KR101171385B1 - RGD peptides which are linked with labeled chilates, and physiolosically acceptable slats thereof, and the preparation method thereof, and the reagent for diagnosis or treatment of cancer containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a cyclolinked chelate with a linker [Arg-Gly-Asp-dPhe-Cys] and a pharmaceutically possible salt thereof, and a cyclolate linked with a chelate [Arg-Gly-Asp-dTyr-Cys] and a pharmaceutical thereof It is possible to provide a salt, and a method for preparing the same, a compound labeled with the chelate, and a pharmaceutically acceptable salt thereof as an active ingredient, a reagent for diagnosing or treating cancer.

Integrin, RGD, DOTA, maleimido

Description

RGD peptides linked with labeled chelates and pharmaceutically acceptable salts thereof, methods for preparing the same, and reagents for diagnosis or treatment of cancers using the same as an active ingredient RGD peptides which are linked with labeled chilates, and physiolosically acceptable slats , and the preparation method particular, and the reagent for diagnosis or treatment of cancer containing the same as an active ingredient}

The present invention relates to an RGD peptide linked to a labeled chelate and a linker conjugated thereto, a pharmaceutically acceptable salt thereof, a method for preparing the same, and a reagent for diagnosing or treating cancer using the same as an active ingredient.

The integrin family is a cell surface receptor known to induce cell attachment by binding to extracellular matrix (ECM). Recent decades of research show that integrin plays an important role in normal or pathological angiogenesis by inducing cell survival, migration, proliferation, and gene expression as well as simple cell attachment and is involved in tumor growth and metastasis. Appeared to be.

Integrin is a heterodimer consisting of αsubunits (140-180 kD) and βsubunits (approximately 95 kD). To date, 18 subunits and 8 βsubunits have been reported and 24 heterodimers have been identified.

The combination of integrin and ECM is characterized by promiscuity and redundancy. That is, it is known that an integrin binds to several ECMs, and different integrins bind to the same ECM. For example, αvβ3 binds to vitronectin, fibrinogen, fibronectin, thrombospodin, tenascin-C, von Willenbrand Factor (vWF), osteopontin, Del-1, and denatured collagen. αvβ5 binds to vitronectin, Del-1 and α5β1 binds fibronectin as ligand.

Integrin recognizes and binds to specific amino acid sequences of ligands. Representatively, RGD sequences are found in fibrinogen, vitronectin, fibronectin.

Since the availability of RGD peptides for the treatment of cancer by selectively delivering drugs to tumor vessels (Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model, Arap et at., 1998, Science, Vol. 279. 377-380) Actively studied, these RGD peptides are a combination of Arginine (R), Glycine (G), and Aspartic acid (D) and have high affinity for α _V β ₃ Integrin. .

Particularly in the case of tumors, α _V β ₃ Integrin, which plays an important role in the growth of tumor tissue and neovascularization in endothelium, is much more distributed than normal, and various radioisotopes are used for imaging and treatment. ought.

Cyclic- (RGDfK) and Cyclo- (RGDyK) are the major structures of the peptides, which are mainly studied when RGD peptides are present in Cyclic form, and D-Aspartic acid is Lysine e- It has the form of binding Amide with amine side chain. (f: D-Phenylalanine, y: D-Tyrosine, K: Lysine)

In order to use the Cyclic-RGD peptide as an image diagnosis agent, researches using Cu-64, Tc-99m, In-111, F-18, etc. have been made. (Noninvasive Imaging of Osteoclasts in Parathyroid Hormone-induced Osteolysis Using a 64Cu-Labeled RGD Peptide.Jennifer E. Sprague, Hideki Kitaura, Wei Zou, Yunpeng Ye, Samuel Achilefu, Katherine N. Weilbaecher, Steven L. Teitelbaum and Carolyn J. Anderson Journal of Nuclear Medicine Vol. 48 No. 2 311-318, 2007) (n vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha (V) beta (3) integrin for tumor imaging Su ZF, Liu G, Gupta S, Zhu Z, Rusckowski M, Hnatowich DJ, Bioconjug Chem. 14, 2003, 274), (Effects of linker variation on the in vitro and in vivo characteristics of an 111 In-labeled RGD peptide. I. Dijkgraaf, S. Liu, J. Kruijtzer, A. Soede, W. Oyen, R. Liskamp, F. Corstens, O. Boerman, Nuclear Medicine and Biology, 34, 29-35) Hynic-Cyclo- (RGDyK), which combines Hynic with Lysine's e-amine sidechain and Amide for binding to isotopes, was used (In vitro cell studies of technet). ium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands. Nuclear Medicine and Biology, 30, 141-149, ZF Su, J He, M Rusckowski, DJ Hnatowich.

