Method for recovering and removing palladium in preparation of anticancer agent prodrug conjugate

Abstract

The present invention provides, in a method for preparing maleimide-KGDEVD-PABC-doxorubicin that is an anticancer agent prodrug conjugate activated by caspase, a method for effectively removing and recovering palladium remaining in the drug through simple process improvement.

Description

Method for recovering and removing palladium in the manufacture of anticancer prodrug conjugates

[0001] The present invention relates to a method for recovering and removing palladium in the production of an anticancer prodrug conjugate, and more particularly, to a method for recovering and removing palladium in the production of an anticancer prodrug conjugate activated by caspase.

[0002] Anti-cancer chemotherapeutic agents (CCAs) are the most widely used drugs in cancer treatment due to their potent anti-cancer effects. However, their use is often limited due to serious side effects and toxicity. Therefore, there is a need for CCAs to selectively target cancer tissues, sparing normal tissues. To achieve this, antibodies or peptides that recognize and bind to biomarkers that are rarely or absent in normal cells but specifically expressed in tumor cells have been commonly used. These drugs are then selectively delivered to cancer tissues. However, these drugs have limitations, as the genetic makeup of each patient, even within the same cancer type, can vary significantly. Consequently, these drugs can only be used in patients with cancers expressing the targeted biomarkers. This inherently limits their use to a broad patient population. Furthermore, recent research has revealed intratumor heterogeneity, where genetic characteristics differ between different tumor cells within a single cancer tissue. This means that biopsies, which are routinely performed to determine the characteristics of an affected cancer, cannot represent the specific genetic characteristics of that cancer. This means that even if the expression of a targeted biomarker is confirmed through a biopsy, the efficacy of the drug remains unclear. Existing drugs have fundamental limitations in delivering anticancer chemotherapeutic agents to all affected tumor cells, making them less than ideal delivery systems for anticancer chemotherapeutic agents and unsuitable for effective cancer treatment. To address this issue, prodrugs capable of specific activation within tumor cells and cancer tissues have been developed. In this regard, Korean Patent No. 1759261 discloses a multifunctional anticancer agent precursor activated by an inducible phenotype, a method for preparing the same, and its use.The method for manufacturing the prodrug described in the above prior art document induces a reaction by adding tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) and acetic acid for deprotection by removing the allyl group of maleimide-KGDEVD-PABC-doxorubicin.

[0003] However, the above prior art coprecipitates during the dissolution and crystallization process, resulting in a large amount of remaining palladium. To remove the palladium, there are various methods, such as conventional washing-separation, resin treatment, and filtration, but they are not suitable for removing it to the desired level.

[0004] The present invention is intended to solve various problems including the above-mentioned problems, and aims to provide a method for effectively removing and recovering palladium remaining in maleimide-KGDEVD-PABC-doxorubicin, an anticancer prodrug conjugate activated by caspase, through simple process improvement in the manufacturing method of the drug.

[0005] According to one aspect of the present invention, there is provided a method for preparing an allyl group-protected precursor of a prodrug conjugate, wherein a peptide linker cleaved by a protein cleavage enzyme is linked to an anticancer compound;

[0006] A step of preparing a deprotection reaction solution in which the allyl group of the precursor is removed by dissolving the precursor and tetrakistriphenylphosphine palladium (Pd(PPh3)4) in an anhydrous organic solvent;

[0007] A step of adding hydrochloric acid and a catalyst to the above reaction solution, stirring at room temperature, and then filtering to remove bis(triphenylphosphine)palladium dichloride (PdCl2(PPh3)2) produced by the reaction of Pd(PPh3)4 and hydrochloric acid; and

[0008] A method for removing and recovering a high-purity palladium compound in a process for manufacturing a prodrug conjugate is provided, which comprises a precipitation step of adding a mixture containing an organic solvent and a precipitation solvent to the reaction solution to precipitate an amorphous solid.

