KR102403970B1 - Carboxylic acid-having compounds for PSMA-targeting and use thereof - Google Patents

Abstract

The present invention relates to an ¹⁸ F-labeled compound and its use for the diagnosis of prostate cancer, wherein the carboxylic acid-introduced compounds of the present invention have strong ion-pair binding (Salt) with an arginine residue in the PSMA protein binding site. Bridge Interaction) has high binding affinity, and due to the hydrophilic nature of carboxylic acid, it has a rapid in vivo background radiation removal effect and low non-specific binding.

Description

PSMA-targeting compounds into which carboxylic acids are introduced and uses thereof

The present invention relates to ¹⁸ F-labeled compounds and their use for the diagnosis of prostate cancer.

Prostate cancer is the leading cause of cancer mortality in men in the United States, fifth in Korea, and second in the world. Prostate cancer usually occurs in men over the age of 50, but the number of patients rapidly increases with age. It usually progresses slowly, but when it develops into malignancy and metastasizes, it is an extremely difficult disease to treat. Metastasis mainly starts in the lymph nodes, pelvic bones, vertebrae and bladder around prostate cancer and gradually spreads throughout the body.

Methods currently in use for diagnosis include the prostate-specific antigen test (PSA test) and digital rectal examination, and imaging methods such as transrectal ultrasound, CT, MRI, and WBBS (whole body bone scan). is being implemented

However, in most cases, the diagnostic accuracy is low, and it is difficult to determine the early diagnosis of the disease and whether it recurs or metastasizes. In addition, there is a disadvantage that it is difficult to distinguish from benign diseases such as benign prostatic hyperplasia and prostatitis.

Positron Emission Tomography (PET) is an imaging method of the human body using molecular probes that target a metabolic phenomenon or protein specific to a disease. It has the advantage of enabling early diagnosis of disease, treatment evaluation, and confirmation of metastasis/recurrence by observing

Prostate-Specific Membrane Antigen (PSMA) is a cell membrane protein characterized by overexpression specifically in prostate cancer cells, and a compound of the glutamic acid-Urea-lysine (GUL) structure is It is known to selectively bind well. To date, various radiotracers labeled with radioactive isotopes on compounds having a basic structure of GUL are being studied as prostate cancer-specific diagnostic drugs.

Among them, ¹⁸ F-DCFPyL is a GUL compound labeled with an ¹⁸ F isotope, and is evaluated as one of the best among PET tracers for diagnosing prostate cancer reported to date (Patent Document 1). ¹⁸ F-DCFPyL has relatively low lipophilicity compared to the previously developed compound ( ¹⁸ F-DCFBzL), so there is less non-specific binding in vivo and excellent removal through the kidneys. ¹⁸ F-DCFPyL or ¹⁸ F-DCFBzL compounds have pyridine and benzene groups, respectively, and these aryl groups have higher binding strength compared to compounds without aryl groups due to aryl-cation interactions with arginine residues in the PSMA protein binding site. have characteristics. However, such an aryl group tends to induce non-specific binding in vivo and thus show strong uptake in normal tissues such as salivary glands and lacrimal glands.

The compound called ¹⁸ F-YC88 reported later is a compound without an aryl group, and compared to the ¹⁸ F- ^DCFPyL compound, the background radiation is removed faster and gives a clearer image. There is a disadvantage that the intake of prostate cancer significantly falls over time due to a decrease (Patent Document 2).

The present inventors completed the present invention by confirming that compounds having a carboxylic acid introduced at the aryl group position of the existing compound not only have excellent binding ability with PSMA protein, but also exhibit excellent pharmacokinetic properties in vivo due to the hydrophilicity of the carboxylic acid. did

US registered patent US 8,778,305 B2 International Patent Publication WO 2017/027870 A1

An object of one aspect of the present invention is to provide a novel compound that has excellent binding ability with PSMA protein and exhibits excellent pharmacokinetic properties in vivo.

Another object of the present invention is to provide a composition for diagnosing prostate cancer containing the compound as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating prostate cancer containing the compound as an active ingredient.

Another object of the present invention is to provide a method for diagnosing prostate cancer by administering an effective amount of the compound to a subject.

Another object of the present invention is to provide a method for treating prostate cancer by administering to a subject an effective amount of the compound.

Another object of the present invention is to provide a use of the compound for the manufacture of a medicament for the treatment of prostate cancer.

In order to achieve the above object,

One aspect of the present invention provides a compound represented by the following Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

L ₁ is —(CH ₂ ) _a —, wherein a is an integer from 2 to 4;

U is a bond, or -C(=O)-;

V is a bond, -NH-, or -C(=O)-;

W is CH, or N;

L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;

X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;

Y is a bond, N, or CH;

L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;

L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;

R is hydrogen, halogen, or CH ₃ ;

Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;

, 또는

이고;Tz is

, or

ego;

L ₅ is -(CH ₂ ) _f -, -(CH ₂ CH ₂ O) _g (CH ₂ ) _h -, or -(CH ₂ O) _i (CH ₂ CH ₂ O) _j (CH ₂ ) _k - , wherein f, g, h, i, j and k are independently integers from 1 to 5;

m and n are independently integers of 0 or 1,

는 -H, 또는 부재이고,If m is an integer of 0, then

is -H, or absent;

는 -H, 또는 부재이고; 및If n is an integer of 0, then

is -H, or absent; and

F is ¹⁸ F.

Another aspect of the present invention provides a compound represented by the following Chemical Formula 2, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 2]

In Formula 2,

L ₁ is —(CH ₂ ) _a —, wherein a is an integer from 2 to 4;

U is a bond, or -C(=O)-;

V is a bond, -NH-, or -C(=O)-;

W is CH, or N;

L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;

X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;

Y is a bond, N, or CH;

L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;

L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;

R is hydrogen, halogen, or CH ₃ ;

Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;

T is -C≡CH, or -N ₃ ; and

n is an integer of 0 or 1,

는 -H, 또는 부재이다.If n is an integer of 0, then

is -H, or absent.

Another aspect of the present invention provides a composition for diagnosing prostate cancer comprising the compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating prostate cancer containing the compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect of the present invention provides a method for diagnosing prostate cancer by administering an effective amount of the compound to a subject.

Another aspect of the present invention provides a method of treating prostate cancer by administering to a subject an effective amount of the compound.

Another aspect of the present invention provides the use of the compound for the manufacture of a medicament for the treatment of prostate cancer.

The carboxylic acid-introduced compounds of the present invention form a strong salt bridge interaction with an arginine residue in the PSMA protein binding site, thereby having high binding affinity, and due to the hydrophilic nature of carboxylic acid, rapid in vivo It is characterized by a background radiation removal effect and low non-specific binding.

Hereinafter, the present invention will be described in detail.

