Antibody-coupled drug intermediate SET0577 as well as preparation method and application thereof

Abstract

The invention discloses an antibody-coupled drug intermediate SET0577 and a preparation method and application thereof, the chemical structural formula of the antibody-coupled drug intermediate SET0577 is as follows: # imgabs0 # is connected with a structure (namely, the Calichemicin is used as cytotoxin) of Calichemicin through a C-lock connexon, the homogeneity of an ADC (Analog to Digital Converter) drug can be improved, the stability of an antibody is further improved, the enzyme specific splitting decomposition of Linker is improved, and the toxic and side effects of ADC are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemical synthesis and relates to a pharmaceutical intermediate, in particular to an antibody-drug conjugate intermediate SET0577 and a preparation method and application thereof. Background Art

[0002] Antibody drug conjugate (ADC) is a new type of anti-tumor drug. Its principle is to connect cytotoxins to antibodies. Through the recognition of specific antigens on the surface of cancer cells by antibodies, ADCs enter cancer cells through endocytosis, thereby transporting cytotoxins to the target site, achieving the purpose of targeted treatment of malignant tumors. Compared with traditional small molecule anti-tumor drugs, ADCs can take advantage of the targeted recognition of antibodies and the high activity of toxins, so they are more specific and effective.

[0003] ADC consists of three different components, namely antibodies, linkers and cytotoxins. Antibodies achieve targeting; linkers ensure the stability of ADC during blood transport, and after reaching the target site, the toxins play a killing effect on cancer cells. According to different mechanisms of action, toxins suitable for ADC are divided into microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors, etc. At present, the toxins used in ADCs sold on the market and in clinical trials are mainly microtubule inhibitors, mainly including compounds designed based on Dolastatin (such as MMAE, MMAE and MMAD) and compounds designed based on Maytansine (such as DM1 and DM4).

[0004] Traditional ADCs use the amino group of antibody lysine or the thiol group of cysteine ​​obtained by opening the interchain disulfide bond for coupling. The amino group of lysine is connected to the activated carboxylate linker through an amide bond, and the thiol group of cysteine ​​reacts with the maleimide group. An antibody molecule contains 80-90 lysines, and coupling may occur on nearly 40 different lysine residues. Opening the interchain disulfide bond will obtain multiple cysteine ​​residues, while destroying the integrity of the antibody molecule; therefore, traditional ADCs are highly heterogeneous mixtures with poor uniformity (i.e., the drug-to-antibody ratio (DAR) is 1-8) and low stability, which affects the efficacy. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an antibody-drug conjugate intermediate SET0577 in order to overcome the deficiencies of the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is: an antibody-drug conjugate intermediate SET0577, whose chemical structure is:

[0007]

[0008] Another object of the present invention is to provide a method for preparing an antibody-drug conjugate intermediate SET0577, comprising the following steps:

[0009] Under the condition of an ice water bath, adding compound DBBK to a solvent containing compound PDA-VC-PAB-CLM, and then reacting at ≤30°C;

[0010] The chemical structural formula of the compound PDA-VC-PAB-CLM is:

[0011]

[0012] Further, the following steps are included:

[0013] reacting the compound FPDA-VC-PAB-CLM with the compound DIPA in a solvent at a temperature of ≤30° C.;

[0014] The chemical structural formula of the compound FPDA-VC-PAB-CLM is:

[0015]

[0016] Furthermore, the following steps are included:

[0017] Compound FPDA-VC-PAB-PNP, compound LND1093-Int-14 and compound HoBt were added to the solvent and mixed evenly, and then compound DIPEA was added to react under nitrogen protection and ≤30°C;

[0018] The chemical structural formula of the FPDA-VC-PAB-PNP is:

[0019]

[0020] The chemical structural formula of the LND1093-Int-14 is:

[0021]

[0022] Furthermore, after the reaction is completed, a reverse phase column is used for high pressure purification, wherein the mobile phase of the reverse phase column is a mixture of acetonitrile and water containing 0.05% TFA in a volume ratio of 5:95.

[0023] Another object of the present invention is to provide an application of an antibody-drug conjugate intermediate SET0577, characterized in that it is conjugated with an antibody to form an ADC drug.

