KR101551232B1 - Novel N3S1 chelator-folate derivatives, preparation method thereof and composition for diagnosis or treatment of cancer containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a folate derivative to which a novel chelator of the N ₃ S ₁ type is conjugated, a method for producing the same, and a composition for diagnosing and treating cancer containing the same as an effective ingredient. The folate derivative to which the novel chelator of the N ₃ S ₁ type according to the present invention is conjugated or a pharmaceutically acceptable salt thereof may be a receptor binding to a tumor in which an α-folate receptor (α-FR) such as ovarian cancer is expressed Into the cell is useful. Therefore, the folate derivative can be widely used in various radiopharmaceutical markers because it can be used for the diagnosis and treatment of tumors using radioactive isotopes such as technetium, rhenium, etc., have.

Description

[0001] The present invention relates to a folate derivative to which a novel chelator of the N3S1 type is conjugated, a method for producing the same, and a composition for diagnosing or treating cancer containing the same as an active ingredient. same as an active ingredient}

The present invention relates to a folate derivative to which a novel chelator of the N ₃ S ₁ type is conjugated, a process for producing the same, and a composition for diagnosing or treating cancer containing the same as an active ingredient.

Folate is introduced into cells by RFC (Reduced Folate Carrier) or endocytosis by folate receptors. In general, α-Folate receptor (α-FR) is overexpressed in a variety of tumors including ovarian, endometrial, breast, lung, renal, colorectal, nasopharyngeal, and colon cancer , And in the case of normal cells, it exists only in a part of kidney (Kidney) and lung (lung) which stores or utilizes vitamins.

Ovarian carcinoma and endometrial carcinoma are the most frequently expressed follicles, which are known to be present in more than 90% of cancer patients. Accordingly, various attempts have been made to transmit drugs to cancer cells effectively by targeting the α-folate receptor (α-FR) over-expressed in tumors and to image cancer.

The α-folate receptor (α-FR) is well known as a membrane-binding tumor-associated antigen with high affinity and is glycosylphosphatidylinositol (GPI) conjugated with a membrane glycoprotein having a molecular weight of 38 to 40 kDa. -Folate receptor (? -FR) binds with high affinity to polylactic acid, with a dissociation constant (K _d ) with poly acid in the α-isoform of the folate receptor being as low as about 0.1 nM, Which is about 10 times lower than the dissociation constant of a derivative such as 5-methyltetrahydrofolate, which is a form of folate.

On the other hand, the polyacid, which is a compound having a high affinity with the folate receptor, can maintain the receptor binding property when it is covalently bound to another molecule through the? -Carboxyl group.

Recently, a renal cell positive α-Folate receptor (α-FR positive renal cell) and ovarian cancer (ovarian cancer) have been identified using Tc-99m-EC20 and In-111-DTPA- carcinoma) successfully terminated clinical phase I and II.

The noninvasive imaging method that can selectively target the tumor can detect the primary tumor or accurately identify the tumor during surgery by acquiring the image, and can accurately determine the stage by detecting the metastatic lesion. Observe and find recurrent lesions.

Therefore, the value will become even greater if radioactive isotopes are used to target and selectively diagnose or treat tumors in a non-invasive manner.

Thus, the present inventors have been studying to synthesize substances for effectively targeting and targeting tumors to the α-folate receptor (α-FR) and for diagnosing and treating α-folate receptor positive tumors in a non-invasive manner (A-FR) characteristic by introducing a chelator in the form of N ₃ S ₁ composed of an amino acid capable of labeling the diagnostic and therapeutic radioactive isotope, The present invention has been accomplished on the basis of these findings.

It is an object of the present invention to provide a folate derivative to which a novel chelator of the N ₃ S ₁ type composed of amino acids is conjugated or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for producing a folate derivative or a pharmaceutically acceptable salt thereof, wherein the novel chelator of the N ₃ S ₁ type is conjugated.

It is still another object of the present invention to provide a complex of a folate derivative and a radioactive isotope to which the novel chelator of the N ₃ S ₁ type is conjugated.

It is another object of the present invention to provide a composition for diagnosing and treating cancer, which comprises a folate derivative to which the novel chelator of the N ₃ S ₁ type is conjugated and a complex of a radioisotope as an active ingredient.

It is still another object of the present invention to provide a kit for forming complexes of folate derivatives and radioactive isotopes to which the novel chelator of the N ₃ S ₁ type is conjugated.

In order to achieve the above object, the present invention provides a folate derivative to which a novel chelator of the N ₃ S ₁ type represented by the following formula (1) is bonded, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

Wherein R, R ¹ , R ² _, R ³ and p are as defined herein.

In addition, the present invention provides a method for producing a folate derivative or a pharmaceutically acceptable salt thereof, wherein the novel chelator of the N ₃ S ₁ type is conjugated.

Furthermore, the present invention provides complexes of radioactive isotopes with folate derivatives to which the novel chelator of the N ₃ S ₁ type is conjugated.

The present invention also provides a composition for diagnosing and treating cancer, which contains, as an active ingredient, a folate derivative to which a novel chelator of the N ₃ S ₁ type is conjugated and a complex of a radioactive isotope.

Further, the present invention provides a kit for forming complexes of folate derivatives and radioactive isotopes to which the novel chelator of the N ₃ S ₁ type is conjugated.

The folate derivative to which the novel chelator of the N ₃ S ₁ type according to the present invention is conjugated or a pharmaceutically acceptable salt thereof may be a receptor binding to a tumor in which an α-folate receptor (α-FR) such as ovarian cancer is expressed Into the cell is useful. Therefore, the folate derivatives can be useful for the diagnosis and treatment of tumors using radiations labeled with technetium, rhenium or the like and imaging the tumors and radiation emitted by the isotopes, so that they are useful for labeling various radiopharmaceuticals .

Fig. 1 is a graph showing LC / MS results of a PKCK-fol compound as a folate derivative to which a novel chelator of the N ₃ S ₁ type is bonded in Example 1 of the present invention.
Figure 2 is a view showing an embodiment 8 ^{99 m} Tc above PKCK-fol-OH ignition cover the water HPLC results by the present invention.
Figure 3 is a view showing the HPLC results after 24 hours of incubation the Example 8 ^{99 m} Tc to the above-PKCK fol-OH complexes and serum markers according to the present invention.
Figure 4 is a view showing the folate receptor-positive KB tumors with a ^{99 m} Tc in the cell cover PKCK-fol-OH complexing water cell affinity and internalization rate by the Experimental Example 1 of the present invention.
Figure 5 is a view of the SPECT image of a nude-lasting in vivo injection KB tumor after 3 hours of ^{99 m} Tc the PKCK-fol-OH complexes (185 Mbq, 5 mCi) labeled according to Example 3 of the present invention the rat .
Figure 6 is a nude rat-lasting a KB tumor was added to the scan in the ^{99 m} Tc is labeled the PKCK-fol-OH complexes (185 Mbq, 5 mCi) and folate 0.25 μmol according to Example 3 of the present invention SPECT image.
7 is a view showing a 3D image of the ^{99 m} Tc is above PKCK-fol-OH complexing tumor after the body of water injection 18 times and height each SPECT / CT markers according to Example 3 of the present invention.
Figure 8 is a view showing an image after an hour after injection of ^{99 m Tc-PKCK-fol-} OH complex of the KB xenograft according to Example 3 of the present invention.
FIG. 9 is a view showing an image after one hour after blocking folate according to Experimental Example 3 of the present invention and injecting ^99m Tc-PKCK-fol-OH complex. FIG.
10 is a view showing an image after 3 hours after the injection of ^{99 m Tc-PKCK-fol-} OH complex of the KB xenograft according to Example 3 of the present invention.
FIG. 11 is a view showing an image after 3 hours after blocking folate according to Experimental Example 3 of the present invention and injecting ^99m Tc-PKCK-fol-OH complex. FIG.
12 is a view showing the image after 18 hours after injection of ^{99 m Tc-PKCK-fol-} OH complex of the KB xenograft according to Example 3 of the present invention.
FIG. 13 is a diagram showing an image after 18 hours after blocking folate according to Experimental Example 3 of the present invention and injecting ^99m Tc-PKCK-fol-OH complex. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a folate derivative to which a novel chelator of the N ₃ S ₁ type represented by the following general formula (1) is bonded.