On the other hand, the embodiment of the present invention can be modified in various other forms, the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art. Furthermore, in the entire specification, "including" a certain element means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

One aspect of the present invention is

Provided are a compound represented by Formula 1 below, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

L ₁ is —(CH ₂ ) _a —, wherein a is an integer from 2 to 4;

U is a bond, or -C(=O)-;

V is a bond, -NH-, or -C(=O)-;

W is CH, or N;

L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;

X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;

Y is a bond, N, or CH;

L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;

L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;

R is hydrogen, halogen, or CH ₃ ;

Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;

, 또는

이고;Tz is

, or

ego;

L ₅ is -(CH ₂ ) _f -, -(CH ₂ CH ₂ O) _g (CH ₂ ) _h -, or -(CH ₂ O) _i (CH ₂ CH ₂ O) _j (CH ₂ ) _k - , wherein f, g, h, i, j and k are independently integers from 1 to 5;

m and n are independently integers of 0 or 1,

는 -H, 또는 부재이고,If m is an integer of 0, then

is -H, or absent;

는 -H, 또는 부재이고; 및If n is an integer of 0, then

is -H, or absent; and

F is ¹⁸ F.

When R is halogen, the halogen may be a radioactive isotope.

The compound represented by Formula 1 may be a compound represented by Formula 1-1 below.

[Formula 1-1]

In Formula 1-1,

L ₁ , U, V, W, L ₂ , X, Z, Tz, L ₅ and F are as defined above; and

Y is a bond, NH, or CH ₂ .

Another aspect of the present invention is

Provided are a compound represented by Formula 2 below, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 2]

In Formula 2,

L ₁ is —(CH ₂ ) _a —, wherein a is an integer from 2 to 4;

U is a bond, or -C(=O)-;

V is a bond, -NH-, or -C(=O)-;

W is CH, or N;

L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;

X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;

Y is a bond, N, or CH;

L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;

L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;

R is hydrogen, halogen, or CH ₃ ;

Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;

T is -C≡CH, or -N ₃ ; and

n is an integer of 0 or 1,

는 -H, 또는 부재이다.If n is an integer of 0, then

is -H, or absent.

The compound represented by Formula 1 is,

It may be any one compound selected from the group consisting of the following formulas 1e, 1f, 1g, 1h, 1i, 1j, 1k and 11l.

,

,

,

,

,

,

,

,

,

,

,

,

및

.

and

.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. get it from Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, a precipitate formed by dissolving a derivative of Formula I in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid It can be prepared by filtration and drying, or by distilling the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

Furthermore, the present invention includes not only the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like, which can be prepared therefrom.

Another aspect of the present invention provides a composition for diagnosing prostate cancer comprising a compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound selectively binds to a prostate-specific membrane antibody (PSMA) overexpressed in prostate cancer cells to diagnose prostate cancer.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating prostate cancer, comprising the compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

A pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration is administered by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. depending on how

At this time, in order to formulate a formulation for parenteral administration, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or buffer to prepare a solution or suspension, which is an ampoule or vial unit dosage form. can be manufactured with The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for regulating osmotic pressure, and other therapeutically useful substances, and mixing, granulation, in the usual manner It can be formulated according to the method of formulation or coating.

Another aspect of the present invention provides a method for diagnosing prostate cancer by administering an effective amount of the compound to a subject.

Another aspect of the present invention provides a method of treating prostate cancer by administering to a subject an effective amount of the compound.

Another aspect of the present invention provides the use of the compound for the manufacture of a medicament for the treatment of prostate cancer.

The carboxylic acid-introduced compounds of the present invention form a strong salt bridge interaction with an arginine residue at the PSMA protein binding site, and thus have high binding affinity, and fast in vivo due to the hydrophilic nature of carboxylic acid. There is a characteristic having a background radiation removal effect and low non-specific binding, which is directly supported by the Examples and Experimental Examples to be described later.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples to be described later.

However, the Examples and Experimental Examples to be described later only partially exemplify the present invention, and the present invention is not limited thereto.

< Example 1> Preparation of compounds 3b and 3c

Preparation of compound 3b

Compound 3a (5.2 g, 10.7 mmol) in dichloromethane (100 mL), cooled to 0 °C, and then slowly added tert-butyl bromoacetate (1.9 mL, 12.8 mmol). While maintaining 0 °C, triethylamine (2.2 mL, 16.0 mmol) was slowly added thereto, and the mixture was stirred while slowly raising the temperature to room temperature. After stirring for 3 hours, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (50 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 3b (3.36 g, 52%).

MS (ESI) m/z 602 [M+H] ⁺

Preparation of compound 3c

Compound 3a (150 mg, 0.31 mmol) was dissolved in ethanol (2.0 mL), and tert-butyl acrylate (0.05 mL, 0.034 mmol) was slowly added thereto. After stirring for 12 hours, the mixture was concentrated under reduced pressure and the concentrate was separated by column chromatography (4% methanol/dichloromethane) to obtain compound 3c (73 mg, 39%).

MS (ESI) m/z 616 [M+H] ⁺

< Example 2> Preparation of compounds 1a, 1b, 1c

Preparation of compound 1a

Step 1:

Compound 3a (300 mg, 0.62 mmol) was dissolved in dichloromethane (6.0 mL), cooled to 0 °C, and then acetyl chloride (0.05 mL, 0.68 mmol) was slowly added thereto. While maintaining 0 °C, triethylamine (0.26 mL, 1.85 mmol) was slowly added thereto, and the mixture was stirred while slowly raising the temperature to room temperature. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 4a (265 mg, 81%).

MS (ESI) m/z 552 [M+Na] ⁺

Step 2:

Compound 4a (100 mg, 0.19 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (1.5 mL) and stirred at room temperature for 3 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated and obtained using a centrifuge, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1a (60 mg, 88%) as a white solid.

MS (ESI) m/z 362 [M+H] ⁺

Preparation of compound 1b

Step 1:

Compound 3b (100 mg, 0.17 mmol) obtained in Example 1 was dissolved in dichloromethane (30 mL), cooled to 0 °C, and then acetyl chloride (0.02 mL, 0.26 mmol) was slowly added thereto. While maintaining 0 °C, triethylamine (0.07 mL, 0.51 mmol) was slowly added thereto, and the mixture was stirred while slowly raising the temperature to room temperature. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (4% methanol/dichloromethane) to obtain compound 4b (79 mg, 74%).

MS (ESI) m/z 666 [M+Na] ⁺

Step 2:

Compound 4b (59 mg, 0.09 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 3 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated and obtained using a centrifuge, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1b (20 mg, 52%) as a white solid.

MS (ESI) m/z 442 [M+Na] ⁺

Preparation of compound 1c

Step 1:

Compound 3c (60 mg, 0.10 mmol) obtained in Example 1 was dissolved in dichloromethane (1.2 mL), cooled to 0 °C, and then acetyl chloride (0.01 mL, 0.15 mmol) was slowly added thereto. While maintaining 0 °C, triethylamine (0.04 mL, 0.30 mmol) was slowly added thereto, and the mixture was stirred while slowly raising the temperature to room temperature. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (4% methanol/dichloromethane) to obtain compound 4c (63 mg, 98%).

