Methods for preparing linker-drug conjugates

Abstract

The present invention relates to a method for preparing the linker-drug conjugate of Formula 1, which has a simple, safe and economical manufacturing process.

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0160324, filed November 25, 2022, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

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

[0003] Antibodies are immunological proteins that bind to specific antigens, and many monoclonal antibodies are currently being developed as anticancer drugs or used in cancer treatment. However, while almost all antibodies inhibit the proliferation of cancer cells and prevent cancer progression, their effectiveness in cancer treatment is severely limited. To overcome these limitations, antibody-drug conjugates (ADCs), in which drugs are conjugated to antibodies, are being developed as a new type of antibody therapeutic agent.

[0004] An antibody-drug conjugate is a compound in which a cytotoxic drug or toxin is conjugated to a monoclonal antibody (mAb) that can selectively deliver the drug to the interior of target tumor cells. When administered to a patient, the antibody-drug conjugate binds to the target cell via its antibody moiety and is delivered into the cell, whereupon the cytotoxic drug or toxin separates from the antibody-drug conjugate and exerts its own efficacy.

[0005] To effectively link an antibody and a cytotoxic drug for the preparation of an antibody-drug conjugate, a method using a linker of an appropriate type is commonly used. Representative examples of linkers used in this case include hydrazone, disulfide, peptide linker, etc. For an antibody-drug conjugate to function effectively, all three components of the antibody-drug conjugate, namely, the antibody, linker, and cytotoxic drug, must function at a certain level or above.

[0006] Antibody-drug conjugates in which the cytotoxic drug MMAE (Monomethyl auristatin E) is bound to an antibody are produced through a multi-step process. The usual method involves covalently linking a linker and MMAE in liquid to form a linker-drug conjugate, followed by thiol or amino coupling with the antibody to form the antibody-drug conjugate. Conventional production methods have the drawback of requiring significant care and constraints to handle toxicity by binding the toxic substance MMAE to the linker at an early stage during the formation of the linker-drug conjugate, making them uneconomical.

[0007] Therefore, there is a need for a method for producing a linker-drug conjugate that can solve the above problems. Summary of the Invention [Problem to be solved by the invention]

[0008] As a result of extensive research to solve the above problems, the present inventors have found that using bis(perfluorophenyl)carbonate as a reactant in a method for preparing a linker-drug conjugate of the following Chemical Formula 1 can reduce side reactions and improve reactivity, and that adding monomethylauristatin E (MMAE), a drug with extremely strong cytotoxicity, at the later stage of the preparation process can prevent long-term exposure to the toxicity of monomethylauristatin E and simplify the preparation process, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide a method for preparing a linker-drug conjugate of the following Chemical Formula 1: [Means for solving the problem]

[0010] In order to achieve the above purpose, The present invention relates to a method for producing a compound represented by the following formula 4 by subjecting a compound represented by the following formula 2 to a condensation reaction with a compound represented by the following formula 3: (2) reacting a compound of the following formula 4 with bis(perfluorophenyl)carbonate to obtain a compound of the following formula 5: (3) subjecting a compound of the following formula 5 to a condensation reaction with monomethylauristatin E to obtain a compound of the following formula 6: (4) reacting a compound of the following formula 6 with a base to obtain a compound of the following formula 7: (5) reacting a compound of the following formula 7 with an acid to obtain a compound of the following formula 1:

[0011] [ka]

[0012] [ka]

[0013] [ka]

[0014] [ka]

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] In the above chemical formulas 1 to 7, The p is an integer of 0 to 2, The m's may be the same or different and are integers of 1 to 6, The n is an integer of 1 to 6, The MMAE is a compound represented by the following formula 8:

[0019] [ka] [Effects of the Invention]

[0020] The method for preparing the linker-drug conjugate of Formula 1 of the present invention has a simple preparation process, can reduce the exposure time to toxicity, and is safe and economical, and can also achieve excellent yield. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will now be described in more detail.

[0022] A conventional method for preparing a linker-drug conjugate of Chemical Formula 1 below is shown in Reaction Scheme 1 below.

