KR20240159447A - Maleimide-containing linker-payload conjugate - Google Patents

Abstract

The present invention provides a linker-payload conjugate comprising a Maleimide unit used in the manufacture of an antibody-drug conjugate (ADC).

Description

{Maleimide-containing linker-payload conjugate}

The present invention relates to a linker-payload conjugate comprising a maleimide unit.

Antibody-Drug Conjugate (ADC) is a new drug that combines the specificity of antibodies and the cytotoxicity of drugs to increase TI. About 10 types of ADC have been approved by the FDA so far, but the number is not large. Early ADCs used a method of binding to the amine group of lysine exposed on the surface of antibodies or a bond using cysteine obtained by reducing the inter-chain disulfide bond of native antibodies.

In order to exhibit accurate efficacy as a pharmaceutical, it is important that the antibody and drug are always combined at a constant ratio. In addition, the number of drugs combined also affects the in vivo pharmacokinetic properties of the ADC.

Currently, among the FDA-approved ADCs, two drugs are known to target HER2-positive BC: Kadcyla (T-DM1) and Enhertu. Both ADCs use trastuzumab, an anti-HER2-positive BC IgG1, and use maleimide as a linker terminal functional group that binds to the inter-chain disulfide bond of the antibody.

Kadcyla is an early ADC that uses DM1 as a drug and is a heterogeneous ADC with a DAR of approximately 3.5. Enhertu uses deruxtecan as a drug and has a DAR of approximately 7.7, which is relatively homogeneous, but is still heterogeneous.

Previously, ADC synthesized by reducing inter-chain disulfide bonds was known to have an optimal DAR value of 2 to 4. This is because a higher DAR may show weakness in PK properties even though the efficacy is better. However, if the high DAR of the ADC can be maintained while controlling toxicity and stability, the higher and more uniform the DAR, the better the ADC can be evaluated.

Looking at ADC drugs currently in preclinical and clinical trials with payloads of DM1 and DM4, SMCC, SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate), SPDB (N-succinimidyl-4-(2-pyridyldithio)butanoate) etc. are used, and the structure is to connect the SH at the end of the payload with a linker and then connect it to the lysine residue of the antibody.

As mentioned above, the conjugation of lysine residues causes various problems such as non-specific and diverse DAR generation-induced side effects and regulatory issues, so the strategy of linking DM1 to cysteine residues of antibodies was chosen. However, this also had limitations in producing ADCs with uniform DARs. Maintaining a constant DAR has become a key competitive edge in ADC development, and although various methods are currently being attempted, satisfactory results have not been reported.

The present invention relates to a novel linker-payload that can be used to manufacture ADCs. The present invention was completed by confirming that an ADC having a highly uniform DAR (e.g., DAR = 8.0) can be manufactured when the linker-payload comprising a novel maleimide group of the present invention is used.

Republic of Korea Patent No. 10-2442906 Republic of Korea Publication Patent No. 10-2023-0008723 Republic of Korea Publication Patent No. 10-2019-0038579

Chemical Science (2022), 13(10), 2909-2918 Bioconjugate Chem.　2021, 32, 8, 1525-1534 Clin Cancer Res (2004) 10 (20): 7063-7070 J. Med. Chem.　2014, 57, 16, 6949-6964 Mol. Pharmaceutics 12, 3986-3998

The present invention seeks to provide a linker-payload conjugate comprising a maleimide unit.

In one embodiment of the present invention to achieve such a task, a linker-payload conjugate is provided, represented by the structure of the following chemical formula (I):

(Ⅰ).

In another embodiment of the present invention, a linker-payload conjugate is provided, represented by the structure of the following chemical formula (II):

(Ⅱ).

In another embodiment of the present invention, a linker-payload conjugate is provided, represented by the structure of the following chemical formula (III):

(Ⅲ).

The novel maleimide-containing linker-payload conjugate according to the present invention can be used to prepare an antibody-drug conjugate (ADC), and when the linker of the present invention is used, there is an advantage in that an ADC having a highly uniform DAR (e.g., DAR = 8.0) can be provided.

Figure 1 shows a reaction scheme showing a method for producing a maleimide linker according to one embodiment of the present invention (Example 1).
FIG. 2 illustrates a reaction scheme showing a method for producing a maleimide linker-payload conjugate according to one embodiment of the present invention (Example 2).

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with a meaning that can be commonly understood by a person of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries shall not be ideally or excessively interpreted unless explicitly specifically defined.

As used herein, the singular form may include the plural form unless the context clearly indicates otherwise.

When it is said in this specification that a part “includes” a certain component, this does not exclude other components, but rather may include other components, unless otherwise specifically stated.

In addition, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood as being modified by the term “about” in all cases unless otherwise specified.

Additionally, the experimental procedures specified in this specification are identical to the experimental procedures commonly performed in the art unless specifically described.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a linker-payload conjugate represented by the structure of the following chemical formula (I):

(Ⅰ)

In one embodiment of the present invention, the payload can be DM1, and the linker-payload conjugate can be used to prepare an antibody-drug conjugate (ADC).