In Formula 1,

R is hydroxy or amine;

,

,

,

,

,

,

,

,

또는

이고, 이때 상기 Aaa는 1 내지 3개의 아미노산, D-form 아미노산 및 이들의 유도체로부터 선택되는 어느 하나이고;R < ^{1 >} is a non-

,

,

,

,

,

,

,

,

or

, Wherein Aaa is any one selected from 1 to 3 amino acids, D-form amino acids and derivatives thereof;

l is an integer from 1 to 10;

m is an integer from 1 to 10;

n is an integer from 1 to 10;

o is an integer from 1 to 20;

R ² is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine Amino acid selected;

,

,

,

,

,

,

또는

이고;R ³ is

,

,

,

,

,

,

or

ego;

R < ⁴ > is halogen, hydroxy, amine;

R ⁵ is a C ₁ to C ₄ alkyl; And

p is an integer of 1 to 10;

Preferably,

Wherein R is hydroxy or amine;

,

,

,

,

,

,

,

,

또는

이고, 이때 상기 Aaa는 1 내지 3개의 아미노산, D-form 아미노산 및 이들의 유도체로부터 선택되는 어느 하나이고;R < ^{1 >} is a non-

,

,

,

,

,

,

,

,

or

, Wherein Aaa is any one selected from 1 to 3 amino acids, D-form amino acids and derivatives thereof;

l is an integer from 1 to 4;

m is an integer from 1 to 4;

n is an integer from 1 to 6;

o is an integer from 1 to 10;

R ² is a group consisting of alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine ≪ / RTI >

,

,

,

,

,

,

또는

이고;R ³ is

,

,

,

,

,

,

or

ego;

R < ⁴ > is fluoro, hydroxy, amine;

R < ⁵ > is methyl or ethyl; And

p is an integer of 1 to 4;

N ₃ S new chelators are conjugated folate derivative of type ₁ represented by the general formula (I) of the present invention are pharmaceutically acceptable can be used as a possible form of salts, salt with the pharmaceutically acceptable free acid (free acid ) Is useful as an acid addition salt. The term pharmaceutically acceptable salt means a concentration that is relatively non-toxic to a patient and has a beneficial effect that is harmless to the patient, such that the side effects caused by the salt are any organic or inorganic salt of the base compound of Formula 1 that does not impair the beneficial effects of the base compound of Formula An inorganic addition salt. Examples of the inorganic acid include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, maleic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, succinic acid, tartaric acid, galacturonic acid, embic acid, glutamic acid, aspartic acid, oxalic acid, P-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid. These salts also include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include, but are not limited to, acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, eddylate, , Glucoside, glucoside, glucoside, gluconate, gluconate, glucuronate, hexafluorophosphate, hybene zeit, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, But are not limited to, maleic anhydride, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, Saccharate, stearate, succinate, tartrate, tosylate, trifluoroacetate Diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like, and they may be used alone or in combination of two or more thereof. Preferred is hydrochloride or trifluoroacetate.

In addition, N ₃ new chelators are conjugated folate derivatives of S ₁ type is any salt as well as pharmaceutically acceptable salts, may be prepared by a conventional method, isomers represented by the general formula (I) of the present invention, Hydrates and solvates thereof.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Chemical Formula 1 in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile, etc., Followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

Also, as shown in the following Reaction Scheme 1,

Reacting a polyacid derivative of formula (2) with a compound of formula (3) to produce a folate derivative of formula (4) (step 1); And

By removing a folate derivative in the solid phase support chain resin (resin) of formula (4) prepared in Step 1 in acid conditions, comprising the step (step 2) for preparing a compound of Formula 1, N ₃ S ₁ Type chelator is bonded to a chelate derivative of the present invention.

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

R, R ¹ , R ² , R ³ and p are the same as defined in Formula 1; resin is a solid support; And Trt is a trityl group.)

Hereinafter, the manufacturing method will be described step by step.

First, the step 1 according to the present invention is a step of binding-reacting a polyacid derivative with a compound of the formula (3), and more specifically, bonding a benzoester group of a polyacid derivative with a terminal amine of the compound of the formula 4 < / RTI >

At this time, the coupling reaction is carried out with diisopropylethylamine or triethylamine in the presence of benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (Py-BOP) Benzotriazole-N, N, N ', N'-tetramethyl-euenium-hexafluoro-phosphate (HBTU), 2- , 1,3,3-tetramethyluronium hexafluorophosphate (HATU), hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- ), Carbodiimide (EDC), or carbonyldiimidazole (CDI) can be used, and preferably hydroxybenzotriazole (HOBt) and / or O-benzotriazole-N, N, N ', N '-Tetramethyl-euenium-hexafluoro-phosphate (HBTU) may be used.

In addition, as the usable organic solvent, the reaction can be carried out using methanol, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran, dichloromethane, toluene or the like which does not adversely affect the reaction , And preferably dimethylsulfoxide (DMSO) or dimethylformamide (DMF) can be used.

Next, Step 2 according to the present invention is a step of removing the resin and the protecting group, which are the solid support of the compound of Chemical Formula 4 prepared in Step 1.

In order to remove the resin, it is preferable to use TFA (Trifluoroacetic acid), TIS, EDT, Thioanisole and water to remove the resin. In order to remove the resin effectively, TFA, TIS, EDT, And water are used in combination with TFA: TIS: EDT: thioanisole: water = 90-92: 2.0-2.5: 2.0-2.5: 2.0-2.5: 2.0-2.5.

Further, the present invention provides a compound represented by the following formula (2)

Reacting protected lysine with an Alloc protecting group and protected cysteine with an Fmoc protecting group to produce a compound represented by formula (5) (step 1);

Reacting the compound of Formula 5 and the compound of Formula 6 prepared in Step 1 to prepare a compound of Formula 7 (Step 2);

Reacting the compound of formula (7) and the compound of formula (8) prepared in step 2 to prepare a compound of formula (9) (step 3); And

(3) reacting the compound of formula (9) prepared in step 3 with the compound of formula (10) to prepare a compound represented by formula (3) (step 4) ≪ / RTI >

[Reaction Scheme 2]

(In the above Reaction Scheme 2,

R, R ¹ , R ² , R ³ and p are the same as defined in Formula 1; resin is a solid support; Alloc is allylcarbamate; Fmoc is a fluorenylmethyloxycarbonyl group; And Trt is a trityl group.)

