Preparation method of linker-drug conjugate

Abstract

The invention relates to a preparation method of the linker-drug conjugate of the chemical formula 1. The linker-drug conjugate has the advantages of being simple in preparation process, safe and economical.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0160324, filed on November 25, 2022, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to a method for preparing a linker-drug conjugate. Background Art

[0003] Antibodies are immune proteins that bind to specific antigens. Currently, multiple monoclonal antibodies are being developed as anti-cancer drugs or used in cancer treatment. However, although almost all antibodies prevent cancer progression by inhibiting the proliferation of cancer cells, they have significant limitations in treating cancer. As an alternative to overcome this limitation, antibody-drug conjugates (ADCs), which conjugate antibodies with drugs, are being developed as a new type of antibody therapeutic agent.

[0004] An antibody-drug conjugate is a complex in which a cytotoxic drug or toxin is conjugated to a monoclonal antibody (mAb) that can selectively deliver it inside targeted tumor cells. When administered to a patient, the antibody-drug conjugate conjugates with the target cells through its antibody part and is delivered into the cells, and then the cytotoxic drug or toxin is separated from the antibody-drug conjugate and exerts its own efficacy.

[0005] In the preparation process of antibody-drug conjugates, a linker in an appropriate form is usually used to achieve effective conjugation of the antibody with the cytotoxic drug. Commonly used linkers at this time include hydrazone, disulfide, polypeptide linkers, etc. To ensure that the antibody-drug conjugate functions effectively, basically, all three components of the antibody-drug conjugate - the antibody, the linker, and the cytotoxic drug - need to exhibit functions above a certain level.

[0006] The antibody-drug conjugate binds the cytotoxic drug MMAE (Monomethyl auristatin E) to the antibody and is prepared through multiple-step processes. Typically, the linker is covalently linked to MMAE in the liquid phase to form a linker-drug conjugate, and then conjugated with the thiol or amino group of the antibody to form an antibody-drug conjugate. In the existing preparation methods, when forming the linker-drug conjugate, since the toxic substance MMAE is conjugated to the linker at an early stage, a great deal of attention needs to be paid to toxicity control and there are many restrictions, resulting in poor economy.

[0007] Therefore, a method for preparing a linker-drug conjugate capable of solving the above problems is needed. Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] To solve the above problems, the inventors of the present invention conducted extensive research. After confirmation, in the method for preparing the linker-drug conjugate of Chemical Formula 1 below, using bis(perfluorophenyl) carbonate as a reactant can reduce side reactions and improve reactivity, and introducing the highly cytotoxic drug monomethyl auristatin E (MMAE) in the later stage of the preparation process can prevent long-term toxic exposure caused by monomethyl auristatin E and simplify the preparation process, thus completing the present invention.

[0010] Therefore, an object of the present invention is to provide a method for preparing a linker-drug conjugate of Chemical Formula 1 below.

[0011] Means for Solving the Problems

[0012] To achieve the above object, the present invention provides a method for preparing a linker-drug conjugate of Chemical Formula 1 below, which comprises: (1) subjecting a compound of Chemical Formula 2 below to a condensation reaction with a compound of Chemical Formula 3 below to obtain a compound of Chemical Formula 4 below;

[0013] (2) reacting the compound of Chemical Formula 4 below with bis(perfluorophenyl) carbonate to obtain a compound of Chemical Formula 5 below;

[0014] (3) subjecting the compound of Chemical Formula 5 below to a condensation reaction with monomethyl auristatin E (MMAE) to obtain a compound of Chemical Formula 6 below;

[0015] (4) reacting the compound of Chemical Formula 6 below with a base to obtain a compound of Chemical Formula 7 below; and

[0016] (5) reacting the compound of Chemical Formula 7 below with an acid to obtain a compound of Chemical Formula 1 below.