[0009] As described above, the method for recovering and removing palladium in the production of the anticancer prodrug (prodrug) conjugate of the present invention can realize a method for rapidly recovering and removing palladium remaining in the drug used in the deprotection reaction to remove an allyl group through simple improvement of the production process. Compared to conventional methods, the method simplifies the separation and purification process and increases the removal efficiency, thereby increasing the yield to 35%, making it economical and applicable to mass production processes and the production of low-cost, high-quality prodrugs. Of course, the scope of the present invention is not limited by these effects.

[0010] Figure 1 is a graph showing the IR analysis results of Pd(PPh3)4STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0011] Figure 2 is a graph showing the IR analysis results of PdCl2(PPh3)2STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0012] Figure 3 is a graph showing the IR analysis results of the Pd filtrate, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0013] Figure 4 is a graph showing the IR analysis results of Pd(PPh3)4STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0014] Figure 5 is a graph showing the IR analysis results of PdCl2(PPh3)2STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0015] Figure 6 is a graph showing the IR analysis results of the Pd filtrate obtained by analyzing the filtrate of the by-product in the manufacturing process of the present invention.

[0016] Figure 7 is a graph showing the results of P-NMR analysis of Pd(PPh3)4STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0017] Figure 8 is a graph showing the results of P-NMR analysis of PdCl2(PPh3)2STD, which is an analysis of the filtrate of the by-product in the manufacturing process of the present invention.

[0018] Figure 9 is a graph showing the results of P-NMR analysis of the Pd filtrate obtained by analyzing the filtrate of the by-product in the manufacturing process of the present invention.

[0019] Figure 10 is a drawing showing the chemical formula of maleimide-KGDEVD-PABC-doxorubicin of the present invention.

[0020] Figure 11 is a schematic diagram schematically illustrating a process for manufacturing a prodrug using a conventional manufacturing method. The schematic diagram illustrates a reaction scheme for manufacturing a prodrug comprising a maleimide functional group, a peptide spacer (KGDEVD, SEQ ID NO: 2) having an Asp-Glu-Val-Asp sequence (DEVD, SEQ ID NO: 1), and doxorubicin as an active ingredient. Acetic acid (AcOH) is used during the deprotection reaction of the allylic group of the peptide in the doxorubicin and peptide spacer. Doxorubicin and the peptide spacer are conjugated to maleimide-KGDEVD-PABC-doxorubicin via p-aminobenzylcarbamate (PABC).

[0021] Figure 12 is a schematic diagram schematically illustrating a manufacturing process of a prodrug of the present invention. The schematic diagram illustrates a reaction scheme for manufacturing a prodrug comprising a maleimide functional group, a peptide spacer having an Asp-Glu-Val-Asp sequence (DEVD, SEQ ID NO: 1), and doxorubicin as an active ingredient. Hydrochloric acid (HCl) is used during the deprotection reaction of the allylic group of the peptide in the doxorubicin and peptide spacer. Doxorubicin and the peptide spacer are conjugated to maleimide-KGDEVD-PABC-doxorubicin via p-aminobenzylcarbamate (PABC).

[0022] Figure 13 is a schematic diagram showing the reaction formula of tetrakis triphenylphosphine palladium to bis triphenylphosphine palladium dichloride in a dimethylformamide solvent containing hydrochloric acid.

[0023] Definition of terms:

[0024] The term "palladium" (element symbol Pd) used in this document is a white metal that is used in catalysts and special alloys and is highly hydrogen-absorbing. It was isolated in 1802 by Wollaston of England from platinum-containing ore mined in South America, along with rhodium. Palladium, element number 46, is a white metal like ruthenium (Ru) and rhodium (Rh), which were introduced over the past two weeks. It is usually found in platinum ore in the form of a natural alloy. They are precious metals that are produced in small quantities and are used in various fields. In particular, they are very useful elements used as catalysts in catalytic converters, which are automobile exhaust gas purification devices, and in the chemical industry. They are also precious metals that are widely used in jewelry by themselves (including alloys) or as plating. Palladium, along with Pt, Rh, and Ru, is classified as a route-dependent human toxicant, and must be managed at less than 10 ppm in pharmaceuticals.

[0025] The term "prodrug" used in this document refers to a drug that has no pharmacological effect outside the body, but undergoes a metabolic process by metabolic enzymes once inside the body, resulting in the drug's full effect. This process involves altering the drug's chemical structure to allow the substance to exert its effect on the desired target.