Clelators used to introduce Tc-99m in such compounds require 10 ^-4 M or more ligands to label Tc-99m with a yield of 90% or higher, thus radioactive unconjugated receptors. Ligands have shown long-lasting properties in the liver and kidneys. In order to solve this problem of storage in the body, we developed a compound combining Glucose and iminodiacetic acid in RGD to introduce a small ^99m Tc (CO) ₃ at the time of binding. Favorable Biokinetic and Tumor-Targeting Properties of 99m Tc-Labeled Glucosamino RGD and Effect of Paclitaxel Therapy, 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 Choe1 and Dae Yoon Chi Journal of Nuclear Medicine, 47, 2000-2007)

A successful case of radioactive RGD peptides is currently in clinical application as F-18-labeled-galacto-cyclic- (RGDfK) (PET-Based Human Dosimetry of 18F- Galacto-RGD, a New Radiotracer for Imaging αvβ3 Expression, AJ Beer, R Haubner, I Wolf, M Goebel et al., Journal of Nuclear Medicine, 2006, 47: 763-769).

Accordingly, the present inventors consider Cyclic- (RGDfC) and Cyclic- (RGDyC) based on Cyclic- (CRGDC) and considering F-18-labeled-galacto-cyclic- (RGDfK), which is currently being tried clinically. Derivatives containing the side chain Thiol group were synthesized and labeled with Ga-68, In-111, Y-90 and other known lanthanides such as Lu-177 and Sm-153 and Pseudo lanthanum. DOTA was intended to be modified easily.

Thus, in order to bioconjugate Thiol and DOTA using a linker, DOTA-Maleimido-cyclic- (RGDfC) (Formula 2) and DOTA- are prepared using DOTA-aminoethyl-maleimide (Formula 4), which is an aminoethylmaleimide-coupled material to DOTA. Maleimido-cyclic- (RGDyC) (Formula 3) was prepared.

delete

DOTA-aminoethyl-maleimide

[DOTA-Maleimido-cyclic- (RGDfC)]

[DOTA-Maleimido-cyclic- (RGDyC)]

The synthesized material may be used for diagnosis and treatment using energy emitted by radioisotopes using medical imaging equipment including gamma cameras. In particular, the present invention can combine metal and transition metal ions having properties that can be detected by radiation diagnostic devices such as MRI, CT, gamma camera, PET, SPECT, etc. Such metals include radioisotopes that emit radiation.

Specific examples include Fe, Gd, Mn, Zn, and lanthanide elements, which can be imaged by radiation diagnostic devices such as CT, MRI or gamma cameras, SPECT, PET, and the like. It can be selected from gamma, beta, alpha-emitting radioisotopes with properties. Radioactive isotopes that emit radiation include alpha, gamma, and beta isotopes, specifically Sc-47, Cu-61, Cu-62, Cu-64, Cu-67, Ga-67, and Ga. -68, Y-86, Y-90, Tc-99m, Rh-105, In-111, Sn-117m, Pm-149, Sm-153, Dy-165, Ho-166, Er-169, Yb-169 , Lu-177, Re-186, Re-188, Bi-212, and the like.

It is an object of the present invention to provide chelate-RGD derivatives and their pharmaceutically acceptable salts which are labeled in high yield.

Another object of the present invention is to provide a method for preparing the labeled chelate-RGD derivative and its pharmaceutically acceptable salt.

It is still another object of the present invention to provide labeled chelate-rGD derivatives and their pharmaceutically acceptable salts, which accumulate in tumors at high rates.

Still another object of the present invention is to provide a medicament for tumor diagnosis or treatment, comprising as an active ingredient a labeled chelate-rGD derivative and its pharmaceutically acceptable salt, which accumulate at a high rate in the tumor.

In order to achieve the above object, the present invention provides a cyclo [Arg-Gly-Asp-dPhe-Cys] and a pharmaceutically acceptable salt thereof linked by a linker conjugated with a chelating agent, the preparation method and the compound and Provided are reagents for diagnosing or treating cancer bearing salts.

The present invention also provides a cyclo [Arg-Gly-Asp-dTyr-Cys] and a pharmaceutically acceptable salt thereof connected by a linker conjugated with a chelating agent, and a method for preparing the same and for diagnosing cancer containing the compound and salt. Or a therapeutic reagent.