One aspect of the present invention provides a linker-payload conjugate represented by the structure of the following chemical formula (II):

(II)

In one embodiment of the present invention, the payload can be DM1, and the linker-payload conjugate can be used to prepare an antibody-drug conjugate (ADC).

One aspect of the present invention provides a linker-payload conjugate represented by the structure of the following chemical formula (III):

(Ⅲ).

In one embodiment of the present invention, the payload can be DM1, and the linker-payload conjugate can be used to prepare an antibody-drug conjugate (ADC).

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

<Example 1> Preparation of di-maleimide linker

<1-1> 1,1'-(propane-1,3-diyl)bis(1H-pyrrole-2,5-dione)

A flame-dried 250-mL 3-necked round-bottom flask equipped with nitrogen gas was added maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml). 1,3-Diaminopropane (0.371 mg, 5 mmol) was dissolved in DMF (0.6 ml) and slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 h. Upon mixing, amic acid was formed and white strands were generated, which were redissolved with continuous stirring. Acetic anhydride (1.23 g, 12 mmol) was then added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was purified by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: white solid; 1H NMR (400 MHz, CDCl ₃ ) δ 6.70 (4H, s), 3.53 (4H, t, J = 7.2 Hz); 13C NMR (101 MHz, CDCl ₃ ) δ 170.74, 134.30, 35.46, 27.55.

<1-2> 1,1'-(butane-1,4-diyl)bis(1H-pyrrole-2,5-dione)

A flame-dried 250-mL 3-necked round-bottom flask equipped with nitrogen gas was added maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml). 1,4-Diaminobutane (0.441 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 hour. Upon mixing, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; 1H NMR (400 MHz, CDCl ₃ ) δ 6.8 (4H, s), 3.53 (4H, t, J = 6.0 Hz), 1.58 (4H, m); 13C NMR (100 MHz, CDCl ₃ ) δ 170.90, 134.23, 37.28, 25.90.

<1-3> 1,1'-(pentane-1,5-diyl)bis(1H-pyrrole-2,5-dione)

Maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml) was added to a flame-dried 250 ml 3-necked round bottom flask equipped with nitrogen gas. 1,5-Diaminopentane (0.511 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 hour. When mixed, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; 1H NMR (400 MHz, CDCl ₃ ) δ 6.68 (4H, s), 3.49 (4H, t, J = 7.2 Hz), 1.60 (4H, m), 1.26 (2H, m); 13C NMR (100 MHz, CDCl ₃ ) δ 170.95, 134.19, 37.66, 28.15, 23.99.

<Example 2> Preparation of a conjugate of maleimide linker and DM1

DM1 and the maleimide linker prepared in Example 1 were added in a ratio of 1:1 to 1:2, 1 equivalent of triethylamine was added, dissolved in dichloromethane, and stirred at room temperature. After the reaction was completed, the solvent was removed, and the resultant was purified by column chromatography and analyzed.

<2-1> 1,1'-(propane-1,3-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl ₂ , the linker prepared in Example 1-1 (47.6 mg, 0.203 mmol) and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (600 MHz, CDCl ₃ ) δ 6.81 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 3.5 Hz, 3H), 6.62 (d, J = 3.8 Hz, 1H), 6.42 (dd, J = 15.2, 11.1 Hz, 1H), 6.24 (s, 1H), 5.64 (dd, J = 15.4, 9.4 Hz, 1H), 5.38 (q, J = 6.9 Hz, 1H), 4.79 - 4.73 (m, 1H), 4.27 (t, J = 11.5 Hz, 1H), 3.98 (s, 3H), 3.75 - 3.63 (m, 2H), 3.52 - 3.45 (m, 4H), 3.44 - 3.39 (m, 1H), 3.35 (s, 3H), 3.19 (s, 3H), 3.04 (tdd, J = 18.2, 13.5, 7.7 Hz, 5H), 2.84 (s, 4H), 2.67 - (m, 2H), 2.37 (td, J = 18.5, 3.8 Hz, 1H), 2.17 (d, J = 14.7 Hz, 1H), 1.88 (dq, J = 13.4, 7.1 Hz, 2H), 1.65 (d, J = 12.9 Hz, 4H), 1.55 (d, J = 1) 3.7 Hz, 1H), 1.46 (td, J = 10.0, 5.7 Hz, 1H), 1.32 - 1.18 (m, 7H), 0.79 (s, 3H). ¹³ C NMR (151MHz, Chloroform-d) δ 176.66, 174.46, 170.79, 170.72, 168.87, 156.13, 152.30, 142.31, 141.20, 139.43, 134.32, 133.43, 127.7 8, 125.49, 122.33, 118.91, 113.29, 88.62, 81.00, 78.29, 74.22, 67.29, 60.05, 56.76, 56.71, 52.54, 46.73, 39.89, 39.74, 39.00, 36.60, 36.26, 35.72, 35.66, 35.29, 34.25, 34.05, 32.54, 30.83, 27.22, 26.57, 26.54, 15.63, 14.68, 13.50, 12.26.