[0023] [Reaction Scheme 1] JPEG2025538485000009.jpg109162JPEG2025538485000010.jpg199130

[0024] The conventional method for producing the linker-drug conjugate of Chemical Formula 1 has the problem that the cytotoxic substance monomethylauristatin E (MMAE) is added and conjugated at the beginning of the production process, resulting in continuous exposure to toxicity in subsequent processes. Furthermore, in order to prevent this toxicity, numerous precautions and restrictions must be taken during production, making it uneconomical. Furthermore, the production process is complicated due to the need for multiple steps, and the yield of the compound of Chemical Formula 6 is about 15%, while the yield of the final product, the linker-drug conjugate of Chemical Formula 1, is also very low, at about 15%.

[0025] The present invention has been made to provide a method for preparing a linker-drug conjugate of Formula 1 that can solve the above problems. That is, the method for preparing a linker-drug conjugate of Formula 1 of the present invention (a) shortens the time of exposure to toxicity by adding and reacting monomethylauristatin E (MMAE), a cytotoxic substance, at a later stage of the preparation process. Furthermore, (b) when preparing a precursor for introducing monomethylauristatin 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 improved, thereby (c) increasing the yield of the linker-drug conjugate of Formula 1 by about 60%. Furthermore, (d) the preparation process is simple and therefore economical.

[0026] The method for preparing the linker-drug conjugate of Formula 1 of the present invention comprises: (1) a step of condensing a compound of the following formula 2 with a compound of the following formula 3 to obtain a compound of the following formula 4: (2) reacting a compound of the following formula 4 with bis(perfluorophenyl)carbonate to obtain a compound of the following formula 5: (3) condensing a compound of the following formula 5 with monomethylauristatin E (MMAE) to obtain a compound of the following formula 6: (4) reacting a compound of the following formula 6 with a base to obtain a compound of the following formula 7: (5) reacting a compound of the following Chemical Formula 7 with an acid to obtain a compound of the following Chemical Formula 1:

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] [ka]

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] In the above chemical formulas 1 to 7, The p is an integer of 0 to 2, The m's may be the same or different and are integers of 1 to 6, The n is an integer of 1 to 6, The MMAE is a compound represented by the following formula 8:

[0035] [ka]

[0036] The step (1) is a step of obtaining the compound of the formula 4 by subjecting the compound of the formula 2 and the compound of the formula 3 to a condensation reaction.

[0037] The condensation reaction of the step (1) may be carried out in the presence of a condensing agent containing at least one selected from the group consisting of a carboxylic acid activator, a racemization inhibitor, and a base.

[0038] Specifically, the carboxylic acid activator may include at least one selected from the group consisting of carbodiimides, uronium salts, phosphonium salts, and phosphoric anhydrides.

[0039] The carbodiimides may preferably include at least one selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC).

[0040] The uronium salts are preferably 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, The compound may contain at least one selected from the group consisting of 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).

[0041] The phosphonium salts may preferably include benzotriazol-1-yl-oxytripyrrolidinphosphonium hexafluorophosphate (PyBOP).

[0042] The phosphoric anhydrides may preferably include propylphosphonic anhydride (T3P).

[0043] The racemization inhibitor may preferably include one or more selected from the group consisting of hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).

[0044] The base may preferably include at least one selected from the group consisting of triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and N-methylmorpholine (NMM).

[0045] The condensation reaction may be preferably carried out in the presence of at least one selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine, and most preferably 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 equal equivalent amounts.

[0046] The step (1) includes: (1-1) mixing the compound of Formula 2 and the compound of Formula 3, and then cooling the mixture to -10°C to 5°C; (1-2) adding the condensing agent at the temperature; (1-3) stirring the mixture at room temperature.

[0047] In step (1-1), the compound of Formula 2 and the compound of Formula 3 are mixed in an organic solvent, and the organic solvent may include at least one selected from the group consisting of N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF). Preferably, the organic solvent includes dichloromethane.

[0048] The compound of Chemical Formula 2 and the compound of Chemical Formula 3 may be mixed at an equivalent ratio of 1:1.5 to 1:3, preferably at an equivalent ratio of 1:1.8 to 1:2.5.