The compounds and salts of the present invention are labeled in high yield with radioisotopes and the like, and can be used for diagnosis and treatment of cancer by effectively binding to integrins of cancer cells.

The present invention relates to a cyclo [Arg-Gly-Asp-dPhe-Cys] linked with a chelating agent and a pharmaceutically acceptable salt thereof, or a cyclo [Arg-Gly-Asp-dTyr linked with a chelating agent linked with a linker -Cys] and pharmaceutically acceptable salts thereof. The structure has the structure of Formula 1 below.

C-L-X

Wherein C is a chelating agent, L is a linker, and X is cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys]. In this case, L is a cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys] Cys of sulfur (S) through the sulfide (sulfide) bond to the cyclo (Arg-Gly- Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys].

In this case, the linker is preferably -maleimido-, and the compound is more preferably a bioconjugated cysteine (cys) thiol and DOTA through a linker using a material in which aminoethylmaleimide is bound to DOTA. The chelating agent is preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

The present invention also relates to a cyclo [Arg-Gly-Asp-dPhe-Cys] and a pharmaceutically acceptable salt thereof, or chelating agent linked by a linker conjugated with a chelating agent labeled with a metal, a transition metal ion or a radioisotope. Provided are cyclo [Arg-Gly-Asp-dTyr-Cys] and pharmaceutically acceptable salts thereof linked by conjugated linkers. At this time, it is preferable to label with Fe, Gd, Mn, lanthanide or lanthanum, gamma, beta, or alpha-emitting radioisotope, and Fe, Gd, Mn, Zn, lanthanide or lanthanide, or gamma More preferably, it is labeled with beta, or alpha emitting radioisotopes, Sc-47, Cu-61, Cu-62, Cu-64, Cu-67, Ga-67, Ga-68, Y-86, Y -90, Tc-99m, Rh-105, In-111, Sn-117m, Pm-149, Sm-153, Dy-165, Ho-166, Er-169, Yb-169, Lu-177, Re-186 It is even more preferred to label with Re-188 or Bi-212, especially with Y-90, Lu-177 or Sm-153. In addition, the linker may be cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys] Cys of sulfur (S) through the sulfide (Sulfide) bond to the cyclo [Arg-Gly- Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys], the linker is preferably -maleimido-, more preferably the -maleimido- is DOTA (1,4,7) , 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and aminoethyl maleimide (aminoehtylmaleimide) is prepared by treating a material combined, more preferably using DOTA-aminoethyl-maleimide Are manufactured.

The present invention also relates to a cyclo [Arg-Gly-Asp-dPhe-Cys] and a pharmaceutically acceptable salt thereof, or chelating agent linked by a linker conjugated with a chelating agent labeled with a metal, a transition metal ion or a radioisotope. Provided are reagents for diagnosing or treating cancer comprising cyclo [Arg-Gly-Asp-dTyr-Cys] and pharmaceutically acceptable salts thereof as an active ingredient linked by conjugated linkers. At this time, the diagnosis of cancer is preferably a diagnosis by the target image of the CT, MRI or radiation diagnostic device. In this case, the radiation diagnostic apparatus includes, but is not limited to, a gamma camera, SPECT, and PET. In addition, cancers to be diagnosed or treated include chronic lymphocytic leukemia, breast cancer, cervical cancer, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangiopia cell tumor, and synovial membrane. Carcinoma, mesothelioma, Ewing tumor, smooth sarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon tumor, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, impression cell tumor, Sebaceous gland tumor, papillary tumor, papillary adenocarcinoma, cystic adenocarcinoma, medulla tumor, tracheal support tumor, renal cell tumor, liver cancer, biliary gland tumor, chorionic cancer, normal carcinoma, fetal tumor, Wilm's tumor, testicular cancer, lung tumor , Small cell lung tumor, non-small cell lung tumor, bladder tumor, epithelial tumor, glioma, astrocytoma, medulloblastoma, craniocytoma, cerebral hepatocellular carcinoma, pineal carcinoma, hemangioblastoma, auditory neuroma, dendritic gliomas, hydrocephalus, melanoma Neuroblastoma, retina Cell tumors, leukemia, preferably a lymphoma or Kaposi (Kaposi) sarcoma. In addition, the chelating agent is Sc-47, Cu-61, Cu-62, Cu-64, Cu-67, Ga-67, Ga-68, Y-86, Y-90, Tc-99m, Rh-105, In Labeled as -111, Sn-117m, Pm-149, Sm-153, Dy-165, Ho-166, Er-169, Yb-169, Lu-177, Re-186, Re-188 or Bi-212 desirable. In addition, the linker is preferably -maleimido-, wherein -maleimido- is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and aminoethylmaleimide (aminoehtylmaleimide) is preferably prepared by treating a substance to which it is bound, more preferably using DOTA-aminoethyl-maleimide.