[0026] Detailed description of the invention:

[0027] According to one aspect of the present invention, there is provided a method for preparing an allyl group-protected precursor of a prodrug conjugate, wherein a peptide linker cleaved by a protein cleavage enzyme is linked to an anticancer compound;

[0028] A step of preparing a deprotection reaction solution in which the allyl group of the precursor is removed by dissolving the precursor and tetrakistriphenylphosphine palladium (Pd(PPh3)4) in an anhydrous organic solvent;

[0029] A step of adding hydrochloric acid and a catalyst to the above reaction solution, stirring at room temperature, and then filtering to remove bis(triphenylphosphine)palladium dichloride (PdCl2(PPh3)2) produced by the reaction of Pd(PPh3)4 and hydrochloric acid; and

[0030] A method for removing and recovering a high-purity palladium compound in a process for manufacturing a prodrug conjugate is provided, which comprises a precipitation step of adding a mixture containing an organic solvent and a precipitation solvent to the reaction solution to precipitate an amorphous solid.

[0031] In the above method, a step of further purifying the prodrug conjugate by adding the obtained prodrug conjugate to a reaction solution containing triphenylphosphine, hydrochloric acid, and a precipitation solvent may be additionally included, and the prodrug conjugate may be maleimide-KGDEVD-PABC-doxorubicin, and the catalyst may be selected from the group consisting of 4-methylmorpholine, morpholine, 4-ethylmorpholine, pyrrolidine, properidine, N-methylpiperidine, N-methylpyrrolidine, piperazine, and imidazolidene.

[0032] In the above method, the precipitation solvent may be dichloromethane or chloroform, the anhydrous organic solvent may be dimethylformamide, dichloromethane, or chloroform, and the organic solvent may be ethanol, methanol, or acetone.

[0033] The present invention relates to a method for preparing maleimide-KGDEVD-PABC-doxorubicin, which is an anticancer prodrug conjugate activated by caspase. As described above, the method for preparing prodrugs disclosed in existing prior literature removes the allyl group of Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX and adds tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) and acetic acid to cause a reaction for deprotection. In this process, Pd(PPh3)4 is dissolved and a large amount is coprecipitated during the crystallization process, so that the palladium content of the final product exceeds the standard by 4,000 to 5,000 ppm. Afterwards, when EMC-AcKGDEVD-PABC- DOX is manufactured by N-(ε-maleimidocaproxyloxy)succinimide ester (EMCS) coupling reaction, the palladium is not removed at all from the mother liquor. In addition, when high-purity Ac-Lys(EMC)-Gly-Asp- Glu-Val-Asp-PABC-doxorubicin (red amorphous solid) is obtained by separation and purification using Prep HPLC, but even after purification, 100-300 ppm of palladium remains. If the separation and purification process is repeated at least 3 times, it can be removed to below the standard of 6.6 ppm, but if the yield of separation and purification is 35% once and is repeated 3 times, the recovery rate drops to below 5%, making it unsuitable as a method for removing palladium. Existing methods for removing palladium (Pd) compounds include distillation, recrystallization, adsorption using carbon black, or silica gel column chromatography. However, these methods have low removal efficiency and high cost, making them inefficient for mass production, as they cannot remove palladium to a level (less than 10 ppm). (See Tables 1 to 4.)

[0034] Herein, the inventors of the present invention confirmed that the cause of palladium remaining in the manufactured maleimide-KGDEVD-PABC-doxorubicin was that Pd(PPh3)4 was dissolved during the deprotection reaction by removing the allyl group and coprecipitated during the crystallization process, which resulted in an excess of the palladium residue standard. Therefore, through process improvement, a method was developed to not only prevent palladium from remaining in the drug but also effectively remove and recover palladium through a simple process. This was done by replacing the reaction reagent from acetic acid to hydrochloric acid (HCl), so that Pd(PPh3)4 and hydrochloric acid react during the reaction to produce PdCl2(PPh3)2, which has completely different physical properties. The problem of palladium remaining was solved by selectively crystallizing the by-product and removing it through filtration.