[0049] The cooling temperature may be from -10°C to 5°C, that is, from -5°C to 5°C.

[0050] Step (1-3) is a step of stirring the mixture prepared in step (1-2) at room temperature, which may be performed under a nitrogen (N2) atmosphere, to prepare the compound of Formula 4. Thereafter, washing and purification may be performed. The purification method is not particularly limited as long as it is a method commonly used in the art, and may be performed by chromatography, for example.

[0051] Step (2) is a step of obtaining the compound of Formula 5 by reacting the compound of Formula 4 with bis(perfluorophenyl)carbonate.

[0052] The bis(perfluorophenyl)carbonate has a higher reactivity toward nucleophiles than bis(4-nitrophenyl)carbonate, and can improve the nucleophilic reaction when monomethylauristatin E (MMAE) is introduced in step (3) described below. Therefore, side reactions such as the attachment of only one monomethylauristatin E can be reduced, and the reactivity can be improved, thereby increasing the yield of the linker-drug conjugate of Formula 1.

[0053] Step (2) may be carried out in the presence of an organic base, and the organic base may include at least one selected from the group consisting of pyridine, triethylamine, and N-methylmorpholine, and preferably includes pyridine. In one embodiment, the bis(perfluorophenyl)carbonate and the organic base may be used in equal amounts.

[0054] Step (2) may be performed by dissolving the compound of Formula 4 in an organic solvent and then adding an organic base and bis(perfluorophenyl)carbonate to the organic solvent. The organic solvent may include at least one selected from the group consisting of N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), and preferably includes dichloromethane.

[0055] Furthermore, step (2) may be carried out at room temperature for 10 to 25 hours. Thereafter, washing may be further carried out, and purification may be carried out as necessary. The purification method is not particularly limited as long as it is a method commonly used in the art, and purification may be carried out, for example, by chromatography.

[0056] The step (3) is a step of obtaining the compound of Formula 6 by subjecting the compound of Formula 5 to a condensation reaction with monomethyl auristatin E (MMAE).

[0057] The monomethyl auristatin E may be a compound represented by the following formula 8:

[0058] [ka]

[0059] Therefore, the compound of Formula 6 can be represented by the structure of Formula 6-1 below.

[0060] [ka] The p, m, and n are defined as in Chemical Formula 6.

[0061] Monomethylauristatin E (MMAE) is a cytotoxic substance with strong toxicity. As mentioned above, in the conventional method, MMAE is added at an early stage in the preparation of the linker-drug conjugate of Formula 1, which results in continuous exposure to toxicity in subsequent processes. However, the present invention solves this problem by adding MMAE at a later stage in the preparation process.

[0062] The monomethyl auristatin E can be added in an amount of 2 to 3 equivalents.

[0063] The condensation reaction in step (3) can be carried out in the presence of a condensing agent and an organic base. The condensing agent can include one or more selected from the group consisting of hydroxyazabenzotriazole (HOAt) and hydroxybenzotriazole (HOBt), and preferably includes hydroxyazabenzotriazole. The organic base can include at least one selected from the group consisting of diisopropylethylamine (DIEA), triethylamine, and pyridine, and preferably includes 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 in an equivalent ratio of 1:5.

[0064] Step (3) may be performed by dissolving the compound of Formula 5 in an organic solvent and then adding a condensing agent, an organic base, and monomethyl auristatin E. The organic solvent may include at least one selected from the group consisting of N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), and preferably includes N,N-dimethylformamide.

[0065] In addition, the step (3) may involve adding the compound of Chemical Formula 5, monomethylauristatin E (MMAE), the organic base, and the condensing agent at a temperature of -10°C to 0°C, and reacting them at a temperature of 0°C to 25°C for 10 to 25 hours.

[0066] Thereafter, the product may be further washed and, if necessary, purified by any method known in the art, and may be purified by, for example, chromatography.

[0067] The yield of the compound of Formula 6 was about 60%, which is significantly higher than the 15% yield of the compound of Formula 6 prepared by the conventional method for preparing a linker-drug conjugate of Formula 1, and can improve the yield by more than four times.