The present invention also provides 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) -maleimido-cyclo [Arg-Gly-Asp-dPhe-Cys] and pharmaceuticals thereof 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) -maleimido-cyclo [Arg-Gly-Asp-dTyr-Cys] and It provides a method for preparing a pharmaceutically possible salt thereof. This method comprises the steps of: 1) preparing an cyclo [Arg-Gly-Asp-dPhe-Cys] or a cyclo [Arg-Gly-Asp-dTyr-Cys] through an amino acid pairing reaction; and

2) treating DOTA-aminoethyl-maleimide with cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys] prepared in step 1).

The present invention also provides a method of providing a cyclo [Arg-Gly-Asp-dPhe-Cys] and a pharmaceutically possible salt thereof linked by a linker conjugated with a chelating agent labeled with a metal, transition metal ion or radioisotope. The present invention also provides a method of providing cyclo [Arg-Gly-Asp-dTyr-Cys] and a pharmaceutically possible salt thereof linked by a linker conjugated with a chelating agent labeled with a metal, transition metal ion or radioisotope. This can be achieved by 1) preparing cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys] through an amino acid pairing reaction;

2) treating DOTA-aminoethyl-maleimide with cyclo [Arg-Gly-Asp-dPhe-Cys] or cyclo [Arg-Gly-Asp-dTyr-Cys] prepared in step 1); and

3) DOTA-maleimido-cyclo [Arg-Gly-Asp-dPhe-Cys] or DOTA-maleimido-cyclo [Arg-Gly-Asp-dTyr-Cys] prepared in step 2), metal, transition metal ion or radioactive Labeling with isotopes.

In this case, the chelating agent is preferably labeled with Fe, Gd, Mn, lanthanide or lanthanum, gamma, beta, or alpha-emitting radioisotopes, more preferably Sc-47, Cu-61, Cu- 62, Cu-64, Cu-67, Ga-67, Ga-68, Y-86, Y-90, Tc-99m, Rh-105, In-111, Sn-117m, Pm-149, Sm-153, Labeled Dy-165, Ho-166, Er-169, Yb-169, Lu-177, Re-186, Re-188 or Bi-212. In addition, the linker is preferably -maleimido-, wherein -maleimido- is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and aminoethyl It is preferable that the material is prepared by treating a substance in which maleimide (aminoehtylmaleimide) is bound, and more preferably, by treating DOTA-aminoethyl-maleimide.

Hereinafter, the present invention will be described in more detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Preparation of an RGD Derivative Linked with a Linker Conjugated with a Chelating Agent

(1) Synthesis of DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys)

DOTA-Maleimido-cyclic- (derivative using a thiol-containing derivative in the form of Cyclic- (RGDfC), that is, aminoehtylmaleimide in DOTA, for bioconjugation of Thiol and DOTA. RGDfC) was prepared. Specifically, the following method is performed (see FIG. 11).

Synthesis of Cyclic (RGDfC) Peptides

Wang resin was washed with DMF (N , N- Dimethylformamide) and then with 1.0M HOBt solution dissolved in DMF. Then, a DMF solution containing 3 equivalents of HOBt, 3 equivalents of HBTU, 6 equivalents of DIPEA, and 3 equivalents of Nα-Fmoc-L-aspartic acid-α-Allylester (Fmoc-Asp-OAll) was added to the resin. After stirring for 2 hours the amino acids were paired. Synthesis was confirmed by a negative reaction through a Kaiser test (E. Kaiser et al ., Anal. Biochem. ( 1970 ) 34 , 595). When positive, the resin is washed with DMF and contains 3 equivalents of HOBt, 3 equivalents of HBTU, 6 equivalents of DIPEA, and 3 equivalents of Nα-Fmoc-L-aspartic acid-α-Allylester (Fmoc-Asp-OAll) DMF solution was added and stirred for 3 hours, and the mating reaction was performed until the Kaiser test was negative. When the reaction was completed, the resin was washed with DMF, and the unreacted site of the amine group included in the first resin was placed in a 1: 1 ratio of DMF solution containing 20% acetic anhydride and 20% DIPEA. Stirred for a minute. To remove N ^α -Fmoc, a DMF solution containing 20% piperidine was added and stirred for 10 minutes. The resin was then washed in the order of DMF, DCM and DMF, followed by N ^α -Fmoc amino acids in the order of Glycine with N ^α -Fmoc protecting group, Arg (Ng-Pbf), Cys (S-Trt), D form-Phe The addition reaction was carried out by addition. To this, 3 equivalents of Tetrakis (triphenylphosphine) Paladium (0) and 3 equivalents of 1,3-dimethylbarbituric acid were reacted in DCM solvent to remove and wash ally ester, followed by addition of DMF solution containing 3 equivalents of DIC and HOAT. Stir for 3 hours and proceed with the mating reaction until a Kaiser test is negative.