MS (ESI) m/z 680 [M+Na] ⁺

Step 2:

Compound 4c (50 mg, 0.08 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1c (24 mg, 73%) as a white solid.

MS (ESI) m/z 434 [M+H] ⁺

< Example 3> Preparation of compounds 1d and 1e

Preparation of compound 1d

Step 1:

6-(N-Fmoc-amino)hexanoic acid (72 mg, 0.25 mmol) was dissolved in dimethylformamide (1.0 mL), HOBT (42 mg, 0.31 mmol) and TBTU (99 mg, 0.31 mmol) were added The mixture was stirred at room temperature. After diisopropylethylamine (0.11 mL, 0.62 mmol) was added to the reaction mixture, 3a (100 mg, 0.21 mmol) diluted in dimethylformamide (1.0 mL) was slowly added thereto. After stirring for 1 hour, an aqueous ammonium chloride solution (50 mL) was added, and the organic compound was extracted with ethyl acetate (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (3% methanol/dichloromethane) to obtain compound 5a (147 mg, 87%).

MS (ESI) m/z 845 [M+Na] ⁺

Step 2:

Compound 5a (353 mg, 0.43 mmol) obtained in step 1 was dissolved in 20% piperidine/dichloromethane solution (5.0 mL) and stirred at room temperature for 2 hours. After adding water (50 mL), organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (NH silica gel, FUJI SILYSIA CHEMICAL LTD) (1% methanol/dichloromethane) to obtain compound 6a (260 mg, 99%). .

MS (ESI) m/z 601 [M+H] ⁺

Step 3:

4-(p-tolyl)butanoic acid (11 mg, 0.06 mmol) was dissolved in dichloromethane (5.0 mL), HOBT (11 mg, 0.08 mmol) and TBTU (26 mg, 0.08 mmol) were added and stirred at room temperature. did After diisopropylethylamine (0.03 mL, 0.15 mmol) was added to the reaction mixture, 6a (30 mg, 0.05 mmol) diluted in dichloromethane (2.0 mL) was slowly added thereto. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (20 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 7a (32 mg, 84%).

MS (ESI) m/z 783 [M+Na] ⁺

Step 4:

Compound 7a (32 mg, 0.042 mmol) obtained in step 3 was dissolved in 70% TFA/dichloromethane solution (0.5 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1d (5.4 mg, 22%) as a white solid.

MS (ESI) m/z 593 [M+H] ⁺

Preparation of compound 1e

Step 1:

6-(N-Fmoc-amino)hexanoic acid (212 mg, 0.60 mmol) was dissolved in dichloromethane (5.0 mL), cooled to 0 °C, and then N,N'-dicyclohexylcarbodiimide (DCC, 113) mg, 0.55 mmol) was added, and then 3b (300 mg, 0.50 mmol) was added and stirred at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (2% methanol/dichloromethane) to obtain compound 5b (261 mg, 56%).

MS (ESI) m/z 959 [M+Na] ⁺

Step 2:

Compound 5b (123 mg, 0.13 mmol) obtained in step 1 was dissolved in 20% piperidine/dichloromethane solution (2.0 mL) and stirred at room temperature for 2 hours. After adding water (50 mL), organic compounds were extracted with dichloromethane (20 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (NH silica gel, FUJI SILYSIA CHEMICAL LTD) (1% methanol/dichloromethane) to obtain compound 6b (88 mg, 94%). .

MS (ESI) m/z 715 [M+H] ⁺

Step 3:

4-(p-tolyl)butanoic acid (4 mg, 0.02 mmol) was dissolved in dichloromethane (5.0 mL), HOBT (4 mg, 0.03 mmol) and TBTU (10 mg, 0.03 mmol) were added and stirred at room temperature. did After diisopropylethylamine (0.01 mL, 0.06 mmol) was added to the reaction mixture, 6b (15 mg, 0.02 mmol) diluted in dichloromethane (2.0 mL) was slowly added thereto. After stirring for 1 hour, water (30 mL) was added, and then organic compounds were extracted with dichloromethane (20 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 7b (15 mg, 82%).

MS (ESI) m/z 897 [M+Na] ⁺

Step 4:

Compound 7b (18 mg, 0.02 mmol) obtained in step 3 was dissolved in 60% TFA/dichloromethane solution (0.5 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1e (5.6 mg, 42%) as a white solid.

MS (ESI) m/z 651 [M+H] ⁺

< Example 4> Preparation of compound 1f

Step 1:

4-pentinoic acid (82 mg, 0.83 mmol) in dichloromethane (10 mL), cooled to 0 ℃, N,N'-dicyclohexylcarbodiimide (DCC, 190 mg, 0.91 mmol) was added, and then compound 3b obtained in Example 1 (500 mg, 0.83 mmol) ) and stirred at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (30% ethyl acetate/ n -hexane) to obtain compound 8 (290 mg, 52%).

MS (ESI) m/z 682 [M+H] ⁺

Step 2:

Compound 8 (100 mg, 0.15 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (2 mL) and stirred at room temperature for 3 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 2a (37 mg, 55%) as a white solid.

MS (ESI) m/z 458 [M+H] ⁺ , 456 [MH] ^-

Step 3:

Compound 2a (13 mg, 0.028 mmol) and F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (5 mg, 0.028 mmol) obtained in step 2 were dissolved in ethanol (1.0 mL), and then 1 M CuSO ₄ aqueous solution (6 μL, 0.006 mmol) and 2 M aqueous sodium ascorbate solution (Na-Asc. 5 μL, 0.009 mmol) were sequentially added, and the mixture was stirred for 1 hour. The reaction solution was filtered, concentrated under reduced pressure, and then separated using high performance liquid chromatography (HPLC). The purified solution was lyophilized to obtain compound 1f (8 mg, 44%) as a white solid.

¹ H NMR (400 MHz, D ₂ O) δ 1.20-1.38 (m, 2H), 1.39-1.56 (m, 2H), 1.58-1.71 (m, 1H), 1.72-1.84 (m, 1H), 1.86- 1.95 (m, 1H), 2.06-2.16 (m, 1H), 2.45 (t, J = 7.2 Hz, 2H), 2.68 (t, J = 6.8 Hz, 1H), 2.84 (t, J = 6.4 Hz, 1H) ), 3.00 (q, J = 6.8 Hz, 2H), 3.28-3.36 (m, 2H), 3.59-3.62 (m, 5H), 3.68-3.70 (m, 1H), 3.91 (t, J = 4.4 Hz, 2H), 4.01 (s, 1H), 4.09-4.21 (m, 3H), 4.45 (t, J = 4.0 Hz, 1H), 4.56-4.60 (m, 3H), 7.89 (s, 1H)

MS (ESI) m/z 635 [M+H] ⁺ , 633 [MH] ^-

< Example 5> Preparation of 1 g of compound

Step 1:

Azidoacetic acid (18 mg, 0.18 mmol) was dissolved in dichloromethane (5.0 mL), and then cooled to 0 °C. N,N'-dicyclohexylcarbodiimide (DCC, 41 mg, 0.20 mmol) was added, and compound 3b (109 mg, 0.18 mmol) obtained in Example 1 was added, followed by stirring at room temperature for 1 hour. After filtration of the reaction solution, the solvent was removed under reduced pressure, and column chromatography (40% ethyl acetate/ n -hexane) was performed to obtain compound 9 (80 mg, 64%).