Hereinafter, the above manufacturing method will be described in detail for each step.

First, the step 1 according to the present invention is a step of reacting a lysine protected with an Alloc protecting group and a cysteine protected with an Fmoc protecting group to prepare a compound represented by the general formula (5). More specifically, the terminal amine is protected with an Alloc protecting group The other end amine of the protected lysine and the terminal amine are the Fmoc protecting group, and the thiol is the step of coupling the carboxylic acid of the cysteine protected with the trityl protecting group to prepare the compound of formula (5).

At this time, the coupling reaction is carried out with diisopropylethylamine or triethylamine in the presence of benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (Py-BOP) Benzotriazole-N, N, N ', N'-tetramethyl-euenium-hexafluoro-phosphate (HBTU), 2- , 1,3,3-tetramethyluronium hexafluorophosphate (HATU), hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- ), Carbodiimide (EDC), or carbonyldiimidazole (CDI) can be used, and preferably hydroxybenzotriazole (HOBt) and / or O-benzotriazole-N, N, N ', N '-Tetramethyl-euenium-hexafluoro-phosphate (HBTU) may be used.

In addition, as the usable organic solvent, it is possible to carry out the reaction using methanol, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dichloromethane, toluene or the like which does not adversely affect the reaction, Methyl sulfoxide or dimethyl formamide can be used.

Next, the step 2 according to the present invention is a step of reacting the compound of the formula (5) prepared in the step 1 with the compound of the formula (6) to prepare a compound represented by the formula (7), more specifically, Removing the Fmoc protecting group of the protected terminal amine of the compound represented by the formula (5), and then reacting the unprotected terminal amine with the compound of the formula (6) to prepare the compound of the formula (7).

At this time, in the step 2, the Fmoc protecting group of the formula (5) can be removed by a method commonly used in the art.

The conditions of this reaction are as described in the above step 1.

Next, the step 3 according to the present invention is a step of preparing a compound represented by the formula (9) by reacting the compound represented by the formula (7) and the compound represented by the formula (8) prepared in the step 2, After removing the Fmoc protecting group of the protected terminal amine of the compound represented by the formula (7), the unprotected terminal amine is bonded to the compound represented by the formula (8) protected with the Fmoc protecting group to prepare the compound of the formula .

At this time, the method for removing the Fmoc protecting group represented by the general formula (7) in the above step may be a method commonly used in the art.

The conditions of this reaction are as described in the above step 1.

Step 4 according to the present invention is a step of reacting the compound of Formula 9 and the compound of Formula 10 prepared in Step 3 to prepare a compound of Formula 3, Removing alloc protecting groups of the protected terminal amine of the compound represented by the formula (9), and then reacting the unprotected terminal amine with the compound of the formula (10) to prepare a compound represented by the formula (3).

At this time, in the above step, alloc protecting group of formula (9) can be removed by a method commonly used in the art.

The conditions of this reaction are as described in the above step 1.

The present invention also relates to a process for preparing a compound represented by the following formula (3)

Reacting a phthaloic acid derivative of formula (11) with a compound of formula (12) to prepare a folate derivative of formula (4) (step 1); And

By removing the solid support chain resin (resin) of a folate derivative of formula (IV) prepared in Step 1 in acid conditions, comprising the step (step 2) for preparing a compound of Formula 1, N ₃ S ₁ Type chelator is bonded to a chelate derivative of the present invention.

[Reaction Scheme 3]

(In the above scheme 3,

R, R ¹ , R ² , R ³ and p are the same as defined in Formula 1; resin is a solid support; And Trt is a trityl group.)

Hereinafter, the manufacturing method will be described step by step.

The step 1 of the present invention is a step of reacting a polyamic acid derivative represented by the formula (11) with a compound represented by the formula (12), more specifically, a step of reacting a benzoester group of a polyacid derivative of the formula (11) Is reacted with a terminal amine of the formula (I) to prepare a compound represented by the formula (4).

At this time, the coupling reaction is carried out with diisopropylethylamine or triethylamine in the presence of benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (Py-BOP) Benzotriazole-N, N, N ', N'-tetramethyl-euenium-hexafluoro-phosphate (HBTU), 2- , 1,3,3-tetramethyluronium hexafluorophosphate (HATU), hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- ), Carbodiimide (EDC), or carbonyldiimidazole (CDI) can be used, and preferably hydroxybenzotriazole (HOBt) and / or O-benzotriazole-N, N, N ', N '-Tetramethyl-euenium-hexafluoro-phosphate (HBTU) may be used.

In addition, as the usable organic solvent, it is possible to carry out the reaction using methanol, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dichloromethane, toluene or the like which does not adversely affect the reaction, Methyl sulfoxide or dimethyl formamide can be used.

Next, Step 2 according to the present invention is a step of removing the resin and the protecting group, which are the solid support of the compound of Chemical Formula 4 prepared in Step 1.

In order to remove the resin, it is preferable to use TFA (Trifluoroacetic acid), TIS, EDT, Thioanisole and water to remove the resin. In order to remove the resin effectively, TFA, TIS, EDT, And water are used in combination with TFA: TIS: EDT: thioanisole: water = 90-92: 2.0-2.5: 2.0-2.5: 2.0-2.5: 2.0-2.5.

Further, the present invention relates to a process for preparing a compound represented by the formula

(Step 1) of preparing a compound represented by formula (14) by reacting a compound represented by formula (3) with glutamic acid represented by formula (13); And

And a step (2) of deprotecting the compound of formula (14) prepared in step 1 to prepare a compound represented by formula (12). The novel chelator of the N ₃ S ₁ type Lt; / RTI > derivatives.

[Reaction Scheme 4]

(In the above Scheme 4,

R, R ¹ , R ² , R ³ and p are the same as defined in Formula 1; resin is a solid support; Fmoc is a fluorenylmethyloxycarbonyl group; And Trt is a trityl group.)

Hereinafter, the above manufacturing method will be described in detail for each step.

First, the step 1 of the present invention is a step of preparing a compound represented by the formula (14) by a reaction between a compound represented by the formula (3) and glutamic acid represented by the formula (13), and more specifically, a compound represented by the formula the coupling reaction of carboxylic acid and the terminal R ¹ and formula 13 is a glutamic acid to prepare a compound of formula 14.

At this time, the coupling reaction is carried out with diisopropylethylamine or triethylamine in the presence of benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (Py-BOP) Benzotriazole-N, N, N ', N'-tetramethyl-euenium-hexafluoro-phosphate (HBTU), 2- , 1,3,3-tetramethyluronium hexafluorophosphate (HATU), hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- ), Carbodiimide (EDC), or carbonyldiimidazole (CDI) can be used, and preferably hydroxybenzotriazole (HOBt) and / or O-benzotriazole-N, N, N ', N '-Tetramethyl-euenium-hexafluoro-phosphate (HBTU) may be used.

In addition, as the usable organic solvent, it is possible to carry out the reaction using methanol, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dichloromethane, toluene or the like which does not adversely affect the reaction, Methyl sulfoxide or dimethyl formamide can be used.

Next, the step 2 according to the present invention is a step of reacting the compound of the formula (5) prepared in the step 1 with the compound of the formula (6) to prepare a compound represented by the formula (7), more specifically, Removing the Fmoc protecting group of the protected terminal amine of the compound represented by the formula (5), and then reacting the unprotected terminal amine with the compound of the formula (6) to prepare the compound of the formula (7).