For the removal of the protecting group and the separation from the resin, TFA: TIS: EDT: Thioanisole: Water = 90: 2.5: 2.5: 2.5: 2.5 was added for reaction for 2 hours to separate the desired compound, and the mixed solution thus obtained was refrigerated. Precipitates were formed by treating the stored diethyl ether solvent in excess. The obtained precipitate was centrifuged to completely settle, and excess TFA was first removed and the above procedure was repeated twice to obtain a solidified peptide.

The resulting peptide was purified by HPLC using a C-18 column and using a 5% to 100% acetonitrile / water concentration gradient solvent system containing 0.01% TFA over 50 minutes. The pure purified fractions were lyophilized to give the desired cyclic (Arg-Gly-Asp-dPhe-Cys) as a white powder. Peptides were synthesized.

Conjugation with DOTA Chelating Agent

In order to attach the DOTA chelator, a DMF solution containing 2 equivalents of DOTA-amidoethyl-maleimide and 2 equivalents of DIPEA was added to the peptide synthesized in the above (1) and stirred for 2 hours.

The resulting peptide was purified by HPLC using a C-18 column and using a 5% to 100% acetonitrile / water concentration gradient solvent system containing 0.01% TFA over 50 minutes.

The pure purified fractions were lyophilized to give the desired DOTA-amidoethyl-maleimido-cyclic (Arg-Gly-Asp-dPhe-Cys) as a white powder. Peptides were synthesized.

[DOTA-maleimido-cyclic (RGDfC)]

The synthesized results showed a yield of 99.8% at 6.47 minutes when analyzed by HPLC, and M + 1: 1105 when analyzed by Mass (FIG. 1).

(2) Preparation of DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dTyr-Cys).

DOTA-Maleimido-cyclic- (RGDyC) using a derivative having a thiol group in the side chain, which is in the form of Cyclic- (RGDyC), that is, an aminoehtylmaleimide conjugated to DOTA for bioconjugation of Thiol and DOTA. ) Was prepared (see FIG. 12).

In the same manner as in <Example 1>, except that N ^α -Fmoc-D form-Phe used in (1) was changed to N ^α -Fmoc-D form-Tyr (O-tBu), Peptides and DOTA-amidoethylmaleimido-cyclic (Arg-Gly-Asp-dTyr-Cys) Peptides were synthesized.

 [DOTA-Maleimido-cyclic- (RGDyC)]

The synthesized results showed a yield of 98.3% at 6.61 minutes when analyzed by HPLC, and M + 1: 1121 when analyzed by Mass (FIG. 2).

Example 2 Preparation of RGD Derivatives Containing Radiolabeled Chelating Agents

[DOTA-maleimide-cyclic (RGDfC) labeled with radioisotopes (M)]

Where M is Fe, Gd, Mn, Zn, lanthanide or lanthanum, or gamma, beta, or alpha-emission radioisotope, allowing imaging with radiological diagnostic devices such as MRI, CT or gamma cameras, SPECT, PET Element, more specifically Sc-47, Cu-61, Cu-62, Cu-64, Cu-67, Ga-67, Ga-68, Y-86, Y-90, Tc-99m, Rh-105 , In-111, Sn-117m, Pm-149, Sm-153, Dy-165, Ho-166, Er-169, Yb-169, Lu-177, Re-186, Re-188 and Bi-212 do.

(One) Preparation and Analysis of Lu-177-DOTA-maleimido-cyclic (RGDfC)

50 mg ascorbic aicd (500mg / 1M Na acetate buffer, pH 5.0, 2ml), 6mg dihydroxybenzoic aicd (60mg /) as a stabilizer after taking ~ 100ug from material prepared with RGD derivative (1mg / 1M Na acetate buffer, pH5.0) 1M Na acetate buffer, pH5.0, 1ml) to prepare the vial, put in ¹⁷⁷ LuCl ₃ 1mCi, and reacted for 30 minutes at 90 ℃ and cooled in an ice bath. For HPLC analysis of labeled radioligands, it was analyzed using Waters HPLC.

a C-18 reversed phase X-Terra (5 μM, 4 × 250 mm) column was used . Water (A) and acetonitrile (B) containing 1% trifluoroacetic acid were analyzed at 1 mL / min flow rate. As a result, it was confirmed that it was prepared in 100% label yield (in the case of ¹⁷⁷ LuCl ₃ has a retention time of 3 to 4 minutes) (FIG. 3).