[0017]

Chemical Formula 2

[0018]

[0019]

Chemical Formula 3

[0020]

[0021]

Chemical Formula 4

[0022]

[0023]

Chemical Formula 5

[0024]

[0025]

Chemical Formula 6

[0026]

[0027]

Chemical Formula 7

[0028]

[0029]

Chemical Formula 1

[0030]

[0031] In the Chemical Formulas 1 to 7 described above,

[0032] the p is an integer from 0 to 2,

[0033] the m are the same as or different from each other and are integers from 1 to 6,

[0034] the n is an integer from 1 to 6,

[0035] the MMAE is a compound of the following Chemical Formula 8.

[0036]

Chemical Formula 8

[0037]

[0038] Advantages of the Invention

[0039] The method for preparing the linker-drug conjugate of Chemical Formula 1 of the present invention has a simple preparation process, can reduce the toxic exposure time, and thus not only has safety and economic benefits, but also can exhibit excellent yield effects.

[0040] Hereinafter, the present invention will be described in more detail.

[0041] The method for preparing the existing linker-drug conjugate of the following Chemical Formula 1 is as shown in the following Reaction Formula 1.

[0042]

Reaction Formula 1

[0043]

[0044]

[0045] The problem with the existing method for preparing the linker-drug conjugate of Chemical Formula 1 is that, since the cytotoxic agent monomethyl auristatin E (MMAE) is introduced and conjugated at an early stage of the preparation process, subsequent processes are continuously exposed to a toxic environment. Additionally, to prevent this toxicity exposure problem, not only a great deal of attention needs to be paid during the preparation process, but there are also many restrictions, making it uneconomical. Furthermore, since many steps are required, the preparation process is complex, and the yield of the compound of Chemical Formula 6 is approximately 15%, and the yield of the final product, the linker-drug conjugate of Chemical Formula 1, is also only approximately 15%, presenting a problem of very low yield.

[0046] An object of the present invention is to provide a method for preparing the linker-drug conjugate of Chemical Formula 1 that can solve the above problems. That is, the method for preparing the linker-drug conjugate of Chemical Formula 1 of the present invention can (a) shorten the toxicity exposure time by introducing and reacting the cytotoxic agent monomethyl auristatin E (MMAE) at a later stage of the preparation process. Additionally, (b) when preparing the precursor for introducing monomethyl auristatin E (step (3) of the present invention), by using bis(perfluorophenyl) carbonate instead of bis(4-nitrophenyl) carbonate, side reactions can be reduced and reactivity can be increased, so that (c) the yield of the linker-drug conjugate of Chemical Formula 1 can be increased to approximately 60%. Furthermore, (d) the preparation process is simple and economical.

[0047] The method for preparing the linker-drug conjugate of Chemical Formula 1 of the present invention may include:

[0048] (1) Condensing the compound of the following Chemical Formula 2 with the compound of the following Chemical Formula 3 to obtain the compound of the following Chemical Formula 4;

[0049] (2) Reacting the compound of the following Chemical Formula 4 with bis(perfluorophenyl) carbonate to obtain the compound of the following Chemical Formula 5;

[0050] (3) Condensing the compound of the following Chemical Formula 5 with monomethyl auristatin E (MMAE) to obtain the compound of the following Chemical Formula 6;

[0051] (4) Reacting the compound of the following Chemical Formula 6 with a base to obtain the compound of the following Chemical Formula 7; and

[0052] (5) Reacting the compound of the following Chemical Formula 7 with an acid to obtain the compound of the following Chemical Formula 1.

[0053]

Chemical Formula 2

[0054]

[0055]

Chemical Formula 3

[0056]

[0057]

Chemical Formula 4

[0058]

[0059]

Chemical Formula 5

[0060]

[0061]

Chemical Formula 6

[0062]

[0063]

Chemical Formula 7

[0064]

[0065]

Chemical Formula 1

[0066]

[0067] In the above Chemical Formulas 1 to 7,

[0068] p is an integer from 0 to 2,

[0069] m, which may be the same or different from each other, is an integer from 1 to 6,

[0070] n is an integer from 1 to 6,

[0071] The MMAE is a compound of the following Chemical Formula 8.