[0035] Specifically, various studies have been conducted to identify biomarkers specifically expressed in tumor cells to prevent chemotherapeutic agents from acting on normal tissues and to selectively act only on cancer tissues. However, even if tumor cells expressing the target biomarker account for the majority of all tumor cells in the cancer tissue, the drug does not affect the remaining tumor cells, resulting in cancer recurrence due to the remaining tumor cells even after treatment. Therefore, existing drugs have fundamental limitations in delivering the drug to all tumor cells that have developed, making them not ideal delivery systems for anticancer chemotherapeutic agents and unsuitable for application in effective cancer treatment. Furthermore, when most cancer tissues express the target molecule, anticancer chemotherapeutic agents affect other cells that are close to or in contact with the cancer tissue, causing side effects or toxicity. To address these issues, the present inventors developed a prodrug that enables specific activation within tumor cells and cancer tissues. For example, U.S. Patent No. 7,445,764 discloses a conjugate of a cleavable matrix metalloproteinase (MMP), a cleavable peptide, and the anticancer drug doxorubicin. Furthermore, U.S. Patent Publication No. 2010 / 0111866 discloses a prodrug conjugate having the maleimide-hydrazone-doxorubicin configuration, and U.S. Patent Publication No. 2013 / 0338422 discloses a prodrug conjugate having the peptide sequence-DEVD-doxorubicin configuration. However, these conjugates still suffer from one or more of the following problems: low potency, low selectivity, and / or frequent dosing.

[0036] The present inventors recognized these problems and worked to develop an anticancer chemotherapeutic agent such as a prodrug that selectively acts only on tumor cells, does not affect normal tissues, has minimal side effects, and exhibits excellent efficacy. Accordingly, they developed maleimide-KGDEVD-PABC-doxorubicin, an anticancer prodrug conjugate activated by caspase. The drug is a prodrug-type drug in which a maleimide group capable of binding to a thiol group is derived at the N-terminus of the Asp-Glu-Val-Asp peptide (SEQ ID NO: 1) and an anticancer chemotherapeutic agent, particularly doxorubicin, which is an active ingredient as an anticancer agent, is chemically bonded at the C-terminus. A linker having a maleimide group that binds to a thiol group of endogenous albumin after intravenous administration and an Asp-Glu-Val-Asp peptide sequence (SEQ ID NO: 1) that is enzymatically hydrolyzed by caspase 3 activated in irradiated tumors is bound to doxorubicin, an anticancer chemotherapeutic agent, and the substance is delivered to tumor tissues by endogenous albumin, and then doxorubicin is released by caspase 3 to exhibit a pharmacological effect.

[0037] The above drug is first manufactured by reacting Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-OH, 4-aminobenzyl alcohol, EEDQ under a DMF solvent to obtain Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABOH. Then, it is reacted with 4-nitrophenyl chloroformate, 2,6-lutidine under anhydrous dichloromethane solvent to obtain Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val- Asp(OAll)-PABC. Next, doxorubicin hydrochloride and DIEA are reacted in the presence of DMF solvent to obtain Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX. Next, deprotected Ac-Lys-Gly-Asp- Glu-Val-Asp-PABC-DOX (red amorphous solid) is isolated and obtained by reacting with Pd(PPh3)4 and acetic acid in the presence of DMF solvent. Next, N-(ε-leimidocaproxy)succinimide ester and triethylamine are reacted in the presence of DMF solvent and separated / purified with an ODS-A 5 μm resin column to obtain Ac-Lys(EMC)-Gly-Asp- Glu-Val-Asp-PABC-doxorubicin. After that, the separation / purification process is repeated three or more times to remove palladium.

