RU2825402C1 - COMPLEX OF UREA DERIVATIVE WITH 99mTc RADIONUCLIDE LABEL FOR DIAGNOSIS OF TUMORS EXPRESSING PROSTATE-SPECIFIC MEMBRANE ANTIGEN, AND METHOD FOR PRODUCTION THEREOF - Google Patents

Abstract

FIELD: organic chemistry; medicine; oncology; pharmacology.

SUBSTANCE: invention relates to a complex of a urea derivative with ^99mTc radionuclide label for the diagnosis of tumors expressing a prostate-specific membrane antigen, ^99mTc-(((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butanamido)butanamido)acetamido)hexanamido)octanamido)-3-(4-hydroxyphenyl)propanamido)-3-(naphthalen-2-yl)propane-amido)-1-carboxypentyl)carbamoyl)-L-glutamic acid, presented by the structural formula given below. Also disclosed is a method of producing said complex of a derivative of urea with ^99mTc radionuclide label.

EFFECT: wider range of complexes of urea derivatives with ^99mTc radionuclide label for the diagnosis of tumors expressing a prostate-specific membrane antigen, with simultaneous increase in specific tumor uptake to (30±4) % and tumor:blood ratio to (38±14).

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Description

The inventions relate to organic chemistry, medicine, oncology and pharmacology, in particular to a radiopharmaceutical compound based on a urea derivative tropic to prostate-specific membrane antigen (PSMA) and containing ^99m Tc, and a method for its production for radionuclide diagnostics of prostate cancer.

NO ₂ -DOTA-PSMA is known to be a urea derivative for diagnosing tumors expressing prostate-specific membrane antigen (PSMA) [RU 2730507 C1, IPC (2006.01) C07D 257/02, A61K 49/00, A61K 51/00, published 08/24/2020. A compound for diagnosing tumors expressing PSMA and a composition based on it], which is a conjugate based on a urea derivative of the Glu-Urea-Lys structure and a modified hydrophobic peptide linker comprising a 6-aminohexanoic acid fragment linked to a DOTA chelator (1):

Trivalent metal cations are used as radionuclide labels for compound (1): ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁹⁰ Y, ¹⁰⁹ Pb, ²⁰³ Pb, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ²¹³ Bi, ⁴⁴ Sc, ⁴⁷ Sc, ¹⁵³ Sm, ¹⁶¹ Tb and ²²⁵ Ac.

For in vivo studies _{, labeled complexes with compound (1) were prepared as follows: 20 μl of an aqueous solution of NO 2 -DOTA-PSMA (PSMA - PSMA inhibitor) with a concentration of 0.5 mg/ml, 20 μl of a 0.5 g/ml NaOAc solution pH 5 were added to 760 μl of a solution of [ 90 Y]YCl 3} ^{with an activity} _{of 200} kBq or [ ⁶⁸ Ga]GaCl 3 with an activity ^of 4 MBq in 0.1 M HCl, obtained from radionuclide generators ⁹⁰ Sr/ 90 Y and 68 Ge/ 68 Ga, respectively.

The resulting solution was kept at 95°C for 15 minutes.

A urea derivative PSMA-I&T of formula (2) for visualization and therapy, as well as a method for obtaining its complex with ¹⁷⁷ Lu with PSMA-I&T (PSMA inhibitor for diagnostics and therapy) are known [Wurzer A, Di Carlo D, Schmidt A, et al. Radiohybrid ligands: a novel tracer concept exemplified by ¹⁸ F- or ⁶⁸ Ga-labeled rhPSMA inhibitors. J Nucl Med. 2020; 61:735–742. Abstract/FREE Full TextGoogle Scholar].

In this method, 4 mg of gentisic acid are dissolved in 0.5 ml of highly purified water. PSMA-I&T is dissolved in a solution containing 0.1 mg of gentisic acid (~ 96 nmol). Then, 13 mg of sodium ascorbate and 31 mg of sodium acetate trihydrate are dissolved in 6 ml of 0.04 M acetic acid solution. 4 GBq of [ ¹⁷⁷ Lu]LuCl ₃ are added to the solution. The reaction mixture is heated at 80 °C for 20 min. After heating, the reaction mixture is cooled to room temperature. Then, a mixture containing sodium chloride, DTPA and ethanol is added to the cooled solution (1.2 ml of ethanol (≥99%) is added to 10 ml of DTPA in sodium chloride). The final volume of the reaction mixture is 11 ml. The radiochemical yield (RCY) is over 95%. The disadvantage is that the labeling technology involves the use of a wide range of reagents that protect the radioligand from the radiolysis process. In addition, the labeling technology involves the use of ethanol as a solubilizer due to the high lipophilicity of the resulting radioligand, and the radiochemical purity (RCP) of the resulting radioligand is 90%, which is due to the use of a wide range of auxiliary substances to ensure the stability of the radiocomplex.