[0068] 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.

[0069] The base may include at least one selected from the group consisting of lithium hydroxide, sodium hydroxide, and calcium hydroxide, and preferably includes lithium hydroxide, which may be lithium hydroxide monohydrate.

[0070] Specifically, step (4) may involve dissolving the compound of Formula 6 in an organic solvent, adding the base dropwise thereto at -50°C to -20°C, and then reacting for 2 to 5 hours at -20°C to 5°C under a nitrogen (N2) atmosphere. The organic solvent may include at least one selected from the group consisting of methanol, N,N-dimethylformamide (DMF), dichloromethane, and tetrahydrofuran (THF), preferably methanol and tetrahydrofuran. The base may be an aqueous base solution. Thereafter, prior to step (5), a step of neutralizing the compound of Formula 7 to a pH of 6 to 8 may be further carried out. The neutralization may be carried out using a weak acid such as acetic acid or trifluoroacetic acid.

[0071] Furthermore, since monomethylauristatin E is a compound of Chemical Formula 8, the compound of Chemical Formula 7 can be represented by the structure of Chemical Formula 7-1 below.

[0072] [ka] The p, m, and n are defined as in Chemical Formula 7.

[0073] Step (5) is a step of reacting the compound of Formula 7 with an acid to obtain the linker-drug conjugate of Formula 1, and the acid may include at least one selected from the group consisting of phosphoric acid, trifluoroacetic acid, sulfuric acid, and acetic acid, and preferably includes phosphoric acid.

[0074] Step (5) may involve dissolving the compound of Formula 7 in an organic solvent, adding an acid dropwise thereto at −10° C. to 10° C., and then reacting for 1 to 5 hours at −10° C. to 10° C. The organic solvent may include at least one selected from the group consisting of N,N-dimethylformamide (DMF), dichloromethane, acetonitrile, and tetrahydrofuran (THF), and preferably includes dichloromethane.

[0075] Further purification can then be performed. The purification method is not particularly limited as long as it is a method commonly used in the art, and can be, for example, prep-HPLC purification. After the purification, the linker-drug conjugate of Formula 1 is finally obtained by lyophilization, with a yield of about 25%.

[0076] In addition, since the monomethylauristatin E is a compound of Chemical Formula 8, the linker-drug conjugate of Chemical Formula 1 can be represented by the structure of Chemical Formula 1-1 below.

[0077] [ka] The p, m, and n are the same as those defined in Chemical Formula 1. In addition, when p is 0, the linker-drug conjugate of Formula 1 may be a linker-drug conjugate of Formula 1-2 below.

[0078] [ka] The m and n are the same as defined in Chemical Formula 1. In addition, when p is 1, the linker-drug conjugate of Formula 1 may be a linker-drug conjugate of Formula 1-3 below.

[0079] [ka] The m and n are the same as defined in Chemical Formula 1. In addition, when p is 2, the linker-drug conjugate of Formula 1 may be a linker-drug conjugate of Formula 1-4 below.

[0080] [ka] The m and n are the same as defined in Chemical Formula 1.

[0081] That is, the number of monomethyl auristatin E bound to the linker-drug conjugate of Formula 1 varies depending on the definition of P, and may range from 1 to 3.

[0082] The method for preparing the linker-drug conjugate of Formula 1 of the present invention can be represented by the following Reaction Scheme 2.

[0083] [Reaction Scheme 2] JPEG2025538485000026.jpg196146JPEG2025538485000027.jpg199142

[0084] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope of the scope and technical idea of ​​the present invention. It is to be understood that such changes and modifications also fall within the scope of the appended claims.

[0085] [Preparation of Linker-Drug Conjugate of Formula 1] [Example 1] [Example 1-1] Preparation of Compound of Formula 4 JPEG2025538485000028.jpg112162

[0086] The compound of Formula 2 (5.31 g, 4.03 mmol), the compound of Formula 3 (3.55 g, 7.33 mmol, 1.8 equivalents), and dichloromethane (CH2Cl2, DCM) (27 mL, 5 V) were added and cooled to 0°C.