[0038] In conclusion, the present invention relates to a method for preparing maleimide-KGDEVD-PABC-doxorubicin, wherein Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX deprotection reaction yields Ac-Lys(EMC)-Gly-Asp-Glu-Val-Asp-PABC-doxorubicin (Fig. 10). During the reaction, Pd(PPh3)4 and hydrochloric acid react to produce PdCl2(PPh3)2, and the produced by-product is selectively crystallized in a dimethylformamide solvent and can be easily filtered off. The recovered by-product is a bright yellow, high-purity product that can be reused in other reactions. Next, dichloromethane is injected to crystallize and filtered to obtain the target compound. The product thus obtained has a palladium content of 60 ppm. Afterwards, by injecting a small amount of triphenylphosphine and hydrochloric acid, slurrying without dissolution and filtering, residual palladium is removed to the mother liquor at a level of 11 ppm. As a result, the inventors confirmed that the residual palladium amount was significantly reduced from 4,000-5,000 ppm to less than 20 ppm.

[0039] Another method for removing palladium involves dissolving Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val- Asp(OAll)-PABC-DOX in dimethylformamide, adding Pd(PPh3)4 and hydrochloric acid to cause a deprotection reaction, and then filtering to remove PdCl2(PPh3)2. The resulting mixture is then crystallized using chloroform and filtered to obtain the target compound. Any palladium not removed by filtration is removed into the mother liquor, and the residual palladium content of the target compound thus obtained is less than 13 ppm. When acetic acid is used as a reagent, Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX is dissolved in dimethylformamide, and after the deprotection reaction by adding Pd(PPh3)4 and acetic acid, it is crystallized using chloroform and filtered to obtain the target compound. The residual palladium is removed as a mother liquor, and the residual palladium content of the target compound thus obtained is 100 ppm or less. However, the conventional technology has the disadvantage of being very difficult to process and having a low recovery rate of 5% due to the need for multiple separation and purification processes to remove Pd(PPh3)4, and high manufacturing cost. However, as described above, the method for recovering and removing palladium of the present invention simplifies the separation and purification processes and increases the yield to 35%, so it is possible to produce a low-cost and high-quality prodrug.

[0040] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0041] Reference Example 1: Preparation of maleimide-Lys-Asp-Glu-Val-Asp-PABC-doxorubicin prodrug

[0042] For the preparation of the above prodrug, Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-OH (344 mg, 0.38 mmol), 4-aminobenzyl alcohol (2 eq), and EEDQ (2 eq) were dissolved in anhydrous DMF (11 mL) and stirred at room temperature for 24 hours under argon gas. Thereafter, the solution was concentrated in vacuo and crystallized with ether to obtain Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABOH. After that, Ac-Lys(OAloc)-Gly- Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABOH (322 mg, 0.318 mmol) and 4-nitrophenyl chloroformate (1.2 eq) were dissolved in anhydrous dichloromethane (10 mL), 2,6-lutidine (3 eq) was added, and the mixture was stirred at room temperature. After 2 hours, anhydrous DMF (2 mL) was added to the reaction mixture, and 2,6-lutidine (2 eq) was added. After 24 hours, 27 hours, and 46 hours, 2,6-lutidine (4.75 eq) and 4-nitrophenyl chloroformate (1 eq) were added, respectively, and the mixture was stirred at room temperature. After 84 hours, sodium bicarbonate aqueous solution was added to complete the reaction. The reaction mixture was then extracted three times with ethyl acetate, and the organic layer was washed successively with 0.5 M citric acid, aqueous NaHCO3, and brine. The obtained organic layer was dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure. The concentrate was then purified by semi-preparative HPLC (water / CH3CN, containing 0.1% TFA as an additive, CH3CN 20-100% over 50 min, 10 ml / min) using a C18 reverse-phase column (250 mm X 22 mm) to obtain Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC (77 mg, 20.5%). ESI-MS: m / z 1200.54 [M + Na]+ . After that, the obtained Ac-Lys(OAloc)-Gly- Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC (77 mg, 0.065 mmol) and doxorubicin hydrochloride (1.2 eq) were dissolved in anhydrous DMF (8 mL), DIEA (5.4 eq) was added, and the mixture was stirred at room temperature in a dark environment for 16 hours. Then, the solvent was removed under reduced pressure, and Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (red amorphous solid, 76 mg, 74%) was obtained by separation using preparative HPLC. ESI-MS: m / z 1605.06 [M + Na] +Ac- Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (50 mg, 0.032 mmol) and Pd(PPh3)4 (0.2 eq) were dissolved in anhydrous DMF (7.6 mL), acetic acid (20 eq) and tributyltin anhydride (17.3 eq) were added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the deprotected Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 6 mg, 13.6%) was isolated by preservative HPLC. ESI-MS: m / z 1378.4 [M + H]+. The obtained Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol) and N-(ε-leimidocaproxyloxy)succinimide ester (EMCS; 8.26 mg, 26.8 μmol, 2 eq; Pierce, Rockford, IL) were dissolved in anhydrous DMF (1.5 ml; Sigma-Aldrich, St. Louis, MO), triethylamine (4.64 μl, 2.5 eq; Sigma-Aldrich) was added, and the mixture was stirred at room temperature for 2 hours. The final product was separated by semi-preparative HPLC (Shimadzu, Kyoto, Japan) using an ODS-A 5 μm reversed-phase semi-preparative column (150 mm x 20 mm) in a gradient system (water and CH3CN containing 0.05% TFA as an additive, CH3CN 20-50% over 50 min, 8 ml / min) to obtain Ac-Lys(EMC)-Gly-Asp-Glu-Val-Asp-PABC-doxorubicin (16.7 mg, 73.6%) as a red amorphous solid, and the peak was monitored at 290 nm. The separation of the final product was performed in a gradient system (0.The purity of the final product was confirmed by analytical HPLC (Agilent 1300 series, Agilent Technologies, Santa Clara, CA) using an ODS-A 5 μm analytical column (150 mm x 3 mm; YMC) in water and CH3CN containing 0.5% TFA, CH3CN 20-50% over 50 min, 8 ml / min). Peaks were monitored by UV detector (214 nm) and fluorescence detector (excitation 470 nm, emission 580 nm), and the purity of the final product was confirmed to be greater than 95%. ESI-MS (m / z): 1593.3 [M + Na]. + .