Next, the step 2 according to the present invention is a step of deprotecting the compound represented by the formula (14) prepared in the step 1 to prepare a compound represented by the formula (12). More specifically, the compound represented by the formula (14) And deprotecting the protecting group of the protected terminal amine of the compound to be displayed.

At this time, the method for removing the Fmoc protecting group of the formula (5) may be a method commonly used in the art.

In the method for producing a folate derivative according to the present invention, the solid support is used in combination with a carboxylic acid group of an amino acid introduced into a folate derivative. Preferably, the resin is a Wang resin ) Or Merrifield resin (MBHA) can be used.

At this time, when Wang resin is used, when a resin is desorbed, a hydroxy group is introduced into the R group of the following formula (1), and when a Merrifield resin (MBHA resin) is used, an amine group is introduced when the resin is desorbed .

[Chemical Formula 1]

(Wherein R, R ¹ , R ² , R ³ and p are as defined in the above formula (1)).

The present invention also provides fate complexes to which a novel chelator of the N ₃ S ₁ type labeled with a radioisotope is conjugated.

In a flared complex of a N ₃ S ₁ type labeled with a radioisotope labeled with a novel chelator according to the present invention, the radioactive isotope can be detected by MRI, CT, gamma camera or the like when it is present in a living body And a radioisotope that releases radiation. For example, scandium-47 (Sc-47), copper-64 (Cu-64), gallium-67 (Ga-67), gallium- -90 (Y-90), Technetium-99m (Tc-99m), Indium-111 (In-111), Promethium- 149 (Pm- 149), Samarium- 153 (Sm- 153), Dys- 165), holmium-166 (Ho-166), erbium-169 (Er-169), lutetium-177 (Lu-177), rhenium-186 (Re-186), rhenium- 212 (Bi-212). Preferably, technetium-99m (Tc-99m), rhenium-186 (Re-186) or rhenium-188 (Re-188) can be used.

Furthermore, the present invention provides a method for producing complexes in which a radioactive isotope is labeled with a folate derivative to which a novel chelator of the N ₃ S ₁ type is conjugated.

The labeling of the radioisotope may be performed by adding a chelator-conjugated N ₃ S ₁ type folate derivative to a substrate such as GH, Glucoheptonate, Gluconate, EDTA, Tartrate, Pyrophosphate ) Can be performed by using a ligand exchange reaction kit.

The radioisotope-labeled complexes of the prepared folate derivatives conjugated with the N ₃ S ₁ type of the prepared N ₃ S ₁ type can be prepared at a concentration of 1 nmol at a high yield of 98% or more without purification process (Example 8 Reference).

The present invention also provides a composition for diagnosis or treatment of cancer, which contains, as an active ingredient, a folate derivative to which a novel chelator of the N ₃ S ₁ type is conjugated and a complex of a radioactive isotope.

The folate derivatives to which the N ₃ S ₁ type chelator is bonded have a radiochemical purity of 98% or more and a labeling efficiency of 37 GBq / umol (see FIG. 2). The in vitro stability is 96% or more (See FIG. 3), and cell affinity and internalization rate are time-dependent (see FIG. 4). Therefore, it is useful for diagnosis of cancer using medical imaging equipment or treatment of cancer using energy emitted by radioactive isotopes .

When the composition of the present invention is used as a medicament, a pharmacologically acceptable composition containing the radioactive isotope-labeled N ₃ S ₁ type chelator conjugated fate complex or a pharmaceutically acceptable salt thereof as an active ingredient The composition may be formulated and administered at the time of clinical administration, but is not limited thereto.

The pharmaceutical composition comprising the complex represented by the above formula (1), which is prepared by labeling a radioactive isotope, can be administered parenterally, and parenteral administration can be performed by subcutaneous injection, intravenous injection, intramuscular injection, It depends on the injection method.

At this time, in order to formulate the formulation for parenteral administration, the radioactive isotope-labeled N ₃ S ₁ type chelator conjugated fate complex or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or a buffer Mixed with water to prepare a solution or suspension, which can be prepared into an ampoule or vial unit dosage form. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the pharmaceutical composition containing the fate complex with the novel chelator of the N ₃ S ₁ type represented by the above radioisotope as an active ingredient for the human body depends on the age, body weight, sex, May be varied depending on the health condition and the degree of disease, and preferably at a dose of 0.01 to 200 mg / kg / day, depending on the judgment of the physician or pharmacist, at intervals of several times a day, preferably once to three times a day May be administered by oral or parenteral route.

Further, the present invention provides a kit for forming complexes of folate derivatives and radioactive isotopes to which the novel chelator of the N ₃ S ₁ type is conjugated. Specifically, the kit is a non-heat-sterilized technetium or rhenium-labeled radiopharmaceutical preparation kit, and a technetium or rhenium label using a radioisotope complex is added to a folate derivative to which the novel chelator of the N ₃ S ₁ type is conjugated. , A folate derivative to which a novel chelator of the N ₃ S ₁ type is bonded in advance and a suitable buffer solution are added in a liquid state to a pharmaceutically preferable sterilization vial and sealed or used in a refrigerator, It can be used when necessary.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

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

PREPARATION EXAMPLE 1 Preparation of a polyacid derivative

[Reaction Scheme 5]

The polyamic acid derivative represented by the general formula (2) may be synthesized by a general method commonly used in the field of organic synthesis as shown in Reaction Scheme 5, or a commercially available one may be used.

PREPARATION EXAMPLE 2 Preparation of a Petro Acid Derivative

[Reaction Scheme 6]

The phthaloic acid derivative represented by the general formula (12) can be synthesized by a general method commonly used in the field of organic synthesis or commercially available ones as shown in the reaction scheme (6).

Preparation Example 3 Preparation of P (Boc) -K (Boc) -C (Trt) -K-Wang resin

The Wnag resin was washed with dimethylformamide (DMF) and then washed with 1.0 M hydroxybenzotriazole solution (HOBt, dimethylformamide solution). Thereafter, a solution of 4-amino-5-fluoro-4-fluoro-4-methoxybenzyl chloride (1 g) was added to a mixture of Alloc-Lys (Fmoc) -OH (1 mmol), hydroxybenzotriazole -Phosphate (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) in dimethylformamide and stirred for 2 hours. The resin was then washed with dimethylformamide, dichloromethane and dimethylformamide in that order, dissolved in a solution of dimethylformamide containing 20% piperidine, and stirred for 10 minutes. After stirring was complete, it was washed with dimethylformamide. Next, to the resin was added Fmoc-Cys (Trt) -OH (3 mmol), hydroxybenzotriazole (HOBt, 3 mmol), O-benzotriazole-N, N, N ', N'- (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) were dissolved in dimethylformamide and stirred for 3 hours. Then, Lys (Boc) -OH and Pro (Boc) -OH in which the terminal amine was in turn quenched with an Fmoc protecting group were added in this order to carry out the reaction. When each step is complete, tetrakis (triphenylphosphine) palladium (0) and 1,3-dimethylbabricic acid are added together in dichloromethane, stirred for 2 hours, washed and then purified without further purification To obtain the target compound.