[0087] To the reaction mixture was added a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (1.41 g, 7.33 mmol, 1.8 equiv.), hydroxybenzotriazole (HOBt) (0.99 g, 7.33 mmol, 1.8 equiv.), and N-methylmorpholine phthalate (NMM) (0.81 mL, 7.33 mmol, 1.8 equiv.) in dichloromethane (CHCl, DCM) (27 mL, 5 V) at 0 °C.

[0088] The reaction was terminated by stirring the reaction mixture at a temperature of 20 to 25° C. under a nitrogen atmosphere.

[0089] It was then washed successively with 10% aqueous NH4Cl (53 mmol, 10 V), 10% aqueous NaHCO3 (53 mmol, 10 V), and water (53 mL, 10 V), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0090] The concentrate was purified by column chromatography (SiO2, 2% methanol / dichloromethane→10% methanol / dichloromethane) to obtain the compound of Formula 4 (6.6 g, 81%) as a white solid.

[0091] The volume and NMR results of the compound of formula 4 are as follows:

[0092] EI-MS m / z: [M+H] + 1318.6 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)

[0093] [Example 1-2] Preparation of compound of formula 5 JPEG2025538485000029.jpg139163 Pyridine (Py) (29.2 g, 370 mmol, 5.00 equivalents) and bis(perfluorophenyl) carbonate (146 g, 370 mmol, 5.00 equivalents) were added sequentially to a dichloromethane (CHCl, DCM) (3.5 L, 20 V) solution of the compound of Formula 4 (175 g, 73.9 mmol) under a nitrogen atmosphere at a temperature of 20°C to 25°C, and the reaction was terminated by stirring for 12 hours.

[0094] The reaction mixture was washed with 10% aqueous NH4Cl (3.50 L), brine (3.50 L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0095] 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%).

[0096] The volume and NMR results of the compound of formula 5 are as follows:

[0097] EI-MS m / z: [1 / 2M+H] + 1336.2, [1 / 3M+H] + 891.0 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)

[0098] [Example 1-3] Preparation of compound of formula 6 JPEG2025538485000030.jpg166163 Monomethyl auristatin E (MMAE) (101 g, 141 mmol, 2.50 equivalents), diisopropylethylamine (DIEA) (36.4 g, 281 mmol, 5.00 equivalents), and hydroxyazabenzotriazole (HOAt) (7.67 g, 56.3 mmol, 1.00 equivalents) were added sequentially to a dimethylformamide (DMF) (1.6 L) solution of the compound of Formula 5 (160 g, 56.3 mmol) at -10°C.

[0099] The reaction was completed by stirring the reaction mixture at 0° C. for 12 hours under a nitrogen atmosphere.

[0100] The reaction mixture was then diluted with ethyl acetate (3.60 L) and washed with 10% aqueous NH4Cl (6.40 L), 5% NaHCO3 (6.40 L), and brine (6.40 L), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0101] The concentrate was purified by column chromatography (SiO, 100% ethyl acetate → dichloromethane:methanol (10 / 1, v / v), TLC:SiO, dichloromethane:methanol (10 / 1, v / v), Rf=0.5) to obtain the compound of formula 6 (200 g, 90.4%).

[0102] The volume and NMR results of the compound of Chemical Formula 6 are as follows:

[0103] EI-MS m / z: [1 / 2M+H] + 1870.3, [1 / 3M+H] + 1247.1 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)

[0104] [Example 1-4] Preparation of compound of formula 7 JPEG2025538485000031.jpg131162

[0105] The compound of Formula 6 (200 g, 48.4 mmol) was dissolved in a mixed solvent containing methanol (MeOH) (2.85 L, 15 V) and tetrahydrofuran (THF) (2.85 L, 15 V), and an aqueous solution of LiOHHO (20.3 g, 484 mmol, 10.0 equivalents) in water (2.85 L, 15 V) was added dropwise to the mixed solvent at a temperature of -40°C to -30°C under a nitrogen atmosphere for 30 minutes.