[0024] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: the antibody-drug conjugate intermediate SET0577 of the present invention is connected to the structure of Calicheamicin through a C-lock linker (i.e., Calicheamicin is used as a cytotoxin), which can improve the uniformity of ADC drugs and thus improve the stability of antibodies, improve the enzyme-specific cleavage of Linker, and reduce the toxic side effects of ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 LC-1 spectrum of the antibody-drug conjugate intermediate SET0577 of the present invention;

[0026] Figure 2 This is the LC-2 spectrum of the antibody-drug conjugate intermediate SET0577 of the present invention;

[0027] Figure 3 This is the MS spectrum of the antibody-drug conjugate intermediate SET0577 of the present invention;

[0028] Figure 4 It is the SEC spectrum of antibody conjugate LN488-41-1;

[0029] Figure 5 It is the HIC spectrum of the antibody conjugate LN488-41-1;

[0030] Figure 6 is the HIC spectrum of the antibody conjugate LN474-64;

[0031] Figure 7 It is the SEC spectrum of antibody conjugate LN474-64;

[0032] Figure 8 The activity curves of LN488-41-1 and BY016 cells;

[0033] Fig. 9 is the CE-SDS spectrum of LN488-41-1;

[0034] Fig.10 This is the CE-SDS spectrum of LN474-64. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below.

[0036] Example 1

[0037] This embodiment provides an antibody-drug conjugate intermediate SET0577 (i.e., compound C-lock-VC-PAB-CLM) and a preparation method thereof, as follows:

[0038] Step (1): Add compound FPDA-VC-PAB-PNP (14 mg, 1.0 eq), compound LND1093-Int-14 (20 mg, 1.0 eq), and HoBt (3.4 mg, 2.0 eq) to a 10 ml single-mouth bottle, add DMF (2 ml) and stir to dissolve; then add DIPEA (1.6 mg, 1.0 eq), N2 protection, and stir at room temperature for 2 h. The structural formula of compound FPDA-VC-PAB-PNP is The structural formula of compound LND is

[0039]

[0040] The reaction of the starting material was basically complete as monitored by LCMS.

[0041] After the reaction was completed, the product was purified by medium pressure using a C18 reverse phase column, and ACN and 0.05% TFA water were used as the mobile phase (i.e., water contained 0.05% (volume concentration) TFA, and ACN and 0.05% TFA water were eluted by gradient elution; the same below) to obtain the target product, which was lyophilized to obtain 20 mg of the compound FPDA-VC-PAB-CLM; the structural formula of the compound FPDA-VC-PAB-CLM is

[0042]

[0043] Step (2): Add compound FPDA-VC-PAB-CLM (20 mg, 1.0 eq) to a 10 ml single-necked bottle, add DMF (1 ml) and stir to dissolve, add compound DIPA (0.5 ml), and react at room temperature for 2 h. LCMS monitoring shows that the raw material has basically reacted completely.

[0044] After the reaction was completed, the product was purified by high pressure preparation using a C18 reverse phase column, and ACN and 0.05% TFA water were used as the mobile phase to obtain the target product, which was lyophilized to obtain 10 mg of the compound PDA-VC-PAB-CLM. The structural formula of the compound PDA-VC-PAB-CLM is

[0045]

[0046] Step (3): Add the compound PDA-VC-PAB-CLM (10 mg, 1.0 eq) to a 2 ml centrifuge tube, add DMF (1 ml) and stir to dissolve, add the compound DBBK (2.9 mg, 2.5 eq) in an ice-water bath, and react at room temperature for 10 min.

[0047] After the reaction, the product was purified by high pressure preparation using a C18 reverse phase column, and ACN and 0.05% TFA water were used as the mobile phase to obtain the target product, which was lyophilized to obtain 4 mg of compound C-lock-VC-PAB-CLM (i.e., antibody-drug conjugate intermediate SET0577). Figures 1 to 3 shown.

[0048] The abbreviations of the above substances are shown in Table 1.