Preparation Example 4 Preparation of P (Boc) -K (Boc) -C (Trt) -K-E (Trt) -Wang resin

(Boc) -C (Trt) -K-Wang resin prepared in Preparation Example 3 was dissolved in dimethylformamide, and then Fmoc-Glu (Trt) -OH (3 N, N ', N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU, 3 mmol), dibutylbenzotriazole (HOBt, Add isopropylethylamine (6 mmol) and stir for 3 hours. Thereafter, the reaction product was washed with dimethylformamide, dichloromethane and dimethylformamide in that order, and the desired compound was obtained without further purification.

<Production Example 5> P (Boc) -K ( Boc) -C (Trt) -KE (Trt) -E (Trt) - Wang resin (Wang Article of resin)

(Boc) -C (Trt) -KE (Trt) -K (Boc) -K (Boc) -C (Trt) - The target compound was obtained in the same manner as in Preparation Example 4, except that Wang resin was used.

< Manufacturing example  6> P ( Boc ) - ( FITC ) K ( Boc ) -C ( Trt ) -K-E ( Trt ) - Wang resin Wang resin Manufacturing

Step 1: P ( Boc ) -K ( FITC ) -C ( Trt ) -K- Wang resin ( Wang resin )

Fmoc-Lys (FITC) -OH using Fmoc-Lys (Mtt) -OH in place of Alloc-Lys (Fmoc) -OH and removing the Mtt protecting group of Fmoc-Lys , The target compound was obtained by carrying out the same processes as in Production Example 3 above.

Step 2: P ( Boc ) - ( FITC ) K ( Boc ) -C ( Trt ) -K-E ( Trt ) - Wang resin Wang resin )

G (Trt) -OH (3 mmol) was prepared by dissolving P (Boc) -K (FITC) -C (Trt) -K- Wang resin prepared in step 1 above in dimethylformamide ), Hydroxybenzotriazole (HOBt, 3 mmol), O-benzotriazole-N, N, N ', N'-tetramethyl-euenium-hexafluoro-phosphate (HBTU, Propylethylamine (6 mmol) was added, and the mixture was stirred for 3 hours. Thereafter, the reaction product was washed with dimethylformamide, dichloromethane, and dimethylformamide in that order to obtain the desired compound without further purification.

< Manufacturing example  7> P ( Boc ) -S (t- This ) -C ( Trt ) -K- ( Beta -A) -E ( Trt ) - Wang resin Wang  resin

The Wang resin was washed with dimethylformamide (DMF) and then with 1.0 M hydroxybenzotriazole solution (HOBt, dimethylformamide solution). Thereafter, a solution of 4-amino-5-fluoro-4-fluoro-4-methoxybenzyl chloride (1 g) was added to a mixture of Alloc-Lys (Fmoc) -OH (1 mmol), hydroxybenzotriazole -Phosphate (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) in dimethylformamide and stirred for 2 hours. The resin was then washed with dimethylformamide, dichloromethane and dimethylformamide in that order, dissolved in a solution of dimethylformamide containing 20% piperidine, and stirred for 10 minutes. After stirring was complete, it was washed with dimethylformamide. Next, to the resin were added Fmoc- (Beta-Ala) -OH (6 mmol), hydroxybenzotriazole (3 mmol), O-benzotriazole-N, N, N ', N'- (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) were dissolved in dimethylformamide and stirred for 3 hours. Subsequently, the reaction was carried out by sequentially adding Cys (Trt) -OH, Ser (t-Bu) -OH and Pro (Boc) -OH in which the terminal amine was positively charged with Fmoc protecting group. When each step is complete, tetrakis (triphenylphosphine) palladium (0) and 1,3-dimethylbabricic acid are added together in dichloromethane, stirred for 2 hours, washed and then purified without further purification To obtain the target compound.

< Manufacturing example  8 > P ( Boc ) -D (t- This ) -C ( Trt ) -K- ( miniPEG ) -E ( Trt ) - MBHA  Resin ( MBHA  resin

The MBHA resin was washed with dimethylformamide (DMF) and then washed with 1.0 M hydroxybenzotriazole solution (HOBt, dimethylformamide solution). Thereafter, a mixture of Alloc-Lys (Fmoc) -OH (1 mmol), hydroxybenzotriazole (HOBt, 3 mmol), O-benzotriazole-N, N, N ', N'-tetramethyl- Fluoro-phosphate (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) in dimethylformamide and stirred for 2 hours. The resin was then washed with dimethylformamide, dichloromethane and dimethylformamide in that order, dissolved in a solution of dimethylformamide containing 20% piperidine, and stirred for 10 minutes. After stirring was complete, it was washed with dimethylformamide. Next, to the resin were added Fmoc-miniPEG-OH (3 mmol), hydroxybenzotriazole (3 mmol), O-benzotriazole-N, N, N ', N'- tetramethyl- (HBTU, 3 mmol) and diisopropylethylamine (6 mmol) were dissolved in dimethylformamide, followed by stirring for 3 hours. After that, the reaction was carried out by sequentially adding Cys (Trt) -OH, Asp (t-Bu) -OH and Pro (Boc) -OH in which the terminal amine was positively charged with Fmoc protecting group. When each step is complete, tetrakis (triphenylphosphine) palladium (0) and 1,3-dimethylbabricic acid are added together in dichloromethane, stirred for 2 hours, washed and then purified without further purification To obtain the target compound.

PREPARATION EXAMPLE 9 Preparation of P (Boc) -K (Boc) -C (Trt) -K-MBHA Resin (MBHA Resin)

The procedure of Preparation Example 3 was repeated except that MBHA resin was used instead of Wang resin to obtain the target compound.

PREPARATION EXAMPLE 10 Preparation of P (Boc) -K (Boc) -C (Trt) -K-E (Trt) -MBHA Resin

(Boc) -C (Trt) -K-MBHA resin prepared in Production Example 9 instead of P (Boc) -K (Boc) -C (Trt) (MBHA resin) was used in place of the target compound, to obtain the target compound.

Example 1 Preparation of PKCK-Fol-OH-1

Step 1: Preparation of P (Boc) -K (Boc) -C (Trt) -K-Wang resin-Fol

After dissolving the folate derivative (1 mmol) or folate (1 mmol) prepared in Preparation Example 1 in dimethylsulfoxide (20 ml), P (Boc) -K ) -C (Trt) -K- Wang resin (0.3 mmol), hydroxybenzotriazole (HOBt, 1 mmol), O-benzotriazole-N, N, N ' (HBTU, 1 mmol) and diisopropylethylamine (1 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction product was washed with dimethylformamide, dichloromethane and dimethylformamide in this order, to obtain the desired compound without further purification.

Step 2: Preparation of PKCK-Fol-OH

(Boc) -K (Boc) -C (Trt) -K-Wang resin-Fol prepared in the step 1 was dissolved in a mixture of TFA: TIS: EDT: Thioanisole: water = 90: 2.5 : 2.5: 2.5: 2.5 (20 ml), and the mixture was stirred at room temperature for 2 hours. The mixture was purified using high performance liquid chromatography (HPLC, SHIMASU prominence HPLC). The high performance liquid chromatography used was a Shiseido capcell-pak 18C column, and the mobile phase contained water (A) containing 0.1% trifluoroacetic acid (TFA) and 0.1% TFA Acetonitrile (B). The concentration gradient was applied from 100% A to 90% A for 2 minutes, then 90% to 60% for 10 minutes, 60% to 30% for 2 minutes, 30% for 3 minutes and 30% to 100% for 3 minutes And flowed at a flow rate of 1 ml / min for a minute.