[0072]

Chemical Formula 8

[0073]

[0074] The step (1) is a step of condensing the compound of Chemical Formula 2 with the compound of Chemical Formula 3 to obtain the compound of Chemical Formula 4.

[0075] The condensation reaction in the step (1) can be carried out in the presence of a condensing agent, and the condensing agent includes one or more selected from carboxylic acid activators, racemization inhibitors, and bases.

[0076] Specifically, the carboxylic acid activator may include at least one selected from carbodiimide, uronium salt, phosphonium salt, and phosphoric anhydride.

[0077] The carbodiimides preferably may include at least one selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC).

[0078] The uronium salts preferably may include at least one selected from benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, N,N,N'N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0079] The phosphonium salts preferably may include benzotriazol-1-yl-oxytripyrrolidinphosphonium hexafluorophosphate (PyBOP).

[0080] The phosphoric anhydrides preferably may include propylphosphonic anhydride (T3P).

[0081] The racemization inhibitor preferably may include at least one selected from hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).

[0082] The base preferably may include at least one selected from triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and N-methylmorpholine (NMM).

[0083] The condensation reaction preferably may be carried out in the presence of one or more selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine, and most preferably may be carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine. In one embodiment, the compound of Formula 3, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine may be added in equimolar amounts.

[0084] Step (1) may include: (1-1) mixing the compound of Formula 2 with the compound of Formula 3, and then cooling to -10°C to 5°C;

[0085] (1-2) adding the condensing agent at the said temperature; and

[0086] (1-3) stirring the mixture at room temperature.

[0087] Step (1-1) is a step of mixing the compound of Formula 2 with the compound of Formula 3 in an organic solvent, and the organic solvent includes at least one selected from N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF). Additionally, preferably it may include dichloromethane.

[0088] The compound of Formula 2 and the compound of Formula 3 may be mixed in an equivalent ratio of 1:1.5 to 1:3, and preferably may be mixed in an equivalent ratio of 1:1.8 to 1:2.5.

[0089] Furthermore, the cooling temperature may be -10°C to 5°C, preferably -5°C to 5°C.

[0090] The step (1-3) is a step of stirring the mixture prepared in the step (1-2) at room temperature, which can be carried out under a nitrogen (N2) atmosphere, whereby the compound of Chemical Formula 4 can be prepared. Subsequently, additional washing and purification can also be carried out. The method of purification is not particularly limited as long as it is a method adopted in the art. For example, purification can be carried out by chromatography.

[0091] The step (2) is a step of reacting the compound of Chemical Formula 4 with bis(perfluorophenyl)carbonate to obtain the compound of Chemical Formula 5.

[0092] The bis(perfluorophenyl)carbonate has more excellent nucleophilicity than bis(4-nitrophenyl)carbonate. Thus, when introducing monomethyl auristatin E (MMAE) in the subsequent step (3), the nucleophilic reaction can be enhanced. Therefore, side reactions such as coupling only one monomethyl auristatin E can be reduced, and the reactivity can be improved, thereby increasing the yield of the linker-drug conjugate of Chemical Formula 1.

[0093] The step (2) can be carried out in the presence of an organic base, and the organic base can include at least one selected from pyridine, triethylamine, and N-methylmorpholine, and preferably can include pyridine. In one embodiment, the bis(perfluorophenyl)carbonate and the organic base can be used in equimolar amounts.

[0094] In addition, the step (2) can be carried out by dissolving the compound of Chemical Formula 4 in an organic solvent, and then adding an organic base and bis(perfluorophenyl)carbonate. The organic solvent can include at least one selected from N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), and preferably can include dichloromethane.

[0095] In addition, the step (2) can be carried out at room temperature for 10 hours to 25 hours. Subsequently, additional washing can be carried out, and purification can be carried out as needed. The purification method is not particularly limited as long as it is a method adopted in the art. For example, purification can be carried out by chromatography.

[0096] The step (3) is a step of carrying out a condensation reaction between the compound of Chemical Formula 5 and monomethyl auristatin E (MMAE) to obtain the compound of Chemical Formula 6.