[0043] Example 1: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 1

[0044] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (10.0 g, 6.4 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (200.0 mL), and then hydrochloric acid (27.3 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered to remove bis(triphenylphosphine)palladium dichloride, and the reaction solution was injected into a mixture of MeOH (420.0 mL) and dichloromethane (5,520.0 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys- Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 6.4 g, 73.5%, Pd 60 ppm). The above-obtained Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (6.4 g, 4.0 mmol, Pd 60 ppm) was added to a mixture of PPh3 (0.1 eq) and hydrochloric acid (0.1 eq) in MeOH (420.0 mL) and dichloromethane (5,520.0 mL), stirred for 8 hours, and the remaining Pd was synthesized as PdCl2(PPh3)2, dissolved in MC and MeOH, and filtered to obtain purified Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (6.1 g, 3.9 mmol, Pd 11 ppm).

[0045] Example 2: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 2

[0046] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (10.0 g, 6.4 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (200.0 mL), and acetic acid (27.3 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. After stirring at room temperature for 2 h, the mixture was filtered, and the reaction mixture was poured into a mixture of chloroform (5,520.0 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 6.1 g, 70.0%, Pd 92 ppm).

[0047] Example 3: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 3

[0048] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (10.0 g, 6.4 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (200.0 mL), and acetic acid (27.3 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. After stirring at room temperature for 2 h, the mixture was filtered to remove bis(triphenylphosphine)palladium diacetate, and the reaction mixture was poured into a mixture of dichloromethane (5520.0 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 6.5 g, 74.5%, Pd 975 ppm). The allylic deprotection reaction of the peptide in Examples 2 and 3 and the prodrug manufacturing process using acetic acid (AcOH) are shown in Fig. 11, and the change in the residual palladium amount according to the precipitation solvent used in Examples 2 and 3 is summarized in Table 1 below.

[0049] Changes in palladium residue depending on the precipitation solvent ReagentPrecipitation solventPalladium residue (ppm)Example 2AcOH (Acetic acid)Chloroform 92Example 3Dichloromethane 975

[0050] Example 4: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 4

[0051] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (10.0 g, 6.4 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (200.0 mL), and hydrochloric acid (27.3 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. After stirring at room temperature for 2 h, the mixture was filtered to remove bis(triphenylphosphine)palladium dichloride, and the reaction mixture was poured into a mixture of chloroform (5520.0 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 6.2 g, 71.2%, Pd 13 ppm).