 The resulting mixed solution was treated at 5 DEG C with diethyl ether to form a precipitate. The precipitate thus obtained was centrifuged to remove trifluoroacetic acid, and a precipitate was obtained. This procedure was repeated two more times to obtain solidified PKCK-Fol-OH.

Mass (m / z): Found: 898 (calculated: 898.02).

&Lt; Example 2 > Preparation of PKCK-Fol-OH-2

Step 1: Preparation of P (Boc) -K (Boc) -C (Trt) -KE (Trt) -Wang resin-

(0.15 mmol) or pteroic acid (0.15 mmol) prepared in Preparation Example 2 was dissolved in dimethylsulfoxide (20 ml), and then P (Boc) -K (Boc) -C (Trt) -KE (Trt) -Wang resin (0.05 mmol), hydroxybenzotriazole (HOBt, 0.15 mmol), O- (HBTU, 0.15 mmol) and diisopropylethylamine (0.15 mmol) were added to the solution, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the resin was washed with dimethylformamide, dichloromethane and dimethylformamide in this order, to obtain the desired compound without further purification.

Step 2: Preparation of PKCK-Fol-OH

(Boc) -C (Trt) -KE (Trt) -Wang resin -Petroic acid prepared in step 1 was dissolved in a mixture of TFA: TIS: EDT: Thioanisole: Water: 90: 2.5: 2.5: 2.5: 2.5 (20 ml), and the mixture was stirred at room temperature for 2 hours. The mixture was purified using high performance liquid chromatography (HPLC, SHIMASU prominence HPLC). The high performance liquid chromatography used was a Shiseido capcell-pak 18C column, and the mobile phase contained water (A) containing 0.1% trifluoroacetic acid (TFA) and 0.1% TFA Acetonitrile (B). The concentration gradient was applied from 100% A to 90% A for 2 minutes, then 90% to 60% for 10 minutes, 60% to 30% for 2 minutes, 30% for 3 minutes and 30% to 100% for 3 minutes And flowed at a flow rate of 1 ml / min for a minute.

 The resulting mixed solution was treated at 5 DEG C with diethyl ether to form a precipitate. The precipitate thus obtained was centrifuged to remove TFA, and a precipitate was obtained. This procedure was repeated two more times to obtain solidified PKCK-Fol-OH.

Mass (m / z): Found: 898 (calculated: 898.02).

The mobile phase consisted of water (A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% TFA. The concentration gradient was applied from 100% A to 90% A for 2 minutes, then 90% to 60% for 10 minutes, 60% to 30% for 2 minutes, 30% for 3 minutes and 30% to 100% for 3 minutes And flowed at a flow rate of 1 ml / min for a minute.

< Example  3> PKCK - Fol - NH ₂ Manufacturing

(Boc) -K (Boc) -C (Trt) -K- manufactured by Production Example 9 instead of the P (Boc) -K (Boc) -C ) -K-MBHA resin (MBHA resin) was used as the starting material, the target compound was obtained.

Mass (m / z): measured value: 911 (calculated: 911.06).

Example 4 Preparation of PKCK-E-Fol-OH

(Boc) -K (Boc) -C (Trt) -K- manufactured by Preparation Example 4 instead of the P (Boc) -K (Boc) ) -KE (Trt) -Wang resin was used as the starting material, the target compound was obtained.

Mass (m / z): measured value: 1025 (calculated value: 1027.13).

< Example  5> PK ( FITC ) CK -E- Fol - OH Manufacturing

(Boc) - (FITC) K (Boc) - K (Boc) -K (Boc) -C (Trt) -K-Wang resin prepared in Production Example 6 instead of P C (Trt) -KE (Trt) -Wang resin was used as the starting material, the target compound was obtained.

Mass (m / z): measured value: 1415 (calculated value: 1416.51).

< Example  6> PSCK - ( Beta -A) - Fol - OH Manufacturing

(Boc) -S (t-Bu) -S (t-Bu) prepared in Preparation Example 7 instead of the P (Boc) -C ) - C (Trt) -K (Beta-A) -E (Trt) -Wang resin was used as the starting material.

Mass (m / z): Found: 927 (calculated: 927).

< Example  7> PDCK - ( miniPEG ) - Fol - NH ₂ Manufacturing

(Trc) -KE (Trt) -Wang resin prepared in Production Example 4 instead of P (Boc) -K (Boc) -C The procedure of Example 2 was repeated except that P (Boc) -D (t-Bu) -C (Trt) -K- (miniPEG) -E (Trt) -MBHA resin To obtain the target compound.

Mass (m / z): Found: 1043 (calculated: 1042).

< Example  8> GKCK - Fol - OH Manufacturing

(Boc) -C (Trt) -KE (Boc) -C (Trt) -KE (Trt) -Wang resin prepared in Preparation Example 4 instead of P (Boc) (Trt) -MBHA resin (MBHA resin) was used in place of the resin (Trt) -MBHA resin.

Mass (m / z): Found: 857 (calculated: 857.95).

Example 9 Preparation of GKC-Dea-Fol-OH

(Boc) -C (Trt) -Dea (Boc) -C (Trt) -Ke (Trt) -Wang resin prepared in Production Example 4 instead of P Was carried out in the same manner as in Example 2, except that Fmoc-Wang resin was used as an initiator to obtain the desired compound (wherein Dea is Ethylenediamine).

Mass (m / z): measured value: 811 (calculated value: 811.92).

Example 10 Synthesis of PKCK-Fol-OH ^99m Tc cover

The compound prepared in Example 1 was labeled with ^99m Tc. A GH kit vial containing 20 mg of glucoheptonate (GH) and 0.45 mg of stannous chloride (SnCl ₂ ) was mixed with 400 μl of a sodium ^99m Tc pertechnetate (Na ^99m TcO ₄ ) and reacted at room temperature for 10 minutes to prepare ^99m Tc-GH. The reaction solution was analyzed by ITLC of a silica gel strip containing saline or MEK as a mobile phase for checking the amount of colloid formation. When mobile phase was used, ^99m TcO ₄ ^- and ^99m Tc-GH migrated to Rf 1.0 and the colloid remained at Rf 0.0. In addition, when MEK was used as the mobile phase, ^99m TcO ₄ ^- migrated to Rf 1.0, while ^99m Tc-GH and colloid remained at Rf 0.0. The compound PKCK-fol-OH ligand prepared in Example 1 was dissolved in distilled water and was prepared at a concentration of 10 ^-3 M (10 ^-6 mol / ml). 100 μl of a ^99m Tc-GH partial sample of the vial (370 MBq, 10mCi) was incubated with various concentrations of PKCK-fol ligand (10 ^-7 mol, 10 ^-8 mol) in 400 μl of 50 mM sodium acetate buffer And 10 ^-9 mol) was added thereto and the mixture was maintained at room temperature for 20 minutes. The radiochemical purity (RCP) and labeling efficiency of the compound were determined using a Waters 2695 pump, a UV detector (Waters 2487), an RI detector (In / US gamma-detector system) was determined by RP-HPLC, a Waters HPLC system equipped with a terra C-18 column (5 [mu] m, 4.6 x 250 mm). The mobile phase consisted of water (A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (B) containing 0.1% TFA. The concentration gradient was applied from 100% A to 90% A for 2 minutes, then 90% to 60% for 10 minutes, 60% to 30% for 2 minutes, 30% for 3 minutes and 30% to 100% for 3 minutes Minute. The purified complex was obtained by RP-HPLC and the chemical purity of the complex prepared was measured and shown in FIG. 2 and FIG.