[0097] The monomethyl auristatin E can be a compound of the following Chemical Formula 8.

[0098]

Chemical Formula 8

[0099]

[0100] Therefore, the compound of Chemical Formula 6 can be represented by the structure of Chemical Formula 6-1 below.

[0101]

Chemical Formula 6-1

[0102]

[0103] The p, m, and n are the same as defined in Chemical Formula 6.

[0104] The monomethyl auristatin E (MMAE) is a cytotoxic substance with strong toxicity. As described above, in the existing methods for preparing the linker-drug conjugate of Chemical Formula 1, there is a problem of continuous exposure to toxicity in the subsequent processes after the initial feeding. However, the present invention can solve the above problem by adding it in the later stage of the preparation process.

[0105] The monomethyl auristatin E can be added in an amount of 2 to 3 equivalents.

[0106] The condensation reaction of step (3) can be carried out in the presence of a condensing agent and an organic base. The condensing agent can include at least one selected from hydroxyazabenzotriazole (HOAt) and hydroxybenzotriazole (HOBt), and preferably can include hydroxyazabenzotriazole. The organic base can include at least one selected from diisopropylethylamine (DIEA), triethylamine, and pyridine, and preferably can include diisopropylethylamine. In one embodiment, the condensing agent and the organic base can be used in an equivalent ratio of 1:1 to 1:5, and most preferably can be used in an equivalent ratio of 1:5.

[0107] In addition, step (3) can be carried out by dissolving the compound of Chemical Formula 5 in an organic solvent, and then adding a condensing agent, an organic base, and monomethyl auristatin E. The organic solvent can include at least one selected from N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), and preferably can include N,N-dimethylformamide.

[0108] In addition, in step (3), the compound of Formula 5, monomethyl auristatin E (MMAE), the organic base, and the condensing agent may be added at a temperature of -10 to 0 °C, and the reaction may be carried out at a temperature of 0 to 25 °C for 10 to 25 hours.

[0109] Subsequently, additional washing may be carried out and purification may be carried out as needed. The purification method is not particularly limited as long as it is a method adopted in the art. For example, purification may be carried out by chromatography.

[0110] The yield of the compound of Formula 6 is about 60%, which is significantly higher than the yield of 15% of the compound of Formula 6 obtained by the aforementioned existing method for preparing the linker-drug conjugate of Formula 1, and the yield can be increased by about 4 times or more.

[0111] Step (4) is a step of reacting the compound of Formula 6 with a base to obtain the compound of Formula 7, and the reaction may be carried out at a low temperature.

[0112] The base may include at least one selected from lithium hydroxide, sodium hydroxide, and calcium hydroxide, preferably may include lithium hydroxide, and the lithium hydroxide may be lithium hydroxide monohydrate.

[0113] Specifically, step (4) may be carried out by dissolving the compound of Formula 6 in an organic solvent, then dropwise adding the base at -50 °C to -20 °C, and reacting for 2 to 5 hours under a nitrogen (N2) atmosphere at -20 °C to 5 °C. The organic solvent may include at least one selected from methanol, N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), preferably may include methanol and tetrahydrofuran. The base may be an aqueous solution of the base. Before carrying out step (5), a step of neutralizing the compound of Formula 7 to a pH value of 6 to 8 may also be carried out, and the neutralization may be carried out using a weak acid such as acetic acid or trifluoroacetic acid.

[0114] In addition, since the monomethyl auristatin E is the compound of Formula 8, the compound of Formula 7 may be represented by the following structure of Formula 7-1.

[0115]

Formula 7-1

[0116]

[0117] p, m, and n are the same as defined in Formula 7.

[0118] Step (5) is a step of reacting the compound of Formula 7 with an acid to obtain the linker-drug conjugate of Formula 1. The acid may include at least one of phosphoric acid, trifluoroacetic acid, sulfuric acid, and acetic acid, and preferably may include phosphoric acid.

