiment of the present invention.
FIG. 3 is an HPLC image of ACH-RGDK-DOTA prepared according to an embodiment of the present invention, and FIG. 4 is a MALDI-TOF-MS image of ACH-RGDK-DOTA prepared according to an embodiment of the present invention.
5 is a TLC image of ACH-RGDK-DOTA- ⁶⁴ Cu prepared according to one embodiment of the present invention.
6 is a graph showing the degree of binding of cRGDyK and ACH-RGD-DOTA to U87MG cells.
Figure 7 is a PET image taken by injecting 0.2 mCi ACH-RGD-DOTA- ⁶⁴ Cu in mice with tumors.
FIG. 8 shows PET / CT images taken from ACH-RGD-DOTA- ⁶⁴ Cu (above) and cRGDyK peptide injected with ACH-RGD after blocking receptors present in tumors without blocking receptors present in tumors. PET / CT image (bottom) taken with -DOTA- ⁶⁴ Cu injection.
9 is a graph showing a comparison of the results obtained by measuring the radioactivity ratio (% ID / g) of tumor tissues relative to the amount of ACH-RGD-DOTA- ⁶⁴ Cu injection of tumors that did not block the tumor and tumors that blocked the receptor.

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 only for better understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Definition of abbreviations

DCM: Dichloromethane

DMF: N, N-dimethylformamide

ACN: Acetonitrile

DIEA: Diisopropylethylamine

DOTA: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid

DIC: diisopropylcarbodiimide

HOBt: N-hydroxybenzotriazole

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

HPLC: High-performance liquid chromatograph

ACH-RGDK: cRGDK containing aminocyclohexanecarboxylic acid

Example 1: Preparation of aminocyclohexanecarboxylic acid-containing cRGDK (cyclo-ACH-RGDK) (Formula 1)

Fmoc-Lys (Boc) -O-trityl resin was filled into a suitable reactor and washed twice with DCM and DMF. After removing Fmoc using a 20% piperidine / DMF solvent, 25 mmol of (1S, 3R) -3-aminocyclohexane-1-carboxylic acid, 25 mmol of tyrosine and HOBT / DIC 2 M were dissolved in DMF And the mixture was agitated for 6 hours. This process was repeated in the order of aspartic acid, glycine, and arginine. After coupling of the final amino acid, resin cleavage was performed using trifluoroacetic acid solution. The separated solution was filtered to obtain an ether, which was recrystallized. The resulting crystals were dried and then cyclized. The prepared aminocyclohexanecarboxylic acid-containing cRGDK (ACH-RGDK) was analyzed by HPLC. Using a SHIMADZU C-18 analysis column (10.0 mm ㅧ 250 mm), 0.1% TFA aqueous solution (A solution) and 0.1% TFA When the ACN solution (B solution) was flowed from 5% B to 65% B at a rate of 1 ml / min for 30 minutes, the peak was confirmed at 12.8 minutes.

FIG. 1 is an HPLC image of a cyclo ACH-RGDK prepared according to an embodiment of the present invention, and FIG. 2 is a MALDI-TOF-MS image of a cyclo ACH-RGDK prepared according to an embodiment of the present invention.

Example 2: Preparation of cyclo-ACH-RGDK-DOTA (Formula 2)

5 mM cyclic ACH-RGDK prepared in Example 1 and 10 mM DOTA were added to the reactor and dissolved in 250 mL of DMSO. 20 mM HOBt and 20 mM DIC were added to the reactor and stirred for one day. After completion of the reaction, some (1 ml) solution was extracted with ether. The prepared cyclo-ACH-RGDK-DOTA was analyzed by HPLC. The ACN solution (solution B) of 0.1% TFA (solution A) and 0.1% TFA was added to the column using SHIMADZU C-18 analytical column (10.0 mm ㅧ 250 mm) Peaks were observed at RT 11.5 min when flow rate was changed from 5% B to 65% B at 1 ml / min for 30 min.

FIG. 3 is an HPLC image of cyclo ACH-RGDK-DOTA prepared according to one embodiment of the present invention, and FIG. 4 is a MALDI-TOF-MS image of cyclo ACH-RGDK-DOTA prepared according to one embodiment of the present invention to be.