[0052] Example 5: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 5

[0053] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (50 mg, 0.032 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (1.0 mL), and then TFA (15.5 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. The mixture was stirred at room temperature for 2 hours and filtered. The filtered reaction solution was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys-Gly-Asp-Glu-Val- Asp-PABC-DOX (red amorphous solid, 21 mg, 48.2%, Pd 960 ppm).

[0054] Example 6: Preparation of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 6

[0055] Ac-Lys(OAloc)-Gly-Asp(OAll)-Glu(OAll)-Val-Asp(OAll)-PABC-DOX (50 mg, 0.032 mmol) and Pd(PPh3)4 (1.3 eq) were dissolved in anhydrous DMF (1.0 mL), and then H2SO4 (11.0 eq) and 4-methylmorpholine (36.0 eq) were added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered to remove bis(triphenylphosphine)palladium disulfate, and the reaction solution was injected into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining deprotected Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 28 mg, 64.3%, Pd 1,270 ppm). The process for manufacturing a prodrug using hydrochloric acid (HCl) for the deprotection reaction of the allylic group of the peptide in Examples 1 and 4 is shown in FIG. 12, and the reaction scheme for tetrakis triphenylphosphine palladium to bis triphenylphosphine palladium dichloride in a dimethylformamide solvent containing hydrochloric acid is shown in FIG. 13, and the change in the residual palladium amount according to the reagent used in Reference Example, Examples 1, 4 to 5 is summarized in Table 2 below.

[0056] Changes in palladium residue depending on reagent Reagent Precipitation solvent Palladium residue (ppm) Reference example AcOH (Acetic acid) - 3,700 Example 1 HCl (hydrochloric acid) Dichloromethane 60 Example 4 HCl (hydrochloric acid) Chloroform 13 Example 5 TFA (Trifluoroacetic acid) Dichloromethane 960 Example 6 H2SO4 (Sulfuric acid) Dichloromethane 1,270

[0057] Comparative Example 1: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 1

[0058] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and ascorbic acid (1.0 eq) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction mixture was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 17.5 mg, 87.5%).

[0059] Comparative Example 2: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 2

[0060] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and EDTA.2Na (1.0 eq) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction mixture was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 18.2 mg, 91.0%).

[0061] Comparative Example 3: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 3

[0062] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and EDTA.4Na (1.0 eq) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction solution was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 18.0 mg, 90.0%).

[0063] Comparative Example 4: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 4

[0064] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and DTPA (1.0 eq) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction mixture was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 16.9 mg, 84.5%). The changes in the residual palladium amount according to the chelating agent used in Examples 7 to 10 are summarized in Table 3 below.

[0065] Changes in palladium residue according to chelating agent Chelating agent Palladium residue (ppm) Comparative example 1 Ascorbic acid 3,570 Comparative example 2 EDTA.2 Na (dehydrated disodium ethylenediaminetetraacetic acid) 3,558 Comparative example 3 EDTA.4 Na (dehydrated disodium ethylenediaminetetraacetic acid) 3,615 Comparative example 4 DTPA (diethylenetriaminepentaacetic acid) 3,458

[0066] Comparative Example 5: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 5

[0067] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and NUCHAR SA-20 (2.0 mg) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction solution was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 18.0 mg, 90.0%).

[0068] Comparative Example 6: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 6

[0069] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and NUCHAR HD (2.0 mg) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction solution was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 17.7 mg, 88.5%).

[0070] Comparative Example 7: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 7

[0071] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and NUCHAR GC (2.0 mg) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction mixture was injected into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 17.8 mg, 89.0%). The change in the residual palladium according to the activated carbon used in Examples 11 to 13 is summarized in Table 4 below.