[0119] In step (5), the compound of Formula 7 can be dissolved in an organic solvent, and the acid is added dropwise at -10°C to 10°C, and then reacted at -10°C to 10°C for 1 hour to 5 hours. The organic solvent may include at least one selected from N,N-dimethylformamide (DMF), dichloromethane, acetonitrile, and tetrahydrofuran (THF), and preferably may include dichloromethane.

[0120] Subsequently, additional purification can also be carried out. The purification method is not particularly limited as long as it is a method used in the art. For example, preparative high performance liquid chromatography (prep-HPLC) purification can be carried out. After the above purification, lyophilization is carried out to finally obtain the linker-drug conjugate of Formula 1, and the yield can be about 25%.

[0121] In addition, since the monomethyl auristatin E is the compound of Formula 8, the linker-drug conjugate of Formula 1 can be represented by the structure of Formula 1-1 below.

[0122]

Formula 1-1

[0123]

[0124] p, m, and n are the same as defined in Formula 1.

[0125] In addition, when p is 0, the linker-drug conjugate of Formula 1 can be the linker-drug conjugate of Formula 1-2 below.

[0126]

Formula 1-2

[0127]

[0128] m and n are the same as defined in Formula 1.

[0129] In addition, when p is 1, the linker-drug conjugate of Formula 1 can be the linker-drug conjugate of Formula 1-3 below.

[0130]

Formula 1-3

[0131]

[0132] The m and n are the same as defined in Chemical Formula 1.

[0133] In addition, when p is 2, the linker-drug conjugate of Chemical Formula 1 may be the linker-drug conjugate of Chemical Formula 1-4 below.

[0134]

Chemical Formula 1-4

[0135]

[0136] The m and n are the same as defined in Chemical Formula 1.

[0137] That is, according to the definition of p, the number of conjugated monomethyl auristatin E in the linker-drug conjugate of Chemical Formula 1 may vary and may include 1 to 3.

[0138] The preparation method of the linker-drug conjugate of Chemical Formula 1 of the present invention is shown in Reaction Formula 2 below.

[0139]

Reaction Formula 2

[0140]

[0141] Detailed Embodiments

[0142] For easy understanding of the present invention, preferred embodiments are given below. However, the following embodiments are only used to describe the present invention, and those skilled in the art should understand that various changes and modifications can be made within the scope and technical concept of the present invention, and these changes and modifications should clearly also fall within the protection scope of the appended claims.

[0143] <Preparation of Linker-Drug Conjugates of Chemical Formula 1>

[0144] Example 1

[0145] Example 1-1, Preparation of the Compound of Chemical Formula 4

[0146]

[0147] Add the compound of Chemical Formula 2 (5.31 g, 4.03 mmol), the compound of Chemical Formula 3 (3.55 g, 7.33 mmol, 1.82 eq) and dichloromethane (CH2Cl2, DCM) (27 mL, 5V), and cool to 0 °C.

[0148] A solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (1.41 g, 7.33 mmol, 1.82 eq), hydroxybenzotriazole (HOBt) (0.99 g, 7.33 mmol, 1.82 eq), and N-methylmorpholine (NMM) (0.81 mL, 7.33 mmol, 1.82 eq) in dichloromethane (CH2Cl2, DCM) (27 mL, 5V) was added to the reactants at 0 °C.

[0149] The reactants were stirred under a nitrogen atmosphere at a temperature of 20 °C to 25 °C to complete the reaction.

[0150] Subsequently, it was washed successively with 10% aqueous NH4Cl solution (53 mL, 10V), 10% aqueous NaHCO3 solution (53 mL, 10V), and water (53 mL, 10V), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0151] The concentrate was purified by column chromatography (SiO2, 2% methanol / dichloromethane → 10% methanol / dichloromethane) to obtain the compound of Chemical Formula 4 (6.64 g, 81%), which was a white solid.

[0152] The mass spectrometry (Mass) and nuclear magnetic resonance (NMR) results of the compound of Chemical Formula 4 are as follows.