A theranostic PSMA-binding ligand PSMA-617 is known based on urea (Glu-Urea-Lys), a chelating part in the form of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and a linker part between them made of 2-naphthyl-L-alanine and tranexamic acid (3) [Schollhammer, Romain, et al. "Comparison of ⁶⁸ Ga-PSMA-617 PET/CT and ⁶⁸ Ga-RM2 PET/CT in Patients with Localized Prostate Cancer Who Are Candidates for Radical Prostatectomy: A Prospective, Single-Arm, Single-Center, Phase II Study." Journal of Nuclear Medicine 64.3 (2023): 379-385]:

The ligand PSMA-617 labeled with ¹⁷⁷ Lu is used for therapeutic purposes, and the ligand labeled with ⁶⁸ Ga is undergoing phase II clinical trials as a PET diagnostic drug.

A method for obtaining a complex of ⁶⁸ Ga with the PSMA-binding ligand PSMA-617 for diagnosing prostate cancer is known [Schollhammer, Romain, et al. "Comparison of ⁶⁸ Ga-PSMA-617 PET/CT and ⁶⁸ Ga-RM2 PET/CT in Patients with Localized Prostate Cancer Who Are Candidates for Radical Prostatectomy: A Prospective, Single-Arm, Single-Center, Phase II Study." Journal of Nuclear Medicine 64.3 (2023): 379-385], corresponding to formula (4):

According to the method, PSMA-617 is dissolved in distilled water at a concentration of 1 μg/μl. About 20-30 mCi ^{of 68} GaCl ₃ is added to 2.5 μg (2.4 nmol) of PSMA-617 and adjusted to pH 3.5-4 with HEPES. The reaction mixture is incubated for 10 min at 95°C. After cooling to room temperature, the mixture is passed through a C ₁₈ Sep-Pak column. The radiochemical purity of the labeled complex is checked by HPLC and thin-layer chromatography. The RHC is over 96%. The uptake of ⁶⁸ Ga-PSMA-617 in PSMA-expressing tumors in distribution experiments in mice is 8.5±4.1%, the tumor:blood ratio is 7.8.

The disadvantages of the ⁶⁸ Ga-PSMA-617 complex are related to the low availability ^{of 68} Ge/ ⁶⁸ Ga generators in the Russian Federation, the short half-life of ⁶⁸ Ga, limited availability and high cost of PET center services.

The closest to the claimed method can be considered to be the method for obtaining technetium and rhenium bis(heteroaryl) complexes with urea derivatives and methods of their use for inhibiting PSMA [RU 2532912 C2, IPC (2006.01) C07C 275/24, C07D 213/56, C07D 403/12, C07D 401/12, C07F 13/00, A61K 49/00, A61K 51/04, published 20.11.2014. Technetium and rhenium bis(heteroaryl) complexes and methods of their use for inhibiting PSMA]. The complexes belong to radiopharmaceutical compounds of the structural formula (5):

where R denotes H or a (C1-C8)alkyl group;

W denotes a bond, -CH(NH ₂ )-, -C(O)-NH-CH(COOH)-, -O-(CH ₂ ) _n -O-(CH ₂ ) _n -, or -(CH ₂ )nO(CH ₂ ) _n O(CH ₂ ) _n ;

Z represents -NHC(O)-, -NH-C(O)-CH(NH ₂ )-, or -C(O)-NH-CH(COOH)-;

e is an integer from 1 to 4;

f is an integer from 0 to 10;

g is an integer from 0 to 10;

n is an integer from 0 to 2;

the values of the radical NR ^a R ^b are given in the invention formula.