As shown in FIG. 2 and FIG. 3, the radiochemical purity (RCP) of the radioactive isotope-labeled N ₃ S ₁ type chelator conjugated fate complex was 98% or more, and the labeling efficiency Was measured at 37 GBq / umol and the retention time was measured at 10.1 minutes. In addition, the in vitro stability of the complex in Salin and Serum was confirmed to be 96% or more when measured after 24 hours at 37 ° C.

< Example  11> PKDK -E- Fol - OH of ^{99 m} TC  sign

In Example 10, ^{99 m} TC for Example 1 and the above embodiment except that cover the PKDK-E-Fol-OH prepared in Example 4 instead of on the cover PKCK-Fol-OH prepared in Example 10 and The same procedure was followed to obtain the complex. The obtained complex had a radiochemical purity of 98% or more and a retention time of 10.4 minutes.

< Example  12> GKC - Dea - Fol - OH of ^{99 m} TC  sign

In Example 10, ^{99 m} TC for Example 1 and the above embodiment except that cover the GKC-Dea-Fol-OH prepared in Example 9 instead of on the cover PKCK-Fol-OH prepared in Example 10 and The same procedure was followed to obtain the complex. The resulting complex had a radiochemical purity of 98% or more and a retention time of 10.0 minutes.

< Experimental Example  1> Measurement of cell uptake and folate receptor binding affinity

In order to evaluate the cell uptake and the binding affinity of the folate receptor of the labeled folate complex according to the present invention, the following experiment was conducted.

Folate receptor-overexpressed folate receptor positive KB tumor cells in RPMI 1640 (human oral epidermoid carcinoma) T-flask without poly acid containing 37 ° C and 10% heat-inactivated fetal bovine serum (FBS) And fused to 80% to 90% under 5% CO ₂ with medium (FFRPMI). Approximately 8 x 10 ⁵ cells were grown in 12 well-plates for 24 hours for in vitro cell uptake and binding affinity measurements. Cells were washed twice with cold PBS buffer and pre-incubated for 30 min at 37 [deg.] C with fresh FFRPMI without 850 [mu] l of FBS. A solution of ^99m Tc labeled complex (18.5 kBq) prepared using the PKCK-fol-OH compound prepared in Example 1 above was added and the well plate was incubated at 37 ° C for 10, 30, 60, 120 and 180 minutes And cultured for time-dependent cell sorption assay. Blocking experiments were also carried out using pre-cultured cells with an excess of cold polylysine (100 [mu] M) for 30 minutes before adding the complexes. After incubation, the well plate was washed twice with cold PBS.

The cells were then washed with an acid wash buffer (50 mM glycine-HCl buffer, pH 2.4) to remove the radioactive ligand attached to the membrane, and the supernatant was attached to the radioactive membrane I gathered to measure the part. The cells were lysed in 1 N NaOH and the migration and measurement of the internalization of the radioactivity with the y-counter was measured. The radioactivity of the membrane-bound portion and the internalization portion can be calculated by summing up the cell uptake. The cell uptake data is expressed as% cell uptake (cell uptake / total added activity). Each data is expressed as an average value of three wells. The binding force between the PKCK peptide and folate was calculated using a relative binding assay. ^Complexes labeled with ^99m Tc in the above compounds were measured using the radioligand in the above assay. Cells were harvested with trypsin and washed with PBS buffer. 8 x 10 ⁵ cells were grown in 12-well plates. A suitable dilution of PKCK-fol-OH and unlabeled folate in ^99m Tc labeled PKCK-fol-OH complex (37 kBq) and 1 ml of FFRPMI was incubated at 37 ° C for 1 hour, Was stopped by the removal of the supernatant and the rapid flushing with FFRPMI. The cells were then collected to measure the radioactivity with a gamma-counter. The IC ₅₀ values of folate and PKCK-fol-OH compounds were calculated by nonlinear degeneration using GraphPad Prism software and were 20 nM and 160 nM, respectively. In addition, each point is calculated by averaging three times and is shown in Fig.

As shown in FIG. 4, it can be confirmed that the cell affinity and internalization rate of the radioactive folate increase in a time-dependent manner.

Therefore, the folate derivative to which the novel chelator of the N ₃ S ₁ type according to the present invention is conjugated not only stably forms a radioactive isotope and a complex, but also has excellent cell affinity and internalization ratio of the formed complex, Can be usefully used as a composition for diagnosing or treating cancer.

<Experimental Example 2> In the KB xenograft model ^99m Distribution of Tc-labeled PKCK-fol-OH complexes in the body

The following experiments were performed to evaluate the absorption of ^99m Tc-labeled PKCK-fol-OH complexes according to the present invention on tumors and organs of the body.

For animal experiments, a ^99m Tc-labeled PKCK-fol-OH complex with an activity of 10 ^-8 mol / ml and 370 MBq was used. The injection solution diluted the mother liquor to obtain the desired volume activity of 3.7 MBq / ml. The distribution of the ^99m Tc-PKCK-fol-OH in the body was performed with KB-related female nude mice (supplied by OrientBio, Korea). The rats maintained rodent chow (Harlan laboratories, Inc., USA) lacking folate by minimizing folate concentration for two weeks. After the tumors grew for 2-3 weeks, the rats were injected with 370 kBq (10 uCi) / 100 μl. At 30 minutes, 1 hour, 3 hours and 24 hours, the animals died.

Table 1 below shows the results of measurement of the body distribution at 30 minutes, 1 hour, 3 hours and 24 hours after the injection.

long time
^99m Tc-PKCK-gamma Glu-folate 30 minutes 1 hours 3 hours 24 hours blood 0.64 ± 0.26 0.36 + 0.06 0.18 ± 0.05 0.06 0.02 liver 7.21 ± 1.58 5.42 ± 1.85 2.83 ± 0.60 1.09 + 0.07 kidney 76.47 + 5.04 77.19 + - 5.43 41.48 ± 12.08 7.49 + - 1.90 spleen 0.47 + 0.13 0.36 + 0.03 0.30 ± 0.07 0.10 0.03 Heart 2.25 0.47 1.73 ± 0.27 1.03 + - 0.32 0.10 0.06 Small intestine (Sm Int) 0.93 + - 0.54 0.43 + - 0.05 0.27 ± 0.12 0.10 ± 0.05 Colon (Lg Int) 2.43 ± 1.38 3.43 ± 0.73 1.48 ± 0.64 1.38 + - 0.86 lungs 1.70 + - 0.35 1.45 ± 0.33 0.63 + 0.26 0.12 + 0.06 top 1.92 + 0.26 1.20 ± 0.05 0.55 + 0.16 0.13 + 0.04 tumor 2.60 ± 0.53 2.96 ± 0.51 2.24 ± 0.27 0.91 + 0.28 Tumor / blood 4.38 ± 0.92 8.35 ± 1.12 13.17 ± 2.78 15.15 + - 4.36 Tumor / kidney 0.03 ± 0.01 0.04 0.01 0.06 ± 0.01 0.12 + 0.02 Tumor / liver 0.38 + 0.14 0.58 + 0.17 0.82 ± 0.20 0.83 + - 0.24