Step 2:

Compound 9 (80 mg, 0.11 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred for 4 hours. A solution was added to ethyl ether to capture the precipitate, and ethyl ether (20 mL) was further added thereto, followed by separation using a centrifuge. The precipitate was dissolved in water, separated using high performance liquid chromatography (HPLC), and then lyophilized to obtain compound 2b (30 mg, 56%).

Step 3:

Compound 2b (8 mg, 0.017 mmol) obtained in step 2 above and 3-(2-(2-fluoroethoxy)ethoxy)prop-1-ene (3 mg, 0.017 mmol) were mixed with ethanol (1 mL) After dissolving in , 1 M CuSO ₄ aqueous solution (4 μL, 0.003 mmol), 2 M aqueous sodium ascorbate solution (Na-Asc. 3 μL, 0.005 mmol) were added, and the mixture was stirred for 1 hour. After filtration of the reaction solution, the solvent was removed under reduced pressure, separated using high performance liquid chromatography (HPLC), and then freeze-dried to obtain 1 g (10 mg, 95%) of the compound.

< Example 6> Preparation of compounds 10a and 10b

Preparation of 10a:

Dissolve propargylamine (160 mg, 2.87 mmol) in dichloromethane (10 mL) and bring it to 0 °C, and then slowly add triethylamine (0.6 mL, 4.3 mmol). tert-Butyl bromoacetate (0.62 g, 3.16 mmol) was dissolved in dichloromethane (10 mL) and slowly added thereto, followed by stirring at room temperature for 18 hours. Water (20 mL) was added to terminate the reaction, and the organic compound was extracted with dichloromethane (20 mL, twice). After dehydration over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure, and the concentrate was subjected to column chromatography (2% methanol/dichloromethane) to obtain compound 10a (0.46 g, 55%).

Preparation of 10b:

2-(2-azidoethoxy)ethylamine (100 mg, 0.77 mmol) was dissolved in dichloromethane (30 mL), cooled to 0 °C, and tert-butyl bromoacetate (0.14 mL, 0.92 mmol) was added thereto. gave. While maintaining 0 °C, triethylamine (0.32 mL, 2.31 mmol) was slowly added, and the reaction solution was stirred while slowly raising the temperature to room temperature. After stirring for 1 hour, the reaction solution was concentrated under reduced pressure, and the concentrate was separated by column chromatography (3% methanol/dichloromethane) to obtain compound 10b (78 mg, 42%).

< Example 7> Preparation of compounds 10c and 10d

Preparation of 10c

After dissolving propargylamine (500 mg, 9.07 mmol) in ethanol (20 mL) and cooling to 0 °C, tert-butyl acrylate (1.16 g, 9.07 mmol) was slowly added and stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the concentrate was subjected to column chromatography (2% methanol/dichloromethane) to obtain compound 10c (1.4 g, 84%).

Manufacturing of 10d

2-(2-azidoethoxy)ethylamine (1.0 g, 7.68 mmol) was dissolved in ethanol (200 mL), and tert-butyl acrylate (1.23 mL, 8.45 mmol) was slowly added thereto. After stirring for 12 hours, the mixture was concentrated under reduced pressure and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 10d (1.07 g, 54%).

< Example 8> Preparation of compound 11

H-Glu(OtBu)-OtBu.HCl (500 mg, 1.69 mmol) was dissolved in dichloromethane (25 mL) and cooled to -10 °C. Diisopropylethylamine (0.7 mL, 4.05 mmol) was added, and 4-nitrophenyl chloroformate (340 mg, 1.69 mmol) was slowly added thereto. After stirring at -10 °C for 10 minutes, the mixture was further stirred at room temperature for 20 minutes. After confirming the reaction by thin plate chromatography (TLC), it was cooled to -10 ℃ again, triethylamine (0.56 mL, 4.05 mmol) was added, and H-Glu(OH)-OtBu HCl (330 mg, 1.60 mmol) was added. The mixture was stirred at room temperature for 1 hour. Water (25 mL) was added to the reaction solution, organic compounds were extracted with dichloromethane (25 mL, 2 times), then water was removed with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to column chromatography (5% methanol/dichloromethane) was separated to obtain compound 11 (600 mg, 72%).

MS (ESI) m/z 489 [M+H] ⁺ , 487 [MH] ^-

< Example 9> Preparation of compounds 1h and 1i

Preparation of compound 1h

Step 1:

Compound 11 (300 mg, 0.61 mmol) obtained in Example 8 was dissolved in dichloromethane (30 mL), cooled to 0 °C, and then N,N'-dicyclohexylcarbodiimide (DCC, 139 mg, 0.68 mmol) ), the compound 10b (225 mg, 0.92 mmol) obtained in Example 6 was added and stirred at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (2% methanol/dichloromethane) to obtain compound 12a (413 mg, 94%).

MS (ESI) m/z 751 [M+Na] ⁺

Step 2:

Compound 12a (300 mg, 0.42 mmol) obtained in step 1 was dissolved in methanol (20 mL), palladium (10% Palladium on carbon, 22 mg) was added, and the mixture was stirred under hydrogen at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (NH silica gel, FUJI SILYSIA CHEMICAL LTD) (1% methanol/dichloromethane) to obtain compound 13a (231 mg, 80%).

MS (ESI) m/z 689 [M+H] ⁺

Step 3:

4-(p-tolyl)butanoic acid (16 mg, 0.09 mmol) was dissolved in dichloromethane (5 mL), HOBT (15 mg, 0.11 mmol) and TBTU (35 mg, 0.11 mmol) were added and stirred at room temperature. did. After diisopropylethylamine (0.04 mL, 0.22 mmol) was added to the reaction mixture, 13a (50 mg, 0.08 mmol) obtained in step 2 diluted in dichloromethane (5 mL) was slowly added thereto. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 14a (51 mg, 83%).

MS (ESI) m/z 871 [M+Na] ⁺

Step 4:

Compound 14a (50 mg, 0.06 mmol) obtained in step 3 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1h (18 mg, 49%) as a white solid.

MS (ESI) m/z 625 [M+H] ⁺

Preparation of compound 1i

Step 1:

Compound 11 (300 mg, 0.61 mmol) obtained in Example 8 was dissolved in dichloromethane (30 mL), cooled to 0 °C, and then N,N'-dicyclohexylcarbodiimide (DCC, 139 mg, 0.68 mmol) ), 10d (238 mg, 0.92 mmol) obtained in Example 7 was added and stirred at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (2% methanol/dichloromethane) to obtain compound 12b (327 g, 73%).