[Solvent Flow Conditions Solvent (A): 100 to 90%, 2 min; 90 to 60%, 10 min; 60 to 30%, 2 min; 30%, 3min, 30 to 100%, 3 min]

(2) Preparation and Analysis of Y-90-DOTA-maleimido-cyclic (RGDfC)

50 mg ascorbic aicd (500mg / 1M Na acetate buffer, pH 5.0, 2ml), 6mg dihydroxybenzoic aicd (60mg /) as a stabilizer after taking ~ 100ug from material prepared with RGD derivative (1mg / 1M Na acetate buffer, pH5.0) 1M Na acetate buffer, pH5.0, 1ml) to prepare the vial, put in ⁹⁰ YCl ₃ 1mCi, and reacted for 90 to 30 minutes and then cooled in an ice bath.

In addition, the labeled radioligand was analyzed using Waters HPLC. A C-18 reversed phase X-Terra (5μM, 4 × 250 mm) column was used . Water (A) and acetonitrile (B) containing 1% trifluoroacetic acid were analyzed at 1 mL / min flow rate. As a result, it was confirmed that the labeling yield was 94% or more ( ⁹⁰ YCl ₃ has a retention time of 3 to 4 minutes) (FIG. 4).

[Solvent Flow Conditions Solvent (A): 100 to 90%, 2 min; 90 to 60%, 10 min; 60 to 30%, 2 min; 30%, 3min, 30 to 100%, 3 min]

(3) Preparation and Analysis of Sm-153-DOTA-maleimide-cyclic (RGDfC)

50 mg ascorbic aicd (500mg / 1M Na acetate buffer, pH 5.0, 2ml), 6mg dihydroxybenzoic aicd (60mg /) as a stabilizer after taking ~ 100ug from material prepared with RGD derivative (1mg / 1M Na acetate buffer, pH5.0) 1M Na acetate buffer, pH5.0, 1ml) to prepare the vial, put in ¹⁵³ SmCl ₃ 1mCi, and reacted for 90 to 30 minutes and then cooled in an ice bath.

For HPLC analysis of labeled radioligands, it was analyzed using Waters HPLC.

a C-18 reversed phase X-Terra (5μM, 4 × 250 mm) column was used . Water (A) and acetonitrile (B) containing 1% trifluoroacetic acid were analyzed at 1 mL / min flow rate. As a result, it was confirmed that the labeling yield was 93% or more (the case of ¹⁵³ SmCl ₃ has a retention time of 3-4 minutes) (FIG. 5).

[Solvent Flow Conditions Solvent (A): 100 to 90%, 2 min; 90 to 60%, 10 min; 60 to 30%, 2 min; 30%, 3min, 30 to 100%, 3 min]

Example 3 Labeling of DOTA-Maleimido-cyclic (RGDfC)

In the labeling experiment using Lu-177 (90 ℃ 20 minutes labeling) using a high specific radioactivity, it was confirmed that the initial radioactivity concentration of 100 mCi or more can be labeled even if the ratio of ligand: isotope is about 2.1: 1.

Peptide

usage

Initial radiation dose
(activity)

ratio
(Ligand: RI)
Specific radioactivity
(specific activity)

DOTA-Maleimido-cyclic (RGDfC)
60 μg
104mCi
2.1: 1
1.73 mCi / μg

Example 4 Labeling Yield of RGD Derivatives Containing Radiolabelled Chelating Agents evaluation

(1) Evaluation of label yield of Lu-177-DOTA-Maleimido-cyclic (RGDfC) and Y-90-DOTA-Maleimido-cyclic (RGDfC)

In the case of temperature change, it was confirmed that the label was labeled with Incubation of 10 minutes or more at 90 ° C. or higher when evaluated at 50 ° C., 75 ° C., and 90 ° C. (FIG. 6). The label yield was determined using DOTA-Maleimido-cyclic (RGDfC) at concentrations of 10-6, 10-7, 10-8, and 10-9 mol, and at least 98% label yield at 10-7 mol. It was seen that (Fig. 7).