[0153] EI-MS m / z: [M+H]+ 1318.6

[0154] 1H-NMR (400 MHz, CDCl3), δ: 7.77 - 7.76 (d, J = 6.2 Hz, 2H), 7.50 - 7.47 (m, 2H), 7.25 - 7.23 (d, J = 6.4 Hz, 2H), 5.63 - 5.61 (d, J = 6.2 Hz, 2H), 5.52 - 5.49 (t, J = 5.8 Hz, 2H), 5.36 - 5.34 (t, J = 6.4 Hz, 2H), 5.24 - 5.22 (t, J = 6.2 Hz, 2H), 4.60 (s, 4H), 4.56 - 4.53 (t, J = 8 Hz, 2H), 4.45 - 4.35 (m, 2H), 4.33 - 4.30 (m, 1H), 4.05 (s, 4H), 3.73 - 3.71 (m, 2H), 3.70 - 3.64 (m, 56H), 3.40 - 3.30 (m, 2H), 3.21 - 3.19 (m, 2H), 2.39 - 2.31 (m, 4H), 2.11 - 2.05 (m, 20H), 1.96 - 1.85 (m, 2H), 1.85 - 1.76 (m, 1H), 1.69 - 1.60 (m, 1H), 1.54 - 1.38 (m, 31H)

[0155] Example 1-2, Preparation of Compound of Chemical Formula 5

[0156]

[0157] Under a nitrogen atmosphere at a temperature of 20 to 25 °C, pyridine (Py) (29.2 g, 370 mmol, 5.00 eq) and bis(perfluorophenyl)carbonate (146 g, 370 mmol, 5.00 eq) were successively added to a solution of the compound of Formula 4 (175 g, 73.9 mmol) in dichloromethane (CH2Cl2, DCM) (3.5 L, 20V), and then stirred for 12 hours to complete the reaction.

[0158] The reaction mixture was washed with 10% aqueous NH4Cl solution (3.50 L) and brine (3.50 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0159] The concentrate was purified by column chromatography (SiO2, 100% ethyl acetate → dichloromethane:tetrahydrofuran (1 / 1, v / v)) to obtain the compound of Formula 5 (165 g, 78.6%).

[0160] The mass spectrometry (Mass) and nuclear magnetic resonance (NMR) results of the compound of Formula 5 are as follows.

[0161] EI-MS m / z: [1 / 2M+H] + 1336.2, [1 / 3M+H] + 891.0

[0162] 1 H-NMR (400 MHz, CDCl3), δ: 8.41 (br, 1H), 8.12 (br, 2H), 7.54~7.49 (m, 4H), 7.42 (m, 2H) 7.40 (m, 1H), 7.12~7.09 (m, 3H), 7.03 (m, 1H), 5.44~5.33 (m, 7H), 4.48~4.46 (m, 3H), 4.26~4.24 (m, 2H), 4.04 (br, 6H), 3.76~3.56 (m, 57H), 3.45 (m, 2H), 3.25 (m, 1H), 3.15 (m, 1H), 2.36~2.31 (m, 5H), 2.22 (br, 6H), 2.14 (m, 3H), 2.12 (s, 9H), 2.11 (s, 3H), 2.02 (m, 3H), 1.51 (m, 1H), 1.48 (m, 2H), 1.45 (s, 9H), 1.44 (s, 18H), 1.35 (m, 2H)

[0163] Example 1-3, Preparation of Compound of Chemical Formula 6

[0164]

[0165] At -10 °C, monomethyl auristatin E (MMAE) (101 g, 141 mmol, 2.50 eq), diisopropylethylamine (DIEA) (36.4 g, 281 mmol, 5.00 eq), and hydroxyazabenzotriazole (HOAt) (7.67 g, 56.3 mmol, 1.00 eq) were sequentially added to a solution of the compound of Formula 5 (160 g, 56.3 mmol) in dimethylformamide (DMF) (1.6 L).

[0166] The reaction mixture was stirred for 12 hours under a nitrogen atmosphere at 0 °C to complete the reaction.