Example 1-3 Cyclo ACH-RGDK-DOTA- ⁶⁴ Preparation of Cu (Formula 3)

The ⁶⁴ Cu produced from cyclotron is obtained as a colorless transparent solution dissolved in dilute hydrochloric acid solution. After proper acquisition (0.1 mCi or more) according to the amount of radioactivity required for the experiment, the ⁶⁴ Cu solution is poured into a suitable glass container and blown with nitrogen. Dried at 100 ° C. The dried ⁶⁴ Cu was attached to the glass container in the form of a transparent film, and 0.2-0.3 mL of 1M sodium acetate buffer solution (pH 5-6) was added thereto and dissolved. Subsequently, the cyclo ACH-RGDK-DOTA prepared in Example 1-2 was dissolved in ethanol to obtain 100 μg / 0.01 mL, and mixed with the ⁶⁴ Cu solution, and the labeling reaction was performed at 50 ° C. for 30 minutes. Cyclo ACH-RGDK-DOTA- ⁶⁴ Cu, the final material, was adsorbed on thin layer chromatography to fully develop with a developing solution (0.1 M citrate buffer solution), and then radiochemical yield and purity were confirmed.

5 is a TLC image of cyclo ACH-RGDK-DOTA- ⁶⁴ Cu prepared according to one embodiment of the present invention. From the TLC image of FIG. 5, it can be seen that the radiochemical yield and purity are on average 98% or more.

Experimental Example 1: U87MG cell binding assay of cyclo-ACH-RGDK-DOTA

The experiment was performed to compare the binding capacity of the cyclo ACH-RGD-DOTA prepared in Example 2 to U87MG cells. As a control group, cRGDyK (cyclo-Arg-Gly-Asp-Tyr-Lys) was used. 0.06 nM ¹²⁵ I-labeled echistatin (NEX439025UC, PerkinElmer Co. MA) and various concentrations (1.00-E4-1.00-E13M) of U87MG cells (2X10 ⁶ ) (cRGDyk, ACH-RGD-DOTA) were added to integrin binding buffer. Put and reacted for 1 hour while stirring at room temperature. As the integrin binding buffer, 25 mM Tris pH 7.4, 150 mM NaCl, 1 mM MnCl ₂ and 0.1% BSA (bovine serum albumin) were used. After the reaction was completed, the radioactivity remaining in each tube was measured with a gamma counter (PerkinElmer Co. MA) after two washes with PBS (Phosphate buffed saline). IC ₅₀ values were obtained by nonlinear regression using GrasphPad Prism (GraphPad Software, Inc., CA).

6 is a graph showing the degree of binding of cRGDyK and cyclo ACH-RGD-DOTA to U87MG cells. According to the results of FIG. 6, it can be seen that cyclo ACH-RGD-DOTA has significantly greater binding capacity to U87MG cells than cRGDyK.

Experimental Example 2: Cyclo ACH-RGD-DOTA- ⁶⁴ Measurement of in-vitro serum stability of Cu

The stability in human serum and mouse serum of the compound of formula 3 (cyclo ACH-RGD-DOTA- ⁶⁴ Cu) prepared in Example 3 was measured. ACH-RGD-DOTA- ⁶⁴ Cu (0.1 mCi, 10 mL) was mixed with human serum and mouse serum (0.5 mL) and incubated at 37 ° C. TLC was performed in the time zone (30 minutes, 1 hour, 2 hours, 24 hours) to be measured. The stability measurement results of cyclo ACH-RGD-DOTA- ⁶⁴ Cu in serum by time zone are shown in Table 1 below.

[Table 1]

According to the results, it can be seen that cyclo ACH-RGD-DOTA- ⁶⁴ Cu is stable for 24 hours in human and mouse serum.

Experimental Example 3: Cyclo ACH-RGD-DOTA- ⁶⁴ PET image acquisition of Cu

PET images were taken with a small animal PET imaging system (R4, Concorde Microsystems, Knoxville TN). Tumors were made by subcutaneous injection of U87MG cells, a malignant glioma cell line, into the nude mouse shoulder. PET images were obtained by injecting cyclo ACH-RGD-DOTA- ⁶⁴ Cu 0.2 mCi prepared in Example 3 through the tail vein of the tumor-bearing mouse (nude, 20 g), and the results are shown in FIG. 7.