MS (ESI) m/z 751 [M+Na] ⁺

Step 2:

Compound 12b (300 mg, 0.41 mmol) obtained in step 1 was dissolved in methanol (20 mL), palladium (10% Palladium on carbon, 22 mg) was added, and the mixture was stirred under hydrogen at room temperature for 1 hour. The reaction solution was filtered under reduced pressure, concentrated, and the concentrate was separated by column chromatography (NH silica gel, FUJI SILYSIA CHEMICAL LTD) (1% methanol/dichloromethane) to obtain compound 13b (197 mg, 68%).

MS (ESI) m/z 703 [M+H] ⁺

Step 3:

4-(p-tolyl)butanoic acid (30 mg, 0.17 mmol) was dissolved in dichloromethane (10 mL), HOBT (29 mg, 0.21 mmol) and TBTU (68 mg, 0.21 mmol) were added and stirred at room temperature. did. After diisopropylethylamine (0.07 mL, 0.43 mmol) was added to the reaction mixture, 13b (100 mg, 0.14 mmol) obtained in step 2 diluted in dichloromethane (5 mL) was slowly added thereto. After stirring for 1 hour, water (50 mL) was added, and then organic compounds were extracted with dichloromethane (30 mL, twice). The combined organic layers were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (5% methanol/dichloromethane) to obtain compound 14b (120 mg, 98%).

MS (ESI) m/z 885 [M+Na] ⁺

Step 4:

Compound 14b (56 mg, 0.06 mmol) obtained in step 3 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 1i (28 mg, 68%) as a white solid.

MS (ESI) m/z 639 [M+H] ⁺

< Example 10> Preparation of compounds 1j and 1k

Preparation of compound 1j

Step 1:

Compound 11 (500 mg, 1.02 mmol) obtained in Example 8 was mixed with dichloromethane (10 mL), cooled to 0 °C, N,N'-dicyclohexylcarbodiimide (DCC, 230 mg, 1.12 mmol) was added, and the compound 10a obtained in Example 6 (0.17 g, 1.02 mmol) was added. ) was added and stirred at room temperature for 1 hour. The reaction solution was filtered, concentrated under reduced pressure, and the concentrate was separated by column chromatography (30% ethyl acetate/ n -hexane) to obtain compound 15a (450 mg, 69%).

MS (ESI) m/z 640 [M+H] ⁺

Step 2:

Compound 15a (75 mg, 0.12 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 2c (14 mg, 29%) as a white solid.

MS (ESI) m/z 416 [M+H] ⁺ , 414 [MH] ^-

Step 3:

Compound 2c (14 mg, 0.034 mmol) and F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (6 mg, 0.034 mmol) obtained in step 2 were dissolved in ethanol (1 mL), and then 1 M CuSO ₄ aqueous solution (7 μL, 0.007 mmol) and 2 M aqueous sodium ascorbate solution (Na-Asc. 5 μL, 0.01 mmol) were sequentially added, and the mixture was stirred for 1 hour. The reaction solution was filtered, concentrated under reduced pressure, and then separated using high performance liquid chromatography (HPLC). The purified solution was lyophilized to obtain compound 1j (13 mg, 65%) as a white solid.

¹ H NMR (400 MHz, D ₂ O) δ 1.88-2.01 (m, 2H), 2.09-2.19 (m, 2H), 2.43-2.49 (m, 3H), 2.67-2.73 (m, 1H), 3.61- 3.64 (m, 5H), 3.71 (t, J = 4.0 Hz, 1H), 3.90-3.93 (m, 2H), 4.12 (s, 1H), 4.19-4.24 (m, 2H), 4.27 (d, J = 2.8 Hz, 1H), 4.45-4.48 (m, 1H), 4.56-4.60 (m, 3H), 4.65 (d, J = 6.0 Hz, 1H), 4.74 (s, 1H), 7.94 (s, 0.4H) , 8.02 (s, 0.6H)

MS (ESI) m/z 593 [M+H] ⁺ , 591 [MH] ^-

Preparation of compound 1k

Step 1:

Compound 11 (83 mg, 0.45 mmol) obtained in Example 8 was mixed with dichloromethane (15 mL), then cooled to 0 ^o C, N,N'-dicyclohexylcarbodiimide (DCC, 102 mg, 0.50 mmol) was added, and the compound 10c obtained in Example 7 (220 mg, 0.45) was added. mmol) and stirred at room temperature for 1 hour. The reaction solution was filtered, the solvent was removed under reduced pressure, and column chromatography (30% ethyl acetate/ n -hexane) was performed to obtain compound 15c (190 mg, 64%).

Step 2:

Compound 15c (170 mg, 0.26 mmol) obtained in step 1 was dissolved in 70% TFA/dichloromethane (2.0 mL) and stirred for 4 hours. After adding the reaction solution to ethyl ether to catch the precipitate, ethyl ether (20 mL) was further added, and then the precipitate was separated using a centrifuge. The precipitate was dissolved in water, separated using high performance liquid chromatography (HPLC), and then lyophilized to obtain compound 2d (83 mg, 74%).

Step 3:

Compound 2d (20 mg, 0.046 mmol) and F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (8 mg, 0.046 mmol) obtained in step 2 were dissolved in ethanol (2 mL), and then 1 M CuSO ₄ aqueous solution (9 μL, 0.009 mmol) and 2 M aqueous sodium ascorbate solution (Na-Asc. 7 μL, 0.014 mmol) were added, and the mixture was stirred for 1 hour. After filtration of the reaction solution, the solvent was removed under reduced pressure, followed by separation using high performance liquid chromatography (HPLC) and freeze-drying to obtain compound 1k (20 mg, 70%).

<Example 11> Preparation of compound 11

Step 1:

Compound 3b (500 mg, 0.83 mmol) and Fmoc-Lys(Z)-OH (340 mg, 0.83 mmol) obtained in Example 1 were dissolved in dichloromethane (10 mL), cooled to 0 °C, and N,N' -Dicyclohexylcarbodiimide (DCC, 190 mg, 0.913 mmol) was added and stirred for 1 hour. After filtration of the reaction solution, the solvent was removed under reduced pressure, and column chromatography (50% ethyl acetate/n-hexane) was performed to obtain compound 16 (500 mg, 55%).

MS (ESI) m/z 1087 [M+H] ⁺

Step 2:

Compound 16 (490 mg, 0.45 mmol) obtained in step 1 was dissolved in 20% piperidine/dichloromethane solution (10 mL) and stirred at room temperature for 1 hour. After removing the solvent under reduced pressure, it was separated by column chromatography (NH silica gel) to obtain compound 17 (240 mg, 61%).