[0106] The reaction was terminated by stirring the reaction mixture under a nitrogen atmosphere at a temperature of -10°C to 0°C for 3.5 hours.

[0107] The reaction mixture was neutralized with acetic acid (30.0 mL) at the same temperature, concentrated, and then lyophilized to obtain the crude compound of Formula 7 (190 g) as a white solid.

[0108] The resulting volume of the compound of formula 7 is as follows: EI-MS m / z: [1 / 2M+H] +1730.1, [1 / 3M+H] + 1153.7

[0109] [Examples 1-5] Preparation of Compound 1 JPEG2025538485000032.jpg159162

[0110] To a solution of the unpurified compound of Formula 7 (190 g) in dichloromethane (CH2Cl2, DCM) (3.8 L, 20 V), phosphoric acid (H3PO4) (380 mL, 2 V) was slowly added dropwise at a temperature of 0 to 5°C.

[0111] Then, the reaction mixture was stirred at 0 to 5° C. for 2 hours to complete the reaction.

[0112] The reactant was dissolved in distilled water, purified by prep-HPLC (0.075% trifluoroacetic acid), and lyophilized to obtain the compound of Formula 1 (76 g, yield 45%).

[0113] The volume and NMR results of the compound of Chemical Formula 1 are as follows:

[0114] EI-MS m / z: [1 / 2M+H] + 1623.2, [1 / 3M + H] + 1082.6 1 H-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. (1) performing a condensation reaction between a compound of the following formula 2 and a compound of the following formula 3 to obtain a compound of the following formula 4: (2) reacting a compound of the following formula 4 with bis(perfluorophenyl)carbonate to obtain a compound of the following formula 5: (3) subjecting a compound of the following formula 5 to a condensation reaction with monomethylauristatin E to obtain a compound of the following formula 6: (4) reacting a compound of the following formula 6 with a base to obtain a compound of the following formula 7: (5) A method for preparing a linker-drug conjugate of the following formula 1, comprising: reacting a compound of the following formula 7 with an acid to obtain a compound of the following formula 1: 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Chemistry 1】 In the above Chemical Formulas 1 to 7, wherein p is an integer of 0 to 2; The m's may be the same or different and are integers of 1 to 6, wherein n is an integer of 1 to 6; The MMAE is a compound represented by the following formula 8: 【Transformation 8】

2. 2. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein the condensation reaction of step (1) is carried out in the presence of at least one selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, and N-methylmorpholine.

3. The step (1) includes: (1-1) mixing the compound of Formula 2 and the compound of Formula 3, and then cooling the mixture to -10°C to 5°C; (1-2) adding the condensing agent at the temperature; (1-3) stirring the mixture at room temperature.

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

6. The condensation reaction of the step (3) is carried out by reacting a condensing agent containing one or more selected from the group consisting of hydroxyazabenzotriazoles and hydroxybenzotriazoles with a condensing agent containing one or more selected from the group consisting of hydroxyazabenzotriazoles and hydroxybenzotriazoles.

2. The method for preparing the linker-drug conjugate of claim 1, which is carried out in the presence of an organic base comprising at least one selected from the group consisting of diisopropylethylamine, triethylamine, and pyridine.

7. The step (3) is 2. The method for preparing the linker-drug conjugate of Formula 1 according to claim 1, wherein the compound of Formula 5, monomethyl auristatin E (MMAE), the organic base, and the condensing agent are added at a temperature of 10°C to 0°C, and reacted at a temperature of 0°C to 25°C for 10 to 25 hours.

8. The method for preparing the linker-drug conjugate of Formula 1 according to claim 7, wherein the monomethyl auristatin E (MMAE) is contained in an amount of 2 to 3 equivalents.

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

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

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

12. 2. 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 formula 1-2: [Chemistry 1-2] The m and n are the same as defined in Formula 1.

13. 2. 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 formula 1-3 below. [Chemistry 1-3] The m and n are the same as defined in Formula 1.

14. 2. 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 formula 1-4 below. [Chemistry 1-4] The m and n are the same as defined in Formula 1.

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