It was confirmed that the label yield evaluation results according to the temperature and concentration change were the same in the case of Y-90.

Example 5 Safety Assessment of RGD Derivatives Containing Radiolabeled Chelating Agents

The stability was evaluated up to 72 hours at room temperature, 37 ° C. and serum conditions (performed at 37 ° C.) using Y-90-DOTA-Maleimido-cyclic (RGDfC). 8).

Example 6 Biodistribution Analysis of DOTA-Maleimido-cyclic (RGDfC) Containing Radiolabelled Chelating Agents

Female Balb / C athymic mice ( nu / nu genotype) were raised and maintained in negative pressure isolators. Mice were reared in a barrier facility at a 12 hour light / dark cycle and provided with appropriate sterile feed, water. All procedures were performed in mice at least 7 weeks old and Calu6 tumor xenocraft was established by subcutaneous injection of serum-free medium and 50:50 Matrigel (BD Bioscience) 100 ul of 1 × 10 ⁶ cells into the hind arm. . Two weeks after cell inoculation, RI labeled-DOTA-maleimide-cyclic (RGDfC) 0.185 MBq (5 uCi) was injected intravenously when tumors grew to a volume of approximately 150-200 mm ³ . After injection, mice were killed at the appropriate time and radioactivity of tumors, kidneys, liver, spleen, heart, small intestine, large intestine, lung, stomach and blood was measured with a γ-scintillation counter (Perkin Elmer). The percentage of injections per gram of tissue (% ID / g (cpm organ / g organ) / cpm injected) 100) for 5 animals.

(1) Lu-177-DOTA-Maleimido-cyclic (RGDfC) Biodistribution Analysis

1 × 10 ⁶ cultured Calu ⁶ cells were implanted in the nape of Balb / C nude mouse females (18g-23g). After 10 days of cancer growth, 100 μl of saline solution of Lu-177-DOTA-Maleimido-cyclic (RGDfC) 5 uCi (1.6 μg) was added to the non-small cell lung carcinoma bearing mice (Calu6). injected into the lateral tail vein and confirmed the change in the distribution of ¹ Lu-177-DOTA-Maleimido-cyclic (RGDfC) in the body. 1: 5.24) confirmed the availability as a reagent for tumor diagnosis. In addition, after 24 hours, it was confirmed that the tumor accumulation ratio was 8.16 times accumulated with the persistence in the tumor, and the potential as a tumor treatment agent was also confirmed (FIG. 9).

(2) Biodistribution analysis of Y-90-DOTA-Maleimido-cyclic (RGDfC)

Cultured Calu ⁶ cells 5 × 10 ⁶ were implanted in the nape of Balb / C nude mouse females (18 g-23 g). After 10 days of cancer growth, 100 μl of Y-90-DOTA-Maleimido-cyclic (RGDfC) 2 uCi (0.111 MBq) of saline was added to the non-small cell lung carcinoma bearing mice (Calu6). After injection into the lateral tail vein and the change in the distribution of Y-90-DOTA-Maleimido-cyclic (RGDfC) in the body, it accumulates rapidly and at a high rate after 2 hours (after 2 hours, 1: 14.47) The availability as a reagent for tumor diagnosis was confirmed. In addition, after 24 hours, the tumor accumulation ratio of blood was 13.56 times, and it was confirmed that the present derivative was accumulated in the tumor with sustainability. The potential as a tumor therapeutic agent was also confirmed (FIG. 10).

(3) As a result of analysis, RGD derivatives bound to radioisotope-labeled chelating agents differ slightly depending on the type of metal ion and nuclide used, but the tumor accumulation ratio is significantly higher than other organs in the body, which is high after administration. As a result, it has been confirmed that accumulation in tumors and high ratios are maintained for a considerable time, so that it can be applied as a diagnostic and therapeutic agent for tumors.

The compounds of the present invention and their pharmaceutically possible salts can be used for the diagnosis and treatment of cancer.

1 is a result of HLPC analysis of DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys), yielding 99.8% yield at 6.47 minutes.

Figure 2 shows the results of HLPC analysis of DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dTyr-Cys), yielding 98.3% yield at 6.61 minutes.

Figure 3 shows the results of the HLPC analysis of Lu-177-DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys), it can be seen that Lu-177 is labeled in 100% yield.

Figure 4 shows the results of HLPC analysis of Y-90-DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys), it can be seen that Y-90 was labeled with a yield of 94% or more.

5 shows HLPC analysis of Sm-153-DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys), and it can be seen that Sm-153 is labeled with a yield of 93% or more.