[0072] Changes in palladium residue according to activated carbonActivated carbonPalladium residue (ppm)Comparative example 5NUCHAR SA-20(MEADWESTVACO CORPORATION)3,570Comparative example 6NUCHAR HD(MEADWESTVACO CHINA HOLDING CO LTD)3,458Comparative example 7NUCHAR GC(MEADWESTVACO CHINA HOLDING CO LTD)3,431

[0073] Comparative Example 8: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 8

[0074] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and Celite 545 (2.0 mg) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction mixture was poured into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 15.5 mg, 77.5%).

[0075] Comparative Example 9: Purification of Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX 9

[0076] Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (20 mg, 0.013 mmol, Pd 3,700 ppm) was dissolved in anhydrous DMF (1.0 mL), and silica gel (2.0 mg) was added to the reaction mixture. After stirring at room temperature for 2 hours, the mixture was filtered, and the reaction solution was injected into a mixture of MeOH (2.1 mL) and dichloromethane (27.6 mL) to precipitate a solid, thereby obtaining Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-DOX (red amorphous solid, 14.9 mg, 74.5%). The changes in the residual palladium according to Celite 545 and silica gel used in Examples 14 and 15 are summarized in Table 5 below.

[0077] Changes in palladium residue according to Celite 545 and silica gel - Palladium residue (ppm) Comparative Example 8 Celite 545 3,680 Comparative Example 9 Silica gel 3,696

[0078] In conclusion, as shown in the analysis results of Tables 1 and 2 above, residual palladium can be removed during chloroform crystallization. However, since it is classified as a genotoxic substance, the residual chloroform must be managed to 3 ppm or less. In addition, one-time purification is required to manage the residual palladium to 25 ppm or less. In addition, as shown in the analysis results of Tables 2 to 5 above, it was confirmed that palladium was not removed in all experimental results except when hydrochloric acid was used as a reagent. Furthermore, as shown in the analysis results of Table 5 above, it was confirmed that the palladium filtrate precipitated during the deprotection reaction of the allyl group of the peptide in the doxorubicin and peptide spacer was bistriphenylphosphine palladium dichloride. Bistriphenylphosphine palladium dichloride has infrared absorption spectra at 1480, 1436, 1099, 998, 744, 706, 691, 520, and 509 (cm -1 ) (margin of error is ± 2cm) -1 ) showed a characteristic peak. In addition, the precipitated palladium filtrate and bistriphenylphostine palladium dichloride 31 It shows the same peak in P-NMR (162 MHz, CDC13) (Figs. 1 to 9). [δ23.25 (s)]

[0079] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A step of preparing an allylic group-protected precursor of a prodrug conjugate in which a peptide linker cleaved by a protein cleavage enzyme is linked to an anticancer compound; A step of preparing a deprotection reaction solution in which the allyl group of the precursor is removed by dissolving the precursor and tetrakistriphenylphosphine palladium (Pd(PPh3)4) in an anhydrous organic solvent; A step of adding hydrochloric acid and a catalyst to the above reaction solution, stirring at room temperature, and then filtering to remove bis(triphenylphosphine)palladium dichloride (PdCl2(PPh3)2) produced by the reaction of Pd(PPh3)4 and hydrochloric acid; and A method for removing and recovering a high-purity palladium compound in a process for manufacturing a prodrug conjugate, comprising a precipitation step of adding a mixture containing an organic solvent and a precipitation solvent to the reaction solution to precipitate an amorphous solid.

2. In paragraph 1, A method further comprising a step of purifying the prodrug conjugate by adding the obtained prodrug conjugate to a reaction solution containing triphenylphosphine, hydrochloric acid, and a precipitation solvent.

3. In paragraph 1, The above prodrug conjugate is maleimide-KGDEVD-PABC-doxorubicin.

4. In paragraph 1, A method according to claim 1, wherein the catalyst is selected from the group consisting of 4-methylmorpholine, morpholine, 4-ethylmorpholine, pyrrolidine, properidine, N-methylpiperidine, N-methylpyrrolidine, piperazine, and imidazolidene.

5. In paragraph 1, A method wherein the above precipitation solvent is dichloromethane or chloroform.

6. In paragraph 1, A method wherein the anhydrous organic solvent is dimethylformamide, dichloromethane, or chloroform.

7. In paragraph 1, A method wherein the organic solvent is ethanol, methanol or acetone.

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