[0049] Table 1 Abbreviations of compounds used

[0050]

[0051]

[0052] The sequence table of antibody BY016 is shown in the following table:

[0053]

[0054] Example 2

[0055] This example provides an application of an antibody-drug conjugate intermediate SET0577, specifically the preparation of an antibody conjugate LN488-41-1, as follows:

[0056] Buffer 1 configuration: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, commercially available 0.5 M EDTA (pH 8.0) aqueous solution 10 mL, adjust pH to 7.2 with phosphoric acid or sodium hydroxide, and dilute to 1000.00 mL with purified water;

[0057] Replace the antibody BY016 into the buffer system Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2);

[0058] Take antibody BY016 (10 μmol), add 10.0 equivalents of TCEP (100 μmol), and stir at room temperature for 2 hours;

[0059] The reduced antibody was replaced with Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2) through an ultrafiltration tube 3 to 5 times (equivalent to a total dilution factor of about 1000 times) to remove excess reducing agent;

[0060] The reduced antibody (control concentration 10±5 mg / mL) was taken out, and then 6.0 equivalents of SET0577 (60 μmol, solvent: acetonitrile / water = 60 / 40, volume ratio, the same below) were added, stirred at room temperature for 30 minutes, and detected by HIC-HPLC;

[0061] Post-treatment: The coupled ADC sample was replaced with Buffer 1 3-5 times through an ultrafiltration tube (equivalent to a total dilution factor of about 1000 times) to remove excess small molecules and organic solvents. The target ADC product LN488-41-1 (such as Figure 4 and Figure 5 ), conduct relevant tests, and store at low temperature.

[0062] Comparative Example 1

[0063] This example provides a preparation of an antibody conjugate LN474-64, as follows:

[0064] Buffer 1 configuration: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, commercially available 0.5 M EDTA (pH 8.0) aqueous solution 10 mL, adjust pH to 7.2 with phosphoric acid or sodium hydroxide, and dilute to 1000.00 mL with purified water;

[0065] The antibody BY016 was replaced into the buffer system Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2).

[0066] Take antibody BY016 (10 umol), add 10.0 equivalents of TCEP (100 umol), and stir at room temperature for 2 hours.

[0067] The reduced antibody was replaced with Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2) through an ultrafiltration tube for 3-5 times (equivalent to a total dilution factor of about 1000 times) to remove excess reducing agent.

[0068] The reduced antibody (control concentration 10±5 mg / mL) was taken out, and then 6.0 equivalents of VcMMAE (60 umol, solvent: acetonitrile / water = 60 / 40) were added, stirred at room temperature for 30 minutes, and detected by HIC-HPLC.

[0069] Post-treatment: The ADC sample after the coupling is replaced with Buffer 1 through an ultrafiltration tube for 3-5 times (equivalent to a total dilution factor of about 1000 times) to remove excess small molecules and organic solvents. The target ADC product LN474-64 (such as Figure 6 and Figure 7 ), conduct relevant tests, and store at low temperature.

[0070] Example 3

[0071] This example provides a Trop2 target cell activity test, which is as follows:

[0072] HEK293T (human myeloma cells) are cells that highly express Trop2. After ADC is incubated with these cells, it can bind to the Trop2 antigen on the cell surface, thereby inducing ADC to be internalized into tumor cells, release toxin molecules, and inhibit the proliferation of tumor cells.

[0073] HEK293T cells were plated at 10,000 cells / well (2×10 5 cells / well, 50 μL) on a 96-well cell culture plate and cultured in a 37° C., 5% CO 2 (volume fraction) incubator for 4-6 hours, during which time a sample dilution plate was prepared.

[0074] The samples LN488-41-1 and antibody BY016 to be tested were diluted in 10 2.5-fold gradients using NCI-H929 growth medium (RPMI 1640 medium + 10% FBS + 0.05mM β-mercaptoethanol) at a starting concentration of 3000ng / mL; the samples LN488-41-1, LN474-64 and antibody BY016 to be tested were diluted in 10 2.5-fold gradients using NCI-H929 growth medium at a starting concentration of 20000ng / mL; the dilutions were added to the cell culture plate at 50μL / well and cultured in a 37°C, 5% CO2 incubator for 96±3 hours.