[0167] Subsequently, the reaction mixture was diluted with ethyl acetate (EtOAc) (3.60 L), washed with 10% aqueous NH4Cl solution (6.40 L), 5% aqueous NaHCO3 solution (6.40 L), and brine (6.40 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0168] The concentrate was purified by column chromatography (SiO2, 100% ethyl acetate → dichloromethane:methanol (10 / 1, v / v), TLC: SiO2, dichloromethane:methanol (10 / 1, v / v), Rf = 0.5) to obtain the compound of formula 6 (200 g, 90.4%).

[0169] The mass spectrometry (Mass) and nuclear magnetic resonance (NMR) results of the compound of formula 6 are as follows.

[0170] EI-MS m / z: [1 / 2M+H] + 1870.3, [1 / 3M+H] + 1247.1

[0171] 1 1H-NMR (400 MHz, MeOD), δ: 7.94 (m, 3H), 7.83 (m, 2H), 7.41 - 7.23 (m, 12H), 5.63 (m, 2H), 5.66 - 5.50 (m, 2H), 5.35 (m, 2H), 5.24 - 5.20 (m, 6H), 4.60 - 4.54 (m, 7H), 4.49 - 4.26 (m, 2H), 4.23 (m, 6H), 4.20 - 4.06 (br, 5H), 3.95 (m, 2H), 3.90 (m, 1H), 3.72 - 3.63 (m, 51H), 3.38 - 3.20 (m, 19H), 2.97 - 2.95 (m, 6H), 2.53 - 1.47 (m, 90H), 1.19 (m, 1H), 1.20 - 1.15 (m, 12H), 0.96 - 0.80 (m, 39H)

[0172] Example 1-4, Preparation of Compound of Chemical Formula 7

[0173]

[0174] Under a nitrogen atmosphere at a temperature of -40°C to -30°C, an aqueous solution in which LiOH·H2O (20.3 g, 484 mmol, 10.0 eq) was dissolved in water (2.85 L, 15V) was added dropwise to a mixed solvent containing methanol (MeOH) (2.85 L, 15V) and tetrahydrofuran (THF) (2.85 L, 15V) in which the compound of formula 6 (200 g, 48.4 mmol) was dissolved, and the dropping time was 30 minutes.

[0175] Under a nitrogen atmosphere at a temperature of -10°C to 0°C, the reaction mixture was stirred for 3.5 hours to complete the reaction.

[0176] At the same temperature, the reactant was neutralized with acetic acid (30.0 mL), concentrated and then lyophilized to obtain 190 g of the unpurified compound of Chemical Formula 7, which is a white solid.

[0177] The mass spectrometry (Mass) and nuclear magnetic resonance (NMR) results of the compound of Chemical Formula 7 are as follows.

[0178] EI-MS m / z: [1 / 2M+H] + 1730.1, [1 / 3M+H] + 1153.7

[0179] Example 1-5, Preparation of Compound of Chemical Formula 1

[0180]

[0181] At a temperature of 0 °C to 5 °C, phosphoric acid (H3PO4) (380 mL, 2V) was slowly added dropwise to a solution of dichloromethane (CH2Cl2, DCM) (3.8 L, 20V) containing the unpurified compound of Chemical Formula 7 (190 g).

[0182] Subsequently, the reactant was stirred at 0 °C to 5 °C for 2 hours to complete the reaction.

[0183] The reactant was dissolved in distilled water and then purified by preparative high performance liquid chromatography (prep-HPLC) (0.075% trifluoroacetic acid), and lyophilized to obtain 76 g (yield 45%) of the compound of Chemical Formula 1.

[0184] The mass spectrometry (Mass) and nuclear magnetic resonance (NMR) results of the compound of Chemical Formula 1 are as follows.