<2-2> 1,1'-(butane-1,4-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl ₂ , the linker prepared in Example 1-2 (50.4 mg, 0.203 mmol) and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (400MHz, CDCl ₃ ) δ 6.82 (d, J = 8.2 Hz, 1H), 6.72 - 6.68 (m, 3H), 6.62 (d, J = 4.0 Hz, 1H), 6.42 (dd, J = 15.3, 11.2 Hz, 1H), 6.24 (s, 1H), 5.63 (dd, J = 15.1, 9.1 Hz, 1H), 5.41 - 5.34 (m, 1H), 4.76 (dd, J = 11.9, 2.7 Hz, 1H), 4.28 (t, J = 11.0 Hz, 2H), 3.98 (s, 3H), 3.74 - 3.63 (m , 3H), 3.52 - 3.48 (m, 5H), 3.41 - 3.34 (m, 5H), 3.24 - 3.17 (m, 4H), 3.13 - 2.92 (m, 6H), 2.86 - 2.78 (m, 5H), 2.66 - 2.55 (m, 3H), 2.33 (ddd, J = 18.7, 9.9, 3.8 Hz, 1H), 2.17 (d, J = 14.3 Hz, 1H), 1.67 (s, 2H), 1.63 (s, 3H), 1.56 - 1.43 (m, 7H), 1.33 - 1.18 (m, 8H), 0.79 (s, 3H) . ¹³ C NMR (101 MHz, Chloroform-d) δ 176.91, 176.74, 174.59, 174.56, 170.87, 170.83, 170.80, 170.76, 170.69, 168.86, 168.84, 156.02, 155. 99, 152.37, 142.15, 142.12, 141.14, 141.06, 139.42, 139.38, 134.21, 133.32, 127.75, 125.35, 122.19, 118.72, 113.23, 113.17, 88.54, 80.87, 78.29, 78.23, 74.15, 67.28, 60.01, 56.75, 56.67, 56.66, 52.61, 52.52, 46.66, 39.79, 39.57, 38.88, .40, 38.34, 37.14, 36.22, 35.76, 35.65, 35.59, 34.27, 33.94, 33.42, 32.46, 30.82, 27.36, 27.20, 25.80, 25.78, 24.72, 14.66, 13.46, 13.42, 12.18, 12.16.

<2-3> 1,1'-(pentane-1,5-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl ₂ , the linker prepared in Example 1-3 (53.3 mg, 0.203 mmol) and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.80 (d, J = 8.9 Hz, 1H), 6.70 - 6.65 (m, 3H), 6.61 (d, J = 5.0 Hz, 1H), 6.41 (dd, J = 15.2, 11.4 Hz, 1H), 6.28 (s, 1H), 5.63 (dd, J = 15.3, 8.5 Hz, 1H), 5.37 (q, J = 6.3 Hz, 1H), 4.74 (dd, J = 12.0, 2.6 Hz, 1H), 4.30 - 4.22 (m, 1H), 3.97 (s, 3H), 3.73 - 3.61 (m, 2H), 3.51 - 3.41 (m, 5H), 3.37 - 3.29 (m, 4H), 3.18 (s, 3H), 3.12 - 2.90 (m, 5H), 2.87 - 2.77 (m, 4H), 2.59 (tt, J = 14.6, 11.8, 5.1 Hz, 2H) , 2.31 (ddd, J = 18.0, 14.0, 3.6 Hz, 1H), 2.16 (d, J = 14.2 Hz, 1H), 1.84 (s, 1H), 1.62 (s, 3H), 1.59 - 1.39 (m, 7H), 1.31 - 1.17 (m, 9H), 0.78 (s, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ 176.95, 176.78, 174.62, 170.96, 170.86, 170.81, 170.70, 168.86, 156.04, 152.37, 142.18, 141.14, 141. 07, 139.45, 139.40, 134.19, 133.35, 127.75, 125.36, 122.23, 118.76, 113.23, 88.54, 80.90, 78.31, 74.17, 67.30, 60.02, 56.76, 56.68, 46.68, 39.82, 39.60, 38.91, 38.84, 38.74, 37.50, 36.22, 35.76, 35.66, 35.59, 33.96, 32.48, 30.85, 29.80, 28. 10, 28.07, 27.40, 27.26, 27.06, 23.90, 15.62, 14.67, 13.47, 12.19.

Claims (5)

A linker-payload conjugate represented by the structure of the following chemical formula (Ⅰ):
(Ⅰ).
A linker-payload conjugate represented by the structure of the following chemical formula (II):
(Ⅱ).
A linker-payload conjugate represented by the structure of the following chemical formula (Ⅲ):
(Ⅲ).
A linker-payload conjugate according to any one of claims 1 to 3, wherein the payload is DM1.
A linker-payload conjugate according to any one of claims 1 to 3, wherein the linker-payload conjugate is used for preparing an antibody-drug conjugate (ADC).