MS (ESI) m/z 865 [M+H] ⁺

Step 3:

Pentinoic acid (12 mg, 0.121 mmol) was dissolved in dichloromethane (2.0 mL) and HOBt (16 mg, 0.121 mmol), TBTU (39 mg, 0.121 mmol), diisopropylethylamine (28 μL, 0.162 mmol) were added They were added sequentially and stirred for 10 minutes. Compound 17 (70 mg, 0.081 mmol) obtained in step 2 diluted in dichloromethane (1.0 mL) was added, stirred at room temperature for 1 hour, and then water was added. The organic compound was extracted using dichloromethane, treated with anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (3% methanol/dichloromethane) to obtain compound 18 (70 mg, 91%).

MS (ESI) m/z 945 [M+H] ⁺ , 967 [M+Na] ⁺

Step 4:

Compound 18 (80 mg, 0.0847 mmol) and F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (15 mg, 0.0847 mmol) obtained in step 3 were dissolved in ethanol (1 mL), and then 1 M CuSO ₄ aqueous solution (17 μL, 0.017 mmol) and 2 M aqueous sodium ascorbate solution (13 μL, 0.026 mmol) were sequentially added, and the mixture was stirred for 1 hour. The reaction solution was filtered, concentrated under reduced pressure, and separated by column chromatography (2% methanol/dichloromethane) to obtain compound 19 (76 mg, 80%).

MS (ESI) m/z 1122 [M+H] ⁺

Step 5:

Compound 19 (60 mg, 0.053 mmol) obtained in step 4 was dissolved in methanol (5 mL), palladium (10% Palladium on carbon, 22 mg) was added, and the mixture was stirred under hydrogen at room temperature for 2 hours. The reaction solution was filtered under reduced pressure and concentrated to remove the solvent to obtain compound 20 (35 mg, 67%).

MS (ESI) m/z 988 [M+H] ⁺

Step 6:

Dissolve 4-(p-tolyl)butanoic acid (8 mg, 0.0425 mmol) in dichloromethane (5 mL), HOBt (8 mg, 0.0531 mmol), TBTU (17 mg, 0.0531 mmol), diisopropylethylamine ( 13 μL, 0.0708 mmol) were sequentially added and stirred for 10 minutes. Compound 20 (35 mg, 0.0354 mmol) obtained in step 5 was dissolved in dichloromethane (2 mL) and stirred at room temperature overnight. The solvent was removed under reduced pressure and the mixture was separated by column chromatography (2% methanol/dichloromethane) to obtain compound 21 (40 mg, 98%).

MS (ESI) m/z 1148 [M+H] ⁺

Step 7:

Compound 21 (40 mg, 0.0348 mmol) obtained in step 6 was dissolved in 70% trifluoroacetic acid/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. The reaction solution was slowly dropped into ethyl ether to form a precipitate, and ethyl ether (20 mL) was further added. The resulting precipitate was separated using a centrifugal separator, which was purified by high-performance liquid chromatography (HPLC), and then the separated solution was freeze-dried to obtain Compound 11 (24 mg, 74%) as a white solid.

MS (ESI) m/z 924 [M+H] ⁺ , 922 [MH] ⁺

< Example 12> compound [ ¹⁸ Preparation of F]1f

Distilled water (3 mL) was flowed through Chromafix® (HCO ₃ ^- ), [ ¹⁸ F] fluoride aqueous solution (120 mCi) was passed through, and then ethanol (1 mL) was flowed through. Krytofix222-potassium methanesulfonate (10 mg) was dissolved in ethanol (1 mL), passed through Chromafix®, and the solvent was removed by blowing the solution with nitrogen gas at 100 °C. Dissolve MsO-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (1 mg) in t-butanol (0.5 mL), put it in a reaction vessel with [ ¹⁸ F]fluoride, and react at 100 ° C for 10 minutes (preparation of ¹⁸ F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ ). After the reaction, nitrogen gas was gently blown at 100 °C to remove the solvent, and then ethanol (0.3 mL) was added to dissolve it. To this solution, add compound 2a (3 mg) dissolved in distilled water (0.1 mL), and then add 0.5 M CuSO ₄ aqueous solution (5 μL) and 0.5 M sodium ascorbate aqueous solution (10 μL) sequentially and react at room temperature for 10 minutes. made it Distilled water (2 mL) was added to the reaction mixture, and the solution was filtered and separated by HPLC to obtain compound [ ¹⁸ F] 1f (34 mCi).

< Example 13> compound [ ¹⁸ F] Preparation of 1j

Distilled water (3 mL) was flowed through Chromafix® (HCO ₃ ^- ), [ ¹⁸ F] fluoride aqueous solution (80 mCi) was passed through, and then ethanol (1 mL) was flowed through. Krytofix222-potassium methanesulfonate (10 mg) was dissolved in ethanol (1 mL), passed through Chromafix®, and the solvent was removed by blowing the solution with nitrogen gas at 100 °C. Dissolve MsO-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ (1 mg) in t-butanol (0.5 mL), put it in a reaction vessel with [ ¹⁸ F]fluoride, and react at 100 ° C for 10 minutes (preparation of ¹⁸ F-CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -N ₃ ). After the reaction, nitrogen gas was blown at 100 °C to remove the solvent, and then ethanol (0.3 mL) was added to dissolve it. Compound 2c (3 mg) dissolved in distilled water (0.1 mL) was added to this solution, and then 0.5 M CuSO ₄ aqueous solution (5 μL) and 0.5 M sodium ascorbate aqueous solution (10 μL) were sequentially added, followed by reaction at room temperature for 10 minutes. made it Distilled water (2 mL) was added to the reaction mixture, the solution was filtered, and then separated by HPLC to obtain compound [ ¹⁸ F] 1j (12 mCi).

< comparative example 1> DCFPyL Produce

Step 1:

6-fluoronicotinic acid (100 mg, 0.71 mmol) and 2,3,5,6-tetrafluorophenol (130 mg, 0.78 mmol) were dissolved in dioxane (2.0 mL), followed by N,N-dicyclohexane Silcarbodiimide (DCC, 146 mg, 0.71 mmol) was added to the reaction solution and stirred at room temperature for 2 hours. The precipitate formed after the reaction was filtered and washed with dichloromethane. The filtrate was concentrated under reduced pressure, and then Compound 22 (136 mg, 47%) as a white solid was obtained by column chromatography (5% ethyl acetate/n-hexane).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.09-7.16 (m, 2H), 8.58 (ddd, J = 2.5 Hz, 7.3 Hz, 8.7 Hz, 1H), 9.10 (d, J = 2.4 Hz, 1H);

MS (ESI) m/z 290 [M+H] ⁺

Step 2:

Compound 22 (100 mg, 0.35 mmol) obtained in step 1 above and compound 3a (202 mg, 0.42 mmol) in dichloromethane (5.0 mL) and triethylamine (0.146 mL, 1.1 mmol) was added to the reaction solution, followed by stirring at room temperature for 20 minutes. After the reaction, distilled water was added, and organic compounds were extracted using dichloromethane. The combined organic solutions were treated with anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography (2% methanol/dichloromethane) was used to obtain compound 23 (138 mg, 66%) as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.37 (s, 9H), 1.42 (s, 9H), 1.43 (s, 9H), 1.48-1.62 (m, 3H), 1.65-1.70 (m, 1H), 1.72-1.87 (m, 3H), 1.98-2.07 (m, 1H), 2.25-2.38 (m, 2H), 3.32-3.39 (m, 1H), 3.54-3.62 (m, 1H), 4.13-4.21 (m) , 2H), 5.46 (d, J = 7.6 Hz, 1H), 5.88 (d, J = 8.0 Hz, 1H), 7.00 (dd, J = 3.0 Hz, 8.4 Hz, 1H), 7.93 (s, 1H), 8.42 (m, 1H), 8.84 (d, J = 2.4 Hz, 1H);

MS (ESI) m/z 611 [M+H] ⁺ , 633 [M+Na] ⁺

Step 3:

Compound 23 (100 mg, 0.16 mmol) obtained in step 2 was dissolved in 60% TFA/dichloromethane solution (1.0 mL) and stirred at room temperature for 4 hours. After adding the reaction solution to ethyl ether to catch the precipitate, ethyl ether (20 mL) was further added, and then the precipitate was separated using a centrifuge. The precipitate was dissolved in water, separated using high performance liquid chromatography (HPLC), and then lyophilized to obtain the compound DCFPyL (56 mg, 79%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.41-1.47 (m, 2H), 1.58-1.65 (m, 2H), 1.67-1.75 (m, 1H) 1.79-1.95 (m, 2H), 2.06-2.15 ( m, 1H), 2.06-2.15 (m, 1H), 2.44 (t, J = 7.2 Hz, 2H), 3.37 (t, J = 6.6 Hz, 2H),

4.13-4.18 (m, 2H), 7.17 (d, J = 8.4 Hz, 1H), 8.25 (tt, J = 1.2 Hz, 8.0 Hz, 1H), 8.51 (d, J = 1.6 Hz, 1H);

MS (ESI) m/z 465 [M+Na] ⁺

< comparative example 2> [ ¹²⁵ Synthesis of compound I]20

Step 1:

Triphosgene (21 mg, 0.071 mmol) was dissolved in dichloromethane (5 mL), and 4-iodoaniline (45 mg, 0.21 mmol) dissolved in dichloromethane (5 mL) was slowly added at 0 °C, followed by triethylamine (0.57). mL, 0.41 mmol) was added and stirred at 0 °C for 30 minutes. Compound 3a (100 mg, 0.21 mmol) dissolved in dichloromethane (10 mL) was slowly added at 0 °C, triethylamine (0.57 mL, 0.41 mmol) was added thereto, and the mixture was stirred for 5 hours while slowly raising the temperature to room temperature. The mixture was concentrated under reduced pressure, and column chromatography (2% methanol/dichloromethane) was used to obtain compound 24 (66 mg, 44%) as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.20-1.27 (m, 2H), 1.37 (s, 9H), 1.40 (s, 9H), 1.44 (s, 9H), 1.47-1.57 (m, 2H), 1.71-1.81 (m, 2H), 1.83-1.91 (m, 1H), 2.03-2.11 (m, 1H), 2.37 (sext, J = 8.2 Hz, 2H), 3.01-3.07 (m, 1H), 3.51- 3.56 (m, 1H), 3.97-4.01 (m, 1H), 4.26-4.32 (m, 1H), 5.75 (d, J = 7.2 Hz, 1H), 6.31 (q, J = 3.4 Hz, 1H), 6.40 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.90 (s, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 24.5, 27.1, 27.8, 27.9, 28.0, 29.6, 31.7, 32.0, 39.1, 53.8, 54.9, 81.0, 81.8, 83.6, 83.7, 120.2, 137.5, 140.2, 155.6, 158.5 , 171.8, 172.0, 175.3; MS (ESI) m/z 733 [M+H] ⁺

Step 2:

Compound 24 (50 mg, 0.068 mmol) synthesized in step 1 was dissolved in 1,4-dioxane (1.0 mL), and then hexamethylditin ((Me ₃ Sn) ₂ , 0.043 mL, 0.21 mmol) and bis ( Triphenylphosphine) palladium dichloride (4.8 mg, 5 mol%) was sequentially added and stirred at 110° C. for 90 minutes. The mixture was cooled to room temperature, potassium fluoride aqueous solution (50 mL) was added, stirred for 1 hour, and then filtered. The filtrate was extracted with ethyl acetate to extract organic compounds, and the collected organic solutions were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography (triethylamine:ethyl acetate:n-hexane, 1:40:59) was used. to obtain compound 25 (28 mg, 53%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 0.25 (s, 9H), 1.22-1.29 (m, 2H), 1.38 (s, 9H), 1.41 (s, 9H), 1.43 (s, 9H), 1.48- 1.59 (m, 2H), 1.72-1.78 (m, 1H), 1.81-1.91 (m, 1H), 2.05-2.13 (m, 2H), 2.34-2.43 (m, 2H), 3.04-3.09 (m, 1H) ), 3.51-3.55 (m, 1H), 4.04 (pent, J = 4.9 Hz, 1H), 4.33 (sext, J = 4.5 Hz, 1H), 5.73 (d, J = 6.8 Hz 1H), 6.23 (br s) , 1H), 6.32 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.73 (s, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ -9.5, 24.2, 27.4, 27.8, 27.9, 28.0, 29.7, 31.8, 32.1, 39.1, 53.7, 54.7, 80.9, 81.7, 83.5, 118.4, 133.6, 136.2, 140.4, 155.9, 158.3, 171.9, 172.2, 175.1; MS (ESI) m/z 771 [M+2H] ⁺

Step 3:

Compound 25 (0.1 mg) obtained in step 2 was dissolved in ethanol (0.250 mL), sodium [ ¹²⁵ I] iodide aqueous solution (3.2 mCi, 0.050 mL) was added thereto, and the mixture was stirred at room temperature. 1 N HCl aqueous solution (0.10 mL) was added, and then 3% H ₂ O ₂ was added and stirred at room temperature for 10 minutes. An aqueous sodium thiosulfate solution (0.1 M, 0.20 mL) was added to the reaction mixture, distilled water (18 mL) was added, and the solution was passed through a C-18 Sep-Pak, and then distilled water (20 mL) was flowed. After flowing acetonitrile (2.0 mL) to the C-18 Sep-Pak, nitrogen was blown into the solution to remove acetonitrile.

Step 4:

To the reaction mixture obtained in step 3, dichloromethane (0.2 mL) and trifluoroacetic acid (0.8 mL) were sequentially added and stirred at room temperature for 20 minutes. After nitrogen was blown to remove the reaction solvent, distilled water (2.0 mL) was added, and the solution was separated by high performance liquid chromatography (HPLC) to obtain compound [ ¹²⁵ I] 26 (1.1 mCi, 24%).

HPLC conditions:

column, XTerra MS C18 (250 mm x 10 mm); mobile phase, 30% acetonitrile/water (0.1% TFA); Flow rate, 5 mL/min; UV, 254 mm; hold time, 10.4 min.