FIG. 6 shows 95% or more of Incubation at 90 ° C. over 10-20 minutes as a result of labeling yield according to temperature change of Lu-177-DOTA-Maleimido-Cyclic (Arg-Gly-Asp-dPhe-Cys) It can be confirmed that it is labeled in yield.

7 shows that the labeling yield according to the ligand concentration of Lu-177-DOTA-Maleimido-cyclic (RGDfC) (labeling yield according to RGD concentration) in more than 98% at a concentration of 10-7 mol or more.

8 shows that the stability was maintained as a result of performing the experiment up to 72 hours (3 days) at 37 ° C. and serum conditions of Lu-177-DOTA-Maleimido-cyclic (RGDfC) (test using HPLC) Result data). The stability of RGD after Y-90 labeling was measured.

Figure 9 is a graph comparing the change in the distribution in the mouse of Lu-177-DOTA-Maleimido-cyclic (RGDfC). Biodistribution of 177Lu-DOTA-RGD in Calu6 tumor bearing mice at 2, 24hr after injection is shown 2 hours and 24 hours after administration.

10 is a graph comparing the change in the distribution of mouse in Y-90-DOTA-Maleimido-cyclic (RGDfC). The biodistribution of 90Y-DOTA-RGD in Calu6 tumour bearing mice at 2, 24hr after injection is shown 2 hours and 24 hours after administration.

11 is a schematic diagram showing a method for producing DOTA-maleimido-cyclic (RGDfC).

12 is a schematic diagram showing a method for producing DOTA-maleimido-cyclic (RGDyC).

Claims (15)

A compound of Formula 1 or a pharmaceutically possible salt thereof: <Formula 1> C-L-X Where C is a chelating agent, L is a linker, X is cyclo [Arg-Gly-Asp-dTyr-Cys], L connects C and X with a sulfide bond through sulfur (S) of Cys of X. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the linker is maleimido. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). delete According to claim 1, wherein the compound having a structure of formula (3) or a pharmaceutically acceptable salt thereof <Formula 3>

. 6. A compound according to any one of claims 1 to 3 and 5, or a pharmaceutically acceptable salt thereof, characterized in that the chelating agent is labeled with a metal, transition metal ion or radioisotope. The method according to any one of claims 1 to 3 and 5, wherein the chelate is Fe, Gd, Mn, Zn, lanthanide or lanthanum, but gamma, beta, or alpha-emitting radioisotope is labeled. Compounds or pharmaceutically possible salts thereof. The method according to any one of claims 1 to 3 and 5, wherein the chelate is Sc-47, Cu-61, Cu-62, Cu-64, Cu-67, Ga-67, Ga-68, Y -86, Y-90, Tc-99m, Rh-105, In-111, Sn-117m, Pm-149, Sm-153, Dy-165, Ho-166, Er-169, Yb-169, Lu-177 , Re-186, Re-188 and Bi-212, characterized in that any one selected from the group consisting of compounds or pharmaceutically possible salts thereof. 6. The compound of any one of claims 1-3 and 5, or a pharmaceutically acceptable salt thereof, wherein the chelate is labeled Y-90, Lu-177 or Sm-153. 7. A reagent for diagnosing or treating cancer, comprising the compound of claim 8 or a pharmaceutically acceptable salt thereof. The reagent for diagnosing or treating cancer of claim 10, wherein the cancer is diagnosed by a target image of a CT, MRI, or radiation diagnostic apparatus. The method of claim 10, wherein the cancer is chronic lymphocytic leukemia, breast cancer, cervical cancer, fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endocytoma, synovial membrane Carcinoma, mesothelioma, Ewing tumor, smooth sarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon tumor, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, impression cell tumor, Sebaceous gland tumor, papillary tumor, papillary adenocarcinoma, cystic adenocarcinoma, medulla tumor, tracheal support tumor, renal cell tumor, liver cancer, biliary gland tumor, chorionic cancer, normal carcinoma, fetal tumor, Wilm's tumor, testicular cancer, lung tumor , Small cell lung tumor, non-small cell lung tumor, bladder tumor, epithelial tumor, glioma, astrocytoma, medulloblastoma, craniocytoma, cerebral hepatocellular carcinoma, pineal carcinoma, hemangioblastoma, auditory neuroma, dendritic gliomas, hydrocephalus, melanoma , Neuroblastoma, retinoblastoma, white Disease, lymphoma, and Kaposi (Kaposi) any one of the reagents for the diagnosis or treatment of cancer, characterized selected from the group consisting of sarcoma. delete delete delete

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