[0185] EI-MS m / z: [1 / 2M+H] + 1623.2, [1 / 3M+H] + 1082.6

[0186] 1H-NMR (400 MHz, MeOD), δ: 7.95 (m, 5H), 7.55 (m, 2H), 7.24 - 7.21 (m, 11H), 5.20 - 5.11 (m, 5H), 4.65 - 4.40 (m, 5H), 4.26 - 4.20 (m, 8H), 4.07 - 4.06 (br, 7H), 3.90 - 3.80 (m, 4H), 3.72 - 3.56 (m, 57H), 3.37 - 3.12 (m, 24H), 2.98 - 2.95 (m, 6H), 2.48 - 2.41 (m, 7H), 2.30 - 1.43 (m, 27H), 1.41 - 1.15 (m, 11H), 0.95 - 0.81 (m, 42H)

Claims

1. A method for preparing a linker-drug conjugate of Formula 1, which comprises: (1) Condensing a compound of Formula 2 with a compound of Formula 3 to obtain a compound of Formula 4; (2) Reacting the compound of Formula 4 with bis(perfluorophenyl) carbonate to obtain a compound of Formula 5; (3) Condensing the compound of Formula 5 with monomethyl auristatin E (MMAE) to obtain a compound of Formula 6; (4) Reacting the compound of Formula 6 with a base to obtain a compound of Formula 7; and (5) Reacting the compound of Formula 7 with an acid to obtain a compound of Formula 1, 【Formula 2】 【Formula 3】 【Formula 4】 【Formula 5】 【Formula 6】 【Formula 7】 【Formula 1】 In Formulas 1 to 7, p is an integer from 0 to 2, m are the same or different and are integers from 1 to 6, n is an integer from 1 to 6, the MMAE is a compound of Formula 8. 【Formula 8】 2. The method for preparing a linker-drug conjugate of Formula 1 according to claim 1, wherein the condensation reaction in step (1) is carried out in the presence of a condensing agent, and the condensing agent includes at least one selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine.

3. The method for preparing a linker-drug conjugate of Formula 1 according to claim 2, wherein step (1) includes: (1-1) Mixing the compound of Formula 2 with the compound of Formula 3, and then cooling to -10°C to 5°C; (1-2) Adding the condensing agent at this temperature; and (1-3) Stirring the mixture at room temperature.

4. The method for preparing a linker-drug conjugate of Formula 1 according to claim 3, wherein the compound of Formula 2 and the compound of Formula 3 are mixed in an equivalent ratio of 1:1.5 to 1:

3.

5. The method for preparing a linker-drug conjugate of Formula 1 according to claim 1, wherein step (2) is carried out in the presence of an organic base, and the organic base includes at least one selected from pyridine, triethylamine, and N-methylmorpholine.

6. The method for preparing a linker-drug conjugate of Formula 1 according to claim 1, wherein the condensation reaction in step (3) is carried out in the presence of a condensing agent and an organic base, the condensing agent includes at least one selected from hydroxyazabenzotriazole and hydroxybenzotriazole, the organic base includes at least one selected from diisopropylethylamine, triethylamine, and pyridine.

7. The method for preparing a linker-drug conjugate of Formula 1 according to claim 1, wherein in step (3), the compound of Formula 5, monomethyl auristatin E, the organic base, and the condensing agent are added at a temperature of 10°C to 0°C, and the reaction is carried out at a temperature of 0°C to 25°C.

8. The method for preparing a linker-drug conjugate of Formula 1 according to claim 7, wherein The content of the monomethyl auristatin E is 2 to 3 equivalents.

9. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, the base in the step (4) includes at least one selected from lithium hydroxide, sodium hydroxide, and calcium hydroxide.

10. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, after the step (4) and before the step (5), it further includes: neutralizing the compound of Formula 7 to a pH value of 6 to 8.

11. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, the acid in the step (5) includes at least one selected from phosphoric acid, trifluoroacetic acid, sulfuric acid, and acetic acid.

12. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, when p is 0, the linker-drug conjugate of Formula 1 is a compound of the following Formula 1-2: 【Formula 1-2】 m and n are the same as defined in Formula 1.

13. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, when p is 1, the linker-drug conjugate of Formula 1 is a compound of the following Formula 1-3: 【Formula 1-3】 m and n are the same as defined in Formula 1.

14. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein, when p is 2, the linker-drug conjugate of Formula 1 is a compound of the following Formula 1-4: 【Formula 1-4】 m and n are the same as defined in Formula 1.

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