[0075] After the cell culture was completed, 100 μL of Cell Titer-Glo 2.0 chemiluminescent cell viability detection reagent was added to each well, and the mixture was shaken and mixed for 2 minutes and then allowed to stand for 15 minutes. The chemiluminescence value was read using a SpectraMax M4 microplate reader. The strength of the chemiluminescent signal was proportional to the number of live cells in the culture medium.

[0076] The data were processed using Softmax Pro software, and a four-parameter curve was selected to obtain the relationship between the chemiluminescence value and the concentration. 50 It can be obtained from its concentration response curve (such as Figures 8 to 10 shown).

[0077] SET0577 was coupled with the Trop-2 target antibody (BY016) to obtain the ADC sample LN488-41-1 (TropSET0577). The cell activity was compared with the antibody BY016, and it was found that the antibody had basically no cell killing ability, while the ADC sample showed good tumor cell killing ability. For HEK293T (human myeloma cells that highly express Trop2), LN488-41-1 (Trop SET0577) showed good killing ability, while BY016 (Trop-2 antibody) did not kill, indicating that the antibody significantly improved the anti-tumor effect after coupling with SET0577.

[0078] In the comparative example, we used the random conjugation technology drug linker VcMMAE to conjugate with the Trop2 target antibody (BY016) to obtain the ADC sample LN474-64, and then performed a hydrophobic interaction chromatography (HIC) test. We performed a hydrophobic interaction chromatography (HIC) test on LN488-41-1 and LN474-64. Figure 5 The results showed that the proportions of each peak in LN488-41-1 were DAR=4 (75%) and DAR=5 (25%). Figure 6 The results showed that the peak proportions of LN474-64 were DAR=0 (3%), DAR=2 (15%), DAR=4 (41%), and DAR=6 (39%). The uniformity of the ADC antibody formed by SET0577 coupling was significantly better than that of VcMMAE, showing the advantages of site-specific coupling technology.

[0079] We also performed capillary electrophoresis (CE-SDS) detection on LN488-41-1 and LN474-64. The results showed that the complete antibody (HHLL) of LN488-41-1 accounted for 72%, while the complete antibody (HHLL) of LN474-64 accounted for only 2%, indicating that the integrity of the ADC antibody formed by SET0577 coupling was significantly better than that of VcMMAE, showing the advantages of site-specific coupling technology.

[0080] Comparison of test results showed that the ADC drug SET0577 using the new C-lock Linker was superior to the randomly conjugated VcMMAE in terms of uniformity, safety, cell activity and antibody integrity, demonstrating the technical advantages of SET0577.

[0081] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody-drug conjugate intermediate SET0577, characterized in that: Its chemical structure is:

2. The method for preparing the antibody-drug conjugate intermediate SET0577 according to claim 1, characterized in that: The following steps are involved: Under the condition of an ice water bath, adding compound DBBK to a solvent containing compound PDA-VC-PAB-CLM, and then reacting at ≤30°C; The chemical structural formula of the compound PDA-VC-PAB-CLM is:

3. The method for preparing the antibody-drug conjugate intermediate SET0577 according to claim 2, characterized in that: The following steps are involved: reacting the compound FPDA-VC-PAB-CLM with the compound DIPA in a solvent at a temperature of ≤30° C.; The chemical structural formula of the compound FPDA-VC-PAB-CLM is:

4. The method for preparing the antibody-drug conjugate intermediate SET0577 according to claim 3, characterized in that: The following steps are involved: Compound FPDA-VC-PAB-PNP, compound LND1093-Int-14 and compound HoBt were added to the solvent and mixed evenly, and then compound DIPEA was added to react under nitrogen protection and ≤30°C; The chemical structural formula of the FPDA-VC-PAB-PNP is: The chemical structural formula of the LND1093-Int-14 is:

5. The method for preparing the antibody-drug conjugate intermediate SET0577 according to claim 4, characterized in that: After the reaction is completed, a reverse phase column is used for high pressure purification. The mobile phase of the reverse phase column is a mixture of acetonitrile and water containing 0.05% TFA in a volume ratio of 5:

95.

6. The use of the antibody-drug conjugate intermediate SET0577 according to claim 1, characterized in that: Conjugated with antibodies to form ADC drugs.

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