Radiolabeling with ^99m Tc is performed in two steps using commercial IsoLink™ kits (Covidien) to form the intermediate [ ^99m Tc(CO) ₃ ( _H2O ) ₃ ] ⁺ , which is reacted with the appropriate compound of formula (5) ( ^10-6 M to ^10-4 M) in an equal volume mixture (1:1) of acetonitrile and phosphate buffer. The sealed vessel is heated at 100°C for 30 min.

The disadvantages of the method are the use of a commercially unavailable set of reagents, a two-stage technique, the use of an organic solvent, while a radiochemical yield of over 95% is achieved only at a concentration of compounds greater than 10 ^-4 M. In addition, it has been shown in laboratory animals that specific tumor uptake is up to 12%, and the tumor:blood ratio is up to 10.

The technical result of the claimed inventions is the expansion of the arsenal of complexes of urea derivatives with a radionuclide label ^99M Tc for the diagnosis of tumors expressing prostate-specific membrane antigen, while simultaneously increasing specific tumor uptake and the tumor:blood ratio.

The technical result is achieved by a complex of a urea derivative ((((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butanamido)butanamido)acetamido) hexanamido)octanamido)-3-(4-hydroxyphenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid) with

radionuclide label ^99m Tc with the following structural formula (6):

(6)

The technical result is also achieved by the method of obtaining the claimed complex of urea derivative ((((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butane-amido)butanamido)acetamido)hexanamido)octanamido)-3-(4-hydroxyphenyl)propane-

(amido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid) with a radionuclide label ^99m Tc, which consists of the following: 5-2000 μg of the specified urea derivative, dissolved in phosphate-buffered saline, are added to a lyophilisate containing 4-6 mg of sodium gluconic acid, 50-150 μg of stannous chloride and 50-200 μg of sodium ethylenediaminetetraacetic acid. Then 250-750 MBq ^{of 99m} Tc-pertechnetate is added to the mixture and incubated at a temperature of 70-90°C for 30-90 min.

The radiochemical yield is 95.0-99.8%, therefore purification of the obtained complex is not required. The uptake of the urea derivative complex with the radionuclide label ^99m Tc in the tumor is (30±4)%, the tumor:blood ratio is (38±14).

Figure 1 shows the biodistribution of the urea derivative BQ0413 complex with the radionuclide label ^99m Tc in xenografts of PSMA-expressing tumors at 1, 3, 6 hours after injection, where %ID/g is the percentage of the administered dose of the substance per organ weight. Data are presented as the mean %ID/g ± standard deviation for four mice.

Figure 2 shows the tumor/organ ratio of the urea derivative BQ0413 complex with the radionuclide label ^99m Tc in xenografts of PSMA-expressing tumors at 1, 3, 6 hours after injection. Data are presented as the mean tumor/organ ratio ± standard deviation of four mice.

The synthesis of the starting urea derivative designated as BQ0413 was carried out as described in [Lundmark, Fanny, et al. "Design, synthesis, and evaluation of linker-optimised PSMA-targeting radioligands." Pharmaceutics 14.5 (2022): 1098] in several steps. First, the tri-tert-butyl derivative of the PSMA-binding ligand of formula (7) was prepared by a method known from the state of the art [Ryan P. Murelli, Andrew X. Zhang, Julien Michel, William L. Jorgensen, David A. Spiege. Chemical Control Over Immune Recognition: A Class of Antibody-Recruiting Molecules (ARMs) that Target Prostate Cancer. J. AM. CHEM. SOC. 2009, 131, 17090-17092]:

From intermediate (7), the urea derivative was synthesized using solid-phase peptide synthesis. All amino acid coupling reactions were performed in dimethylformamide (DMF) using a coupling agent (Oxyma Pure; N,N'-diisopropylcarbodiimide and N,N-diisopropylethylamine base. After each coupling reaction, the resin was washed with DMF, and the Fmoc-protecting groups were removed using 20% piperidine in DMF.