< Reference example 1> Prostate cancer cell line and nude mouse preparation

A human prostate cancer cell line (22RV1) was purchased from the American Type Culture Collection (ATCC) and used. Human prostate cancer cell lines, PC3 PIP (PSMA ⁺ ) and PC3 flu (PSMA ^- ) cell lines, were prepared by Dr. Martin G. Pomper (Johns Hopkins Medical School, Baltimore, MD) provided and used. For human prostate cancer cell lines, a culture medium in which 10% fetal bovine serum (FBS) and 1% antibiotic/antifungal agent was added to RPMI1640 medium was used. When culturing PC3 PIP (PSMA ⁺ ) and PC3 flu (PSMA ^- ) cell lines, puromycin at a concentration of 2 μg/mL was additionally added and cultured.

As the experimental animals, 6-week-old male nude mice (Narabio, Seoul, Korea) were used.

< Experimental example 1> binding ability measurement

In order to confirm the binding ability of the compound of the present invention to the PSMA protein, an experiment was performed as follows.

As a buffer solution, 1% BSA (bovine serum albumin) was added to RPMI1640 medium and used.

In a container containing 22RV1 cells (5×10 ⁴ ), put [ ¹²⁵ I] 26 (0.1 nM) obtained in Comparative Example 2, and 9 concentrations (1.00 x 10 ^-4 ~ 1.00 x 10 ^-12 M) of Chemical Formula 1 After adding each of the compounds, the mixture was stirred at 37 °C for 2 hours. After stirring was completed, each was washed three times with PBS solution (2 mL), and then the radiation dose was measured with a gamma counter (2480 WIZARD2 Gamma Counter PerkinElmer Co., MA). The 50% inhibitory concentration (IC ₅₀ ) for each compound was calculated using the GraphPad Prism (GraphPad Software, Inc., CA) program.

Table 1 below is a table showing the binding affinity (IC ₅₀ ) of each compound, and DCFPyL in Table 1 is the compound obtained in Comparative Example 1. (Chen, Y. et al., Clin Cancer Res. 2011, 17(24), 7645-7653)

compound IC ₅₀ (Mean±SD, nM) DCFPyL (Comparative Example 1) 30.71±10.18 1a 136.76±8.47 1b 12.16±0.93 1c 12.46±3.51 1e 13.86±0.55 1f 12.21±0.46 1 g 12.12±0.17 1h 108.21±1.87 1j 78.88±5.36 1k 197.84±28.32 1l 86.06±3.13

As shown in Table 1 above,

Compounds 1b and 1c of Example 2 of the present invention are compounds having a carboxylic acid, and it can be seen that they have about 11 times higher binding force to the PSMA protein than Compound 1a without a carboxylic acid. This is a strong salt bridge interaction between one of three arginine residues (R463) in the binding region of the PSMA protein and the carboxylic acid of the compound of the present invention called an arginine patch (Salt Bridge Interaction). can be interpreted as forming

The DCFPyL compound of Comparative Example 1 is pyridine-bound, and it is known to increase the binding force because it has an aryl-cation interaction with the arginine residue (R463) of the PSMA protein.

However, the compounds of Example 2 of the present invention having a carboxyl group have a binding force about 2.5 times higher than that of DCFPyL of Comparative Example 1, which is more of an ionic bond between an arginine cation and a carboxylate anion than an aryl-positron interaction. It is consistent with the results of previous studies.

In addition, it can be seen that compounds 1e , 1f , and 1g also exhibit a similar level of binding to PSMA protein as compounds 1b and 1c .

That is, the carboxylic acid of the compound of Formula 1 of the present invention not only greatly improves the binding force with PSMA protein, but also increases the polarity of the compound to lower non-specific binding in vivo and to be removed more rapidly.

As mentioned above, although the present invention has been described in detail through preferred examples and experimental examples, the scope of the present invention is not limited to specific examples and should be interpreted by the appended claims. In addition, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (8)

A compound represented by the following formula (1), a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Formula 1,
L ₁ is —(CH ₂ ) ₄ —;
U is a bond;
V is a bond;
W is N;
L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;
X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;
Y is a bond, N, or CH;
L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;
L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;
R is hydrogen, halogen, or CH ₃ ;
Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;
Tz is

, or

ego;
L ₅ is -(CH ₂ ) _f -, -(CH ₂ CH ₂ O) _g (CH ₂ ) _h -, or -(CH ₂ O) _i (CH ₂ CH ₂ O) _j (CH ₂ ) _k - , wherein f, g, h, i, j and k are independently integers from 1 to 5;

m and n are independently integers of 0 or 1,
If m is an integer of 0, then

is -H, or absent;
If n is an integer of 0, then

is -H, or absent; and

F is ¹⁸ F.
The method of claim 1,
When R is halogen, the compound, characterized in that the halogen is a radioactive isotope, a stereoisomer, a hydrate thereof, or a pharmaceutically acceptable salt thereof.
The method of claim 1,
The compound represented by Formula 1 is a compound, characterized in that it is a compound represented by Formula 1-1, a stereoisomer, a hydrate thereof, or a pharmaceutically acceptable salt thereof:
[Formula 1-1]

In Formula 1-1,
L ₁ , U, V, W, L ₂ , X, Z, Tz, L ₅ and F are as defined in claim 1 ; and
Y is a bond, NH, or CH ₂ .
A compound represented by the following formula (2), a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof:
[Formula 2]

In Formula 2,
L ₁ is —(CH ₂ ) _a —, wherein a is an integer from 2 to 4;
U is a bond, or -C(=O)-;
V is a bond, -NH-, or -C(=O)-;
W is CH, or N;
L ₂ is a bond, or —(CH ₂ ) _b —, where b is an integer from 1 to 3;
X is a bond, —CH ₂ CH ₂ O—, or —C(=O)—;
Y is a bond, N, or CH;
L ₃ is —(CH ₂ ) _c —, wherein c is an integer from 2 to 4;
L ₄ is —(CH ₂ ) _d —, where d is an integer from 2 to 4;
R is hydrogen, halogen, or CH ₃ ;
Z is -(CH ₂ ) _e -, or -NH-C(=O)-(CH ₂ ) _e -, wherein e is an integer from 1 to 3;
T is -C≡CH, or -N ₃ ; and

n is an integer of 0 or 1,
If n is an integer of 0, then

is -H, or absent.
The method of claim 1,
The compound represented by Formula 1 is any one compound selected from the group consisting of the following Formulas 1e, 1f, 1g and 1l, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable compound thereof salt:

,

,

and

.
A composition for diagnosing prostate cancer comprising the compound of claim 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
7. The method of claim 6,
The compound is a composition for diagnosing prostate cancer, characterized in that it selectively binds to a prostate-specific membrane antibody (PSMA) overexpressed in prostate cancer cells to diagnose prostate cancer.
A pharmaceutical composition for preventing or treating prostate cancer, comprising the compound of claim 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.