As shown in Table 1 above, the radioactive isotope-labeled PKCK-fol-OH complex is compared with blood having an absorption rate of 0.64 ± 0.26% ID / g at 30 minutes and 0.18 ± 0.05% ID / g at 3 hours Showed a high tumor uptake rate of 2.60 ± 0.53% ID / g at 30 minutes and 2.24 ± 0.27% ID / g at 3 hours. Also, due to the high tumor uptake rate, the tumor / blood ratio was found to be as high as 13.17 ± 2.78% ID / g at 3 hours at 4.35 ± 0.92 at 30 minutes. On the other hand, high radioactivity in the kidney was found in xenograft xenografts in nude mice (77.19 ± 5.43% ID / g and 41.48 ± 12.08% ID / g at 1 hour and 3 hours, respectively). In addition, absorption in liver was 5.42 ± 1.85% ID / g at 1 hour and 2.83 ± 0.60% ID / g at 3 hours. In other organs such as lung, heart, stomach and intestine, it was confirmed that the absorption rate was low.

Therefore, the folate derivative to which the novel chelator of the N ₃ S ₁ type according to the present invention is conjugated forms a stable complex by designating it as a radioactive isotope, and the complex formed is a complex of the lung, heart, It has a low water uptake rate in various organs and has a high absorption rate to tumors, and thus can be usefully used as a composition for diagnosis or treatment of cancer using the same.

Experimental Example 3 In vivo SPECT imaging

The following experiment was conducted to evaluate the SPECT imaging effect for diagnosis of a radioisotope-labeled N ₃ S ₁ type chelator conjugated fate complex according to the present invention.

For in vivo SPECT imaging, the rats in Experimental Example 2 were kept in rodent chow lacking folate for 2 weeks. During storage, the mice were injected with 185 MBq (5 mCi) / 100 μl in vivo. Blocking experiments were performed by injecting polonic acid (100 μg / 100 μl) for 5 min prior to the radiofolate injection. Flat gamma camera imaging was performed at 5 minutes, 1 hour, and 4 hours after injection. SPECT and CT data of the 3D image were taken by software after 18 hours, and the results are shown in Figs. 5 to 13. Fig.

As shown in FIGS. 5 to 13, it can be seen that the fate complex of the N ₃ S ₁ type labeled with a radioisotope according to the present invention is stably SPECT-imaged in vivo .

Accordingly, the novel chelate-folate complex of N ₃ S ₁ labeled with a radioisotope according to the present invention is stably absorbed in vivo, and has excellent effect of being imaged on medical imaging equipment such as SPECT Therefore, it can be usefully used as a composition for diagnosing or treating cancer using the same.

Claims (13)

A novel chelate-folate derivative of the N ₃ S ₁ type represented by the following formula (1): or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(In the formula 1,
R is hydroxy (-OH) or amine (-NH ₂ );
R ¹ is a single bond,

,

or

ego;
R ² is

,

,

or

ego;
R ³ is

or

ego; And
p is an integer of 3).
The method according to claim 1,
The compound represented by the above formula (I) is the following Formula a to g 1 N ₃ S _1-type novel chelator is conjugated folate derivatives or their pharmaceutically acceptable salts can be prepared in, characterized in that species selected from the group consisting of:
(A)

;
[Formula b]

;
(C)

;
[Chemical formula d]

;
(E)

;
[Formula f]

; And
[Formula g]

.
As shown in Scheme 1 below,
Reacting a polyacid derivative of formula (2) with a compound of formula (3) to produce a folate derivative of formula (4) (step 1); And
To prepare a compound of formula (I) to the resin (resin) of a folate derivative of formula (IV) prepared in Step 1 in acidic conditions (step 2), the first term N ₃ S ₁ comprising the Method for producing a folate derivative having a novel chelator bonded thereto:
[Reaction Scheme 1]

(In the above Reaction Scheme 1,
Wherein R, R ¹ , R ² , R ³ and p are as defined in claim 1;
resin is Wang resin or Merrifield resin; And
Trt is a trityl group).
The method of claim 3,
The compound represented by the general formula (3) is a compound represented by the following general formula (2)
Reacting protected lysine with an Alloc protecting group and protected cysteine with an Fmoc protecting group to produce a compound represented by formula (5) (step 1);
Reacting the compound of Formula 5 and the compound of Formula 6 prepared in Step 1 to prepare a compound of Formula 7 (Step 2);
Reacting the compound of formula (7) and the compound of formula (8) prepared in step 2 to prepare a compound of formula (9) (step 3); And
Reacting the compound of formula (9) prepared in step 3 with a compound of formula (10) to prepare a compound represented by formula (3) (step 4).
[Reaction Scheme 2]

(In the above Reaction Scheme 2,
Wherein R, R ¹ , R ² , R ³ and p are as defined in claim 1;
resin is Wang resin or Merrifield resin;
Alloc is allylcarbamate;
Fmoc is a fluorenylmethyloxycarbonyl group; And
Trt is a trityl group).
As shown in Scheme 3 below,
Reacting a phthaloic acid derivative of formula (11) with a compound of formula (12) to prepare a folate derivative of formula (4) (step 1); And
To prepare a compound of formula (I) to the resin (resin) of a folate derivative of formula (IV) prepared in Step 1 in acidic conditions (step 2), the first term N ₃ S ₁ comprising the Method for producing a folate derivative having a novel chelator bonded thereto:
[Reaction Scheme 3]

(In the above scheme 3,
Wherein R, R ¹ , R ² , R ³ and p are as defined in claim 1;
resin is Wang resin or Merrifield resin; And
Trt is a trityl group).
6. The method of claim 5,
The compound represented by the above formula (12)
(Step 1) of preparing a compound represented by formula (14) by reacting a compound represented by formula (3) with glutamic acid represented by formula (13); And
(2) deprotecting the compound of the formula (14) prepared in the step (1) to prepare a compound represented by the formula (12).
[Reaction Scheme 4]

(In the above Scheme 4,
Wherein R, R ¹ , R ² , R ³ and p are as defined in claim 1;
resin is Wang resin or Merrifield resin;
Fmoc is a fluorenylmethyloxycarbonyl group; And
Trt is a trityl group).
delete A complex with a novel chelator-bonded folate derivative of N ₁ S ₁ of claim ₁ labeled with a radioactive isotope of technetium (99 ^m Tc).
delete The first term N ₃ S using a ligand exchange reaction kit in the novel chelator is conjugated folate derivative of type ₁ trans keel producing ignition of water indicated a radioactive isotope of illustration technetium (99 ^m Tc), comprising a step of Way.
The first term N ₃ S new type ₁ a chelator is conjugated folate derivatives to technetium (99 ^m Tc) radioisotope is ovarian cancer or endometrial cancer diagnostic composition containing a labeled complex of the active ingredient of.
delete The ovarian cancer or endometrial cancer diagnostic imaging apparatus containing a complex of a radiolabeled of claim 1. N ₃ S novel chelator is conjugated folate derivative of type ₁ and technetium (99 ^m Tc) as an active ingredient Used in radioactive medicines.

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