Figure 7 is a PET image taken by injecting 0.2 mCi cyclo ACH-RGD-DOTA- ⁶⁴ Cu in mice with tumors.

According to FIG. 7, after the cyclo ACH-RGD-DOTA- ⁶⁴ Cu is administered, an increased signal (lightening of color) can be observed at the tumor site.

Experimental Example 4: Cyclo ACH-RGD-DOTA- ⁶⁴ Confirmation of Tumor Selectivity of Cu

An experiment was performed to confirm the selective specificity for the receptor expressed in tumor cells of cyclo ACH-RGD-DOTA- ⁶⁴ Cu prepared in Example 3. Tumors were made by subcutaneous injection of U87MG cells, a malignant glioma cell line, into nude mouse shoulders. First, cRGDyK peptide (10 mg / kg) was injected through the tail vein of anesthetized mice for receptor blocking. After 30 minutes, the cyclo ACH-RGD-DOTA- ⁶⁴ Cu 0.2 mCi was injected to obtain PET / CT images. PET / CT images were taken with a PET / SPECT / CT system (INVEON, Simens Medical Solutions). The results are shown in Fig.

Figure 8 is a PET / CT image (top) taken by injection of cyclo ACH-RGD-DOTA- ⁶⁴ Cu and cRGDyK peptide injection to block the receptor present in the tumor after blocking the receptor present in the tumor without blocking the receptor present in the tumor. PET / CT image (bottom) taken with -RGD-DOTA- ⁶⁴ Cu.

According to FIG. 8, it can be seen that the signal at the tumor site where the receptor is blocked is lower than the signal at the tumor site where the receptor is not blocked. From these results, it can be seen that cyclo ACH-RGD-DOTA- ⁶⁴ Cu has a selective specificity for a receptor expressed in tumor cells.

In addition, after obtaining the PET image in Experiment 3, the tumor region was extracted to measure the gamma counter, and the contrast agent accumulation in tissues that did not block the receptor (Experimental Example 3) and tissues that blocked the receptor (Experimental Example 4) The result of comparing the degree is shown in FIG.

9 is a graph showing a comparison of the results obtained by measuring the radioactivity ratio (% ID / g) of tumor tissue to the amount of cyclo ACH-RGD-DOTA- ⁶⁴ Cu injected between tumors that did not block the tumor and tumors that blocked the receptor; .

According to Figure 9 it can be seen that the group that does not block the receptor shows higher radioactivity in the tissue than the group that blocked the receptor. These results show that cyclo ACH-RGD-DOTA- ⁶⁴ Cu has specific selectivity for receptors expressed in tumor cells.

Claims (11)

A compound of formula
[Formula 1]

A compound of formula
(2)

Wherein the ligand is DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA, Cyclen, or DFOM (Deferrioxamine)
The structure of the cyclic aminocyclohexane carboxylic acid-Arg-Gly-Asp-Lys (cyclo ACH-RGDK) corresponding to the compound of formula 1 of claim 1 is amide-bonded with the ligand via lysine. The compound of claim 2 having the structure of formula 2a:
(2a)

A compound of formula
(3)

The ligand is DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA, cyclone, or DFOM,
The ⁶⁴ Cu ^{+ 2} may be coordinated to the ligand,
The structure of the cyclo ACH-RGDK is amide-bonded with the ligand via lysine. The compound of claim 4 having the structure of formula 3a:
[Chemical Formula 3]

Lysine and (1S, 3R) -3-aminocyclohexane-1-carboxylic acid were peptide-bound by Fmoc solid phase synthesis, followed by peptide binding by Fmoc solid phase synthesis in the order of aspartic acid, glycine and arginine. A process for preparing the compound of formula 1 according to claim 1. A compound according to claim 2 comprising subjecting a compound of formula 1 according to claim 1 to a ligand binding reaction with a ligand selected from DOTA, DTPA, DO3A, NOTA, NODAGA, TETA, TE3A, TE2A, PCTA, cyclone, and DFOM Method for preparing the compound of 2. A process for preparing the compound of formula 3 according to claim 4 comprising reacting the compound of formula 2 according to claim 2 with a ⁶⁴ Cu solution. The method of claim 7 or 8, wherein the ligand is DOTA. PET contrast agent comprising a compound according to claim 4. The PET contrast agent according to claim 10 for use in diagnosing cancer.

Images