The Fmoc-Nal-OH moiety was coupled to the free amino group in (7) by coupling, and subsequent amino acids were coupled (Fmoc-Tyr-OH, Fmoc-8Aoc, and Fmoc-Lys(Alloc)-OH). After deprotection of lysine from Fmoc, the free amine was acetylated in pyridine. After washing with DMF and dichloromethane, the Alloc on the lysine side chain was deprotected with PhSiH ₃ and Pd(PPh ₃ ) ₄ in dichloromethane. Glycine, three glutamate residues, and 2-mercaptoacetic acid were coupled using the standard coupling procedure described above. The final product was isolated using 5% aqueous trifluoroacetic acid in the presence of the carbocation scavenger triisopropylsilane. The final product was precipitated in cold diethyl ether and purified by reversed-phase high-performance liquid chromatography. Thus, the compound - a urea derivative containing three residues of glutamic acid and one cysteine residue and designated as BQ0413, ((((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butanamido)butanamido)acetamido)hexanamido) octanamido)-3-(4-hydroxyphenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-

1-carboxypentyl)carbamoyl)-L-glutamic acid) with the following structural formula (8):

Example 1. Preparation of a complex of the urea derivative BQ0413 with the radionuclide label ^99m Tc.

10 μg of the urea derivative BQ0413 dissolved in phosphate-buffered saline (pH 7.4) were added to a lyophilisate containing 5 mg of sodium gluconic acid, 75 μg of stannous chloride and 100 μg of sodium ethylenediaminetetraacetic acid. A solution of ^99m Tc-pertechnetate with an activity of 400 MBq was then added to the mixture, after which incubation was carried out at 90°C for 60 min.

The radiochemical yield was 99.8%, the radiochemical purity was over 99%.

For the obtained complex of the urea derivative BQ0413 with the radionuclide label ^99m Tc, the biodistribution profile was determined in PSMA-expressing PC-3.pip xenografts 1, 3, and 6 hours after injection. BALB/C nu/nu mice were intravenously injected with 40 pmol (60 kBq) of the complex of the urea derivative BQ0413 with the radionuclide label ^99m Tc in a volume of 100 μl in 1% bovine serum albumin in phosphate buffered saline. After 1, 3, and 6 h, respectively, the mice were euthanized by injection of a ketalar-rompun solution (10 mg/ml ketalar and 1 mg/ml rompun; 20 μl of solution/g body weight) and dissection was performed followed by weighing of organs and tumor tissue. The activity absorbed by the organs and tumor tissue was then measured using a gamma counter and the percentage of the administered dose per gram (%ID/g) was calculated (Fig. 1). The uptake of the urea derivative complex BQ0413 with the radionuclide label ^99mTc in the tumor was (30±4)%, the tumor:blood ratio (38±14) 1 hour after injection (Fig. 2).

Other examples of obtaining a complex of the urea derivative BQ0413 with a radionuclide label ^{99 m} Tc for the diagnosis of tumors expressing prostate-specific membrane antigen are presented in Table 1.

From the experimental data presented in Table 1 it follows that the stated technical result is achieved in all the examples given.

Thus, the complex of the urea derivative BQ0413 with the radionuclide label ^99m Tc, obtained by the claimed method, makes it possible to expand the arsenal of promising complexes of urea derivatives with the radionuclide label ^99M Tc for diagnosing prostate cancer, providing specific capture in tumors with PSMA hyperexpression (capture in the tumor is (30±4)%, the tumor:blood ratio is (38±14)), and the method for obtaining the proposed complex ^{of 99m} Tc with the urea derivative BQ0413 provides a radiochemical yield of up to 99.8%.

Claims (3)

1. A complex of a urea derivative with a radionuclide label ^99m Tc for the diagnosis of tumors expressing prostate-specific membrane antigen, with the general formula: ^99m Tc-(((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butanamido)butanamido)acetamido)hexanamido)octanamido)-3-(4-hydroxyphenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid, represented by the following structural formula: 2. A method for producing a complex of a urea derivative ((((1S)-5-((2S)-2-((2S)-2-(8-((2S)-2-acetamido-6-(2-(4-carboxy-2-(4-carboxy-2-(4-carboxy-2-(2-mercaptoacetamido)butanamido)butanamido)butanamido)acetamido)hexanamido)octanamido)-3-(4-hydroxyphenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid) with a radionuclide label ^99m Tc according to claim 1, characterized in that 5-2000 μg of the said urea derivative, dissolved in phosphate buffered saline, are added to lyophilisate containing 4-6 mg sodium salt of gluconic acid, 50-150 μg tin chloride and 50-200 μg sodium salt of ethylenediaminetetraacetic acid, then ^99m Tc-pertechnetate 250-750 MBq is added to the mixture and incubated at 70-100°C for 30-90 min.