Antibody drug conjugate intermediate, and preparation method therefor and use thereof

Abstract

An antibody drug conjugate intermediate, and a preparation method therefor and the use thereof. An antibody-drug conjugate prepared by using a provided intermediate compound represented by formula IM2' as an intermediate exhibits a good biological activity and stability. Moreover, compared with a conventional condensation method or a method involving first preparing an active ester followed by condensation, the preparation method of the present invention can avoid the formation of racemic products and achieve a high yield of a target structure, thereby enabling the subsequent preparation of the antibody-drug conjugate.

Description

An antibody-drug conjugate intermediate, its preparation method and application

[0001] This application claims priority to the following earlier application: Patent application No. 202411822992.6, filed with the China National Intellectual Property Administration on December 11, 2024, entitled "An antibody-drug conjugate intermediate and its preparation method and application". The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of organic synthesis technology, specifically relating to an antibody-drug conjugate intermediate, its preparation method, and its application. Background Technology

[0003] Antibody drug conjugates (ADCs) use antibodies as carriers to covalently deliver cytotoxic molecules into tumor cells. The conjugates then dissociate into small molecules that kill tumor cells in the specific tumor environment. Compared with traditional cytotoxic drugs, ADCs have advantages such as strong targeting and fewer side effects, showing good therapeutic potential in clinical practice and are a current strategy for cancer treatment.

[0004] Camptothecin (CPT) analogues and derivatives are small molecules with cytotoxic properties. They exert antitumor effects by inhibiting topoisomerase I (Topo I) and have shown significant activity against many tumor types. Ixatecan, a water-soluble CPT derivative, has strong inhibitory activity against Topo I and has demonstrated excellent antitumor effects against various tumor cells in vitro.

[0005] Patent document WO2022053650A1 discloses SG3932 and its preparation method. The structure of SG3932 is as follows:

[0006] Currently, many ADC drugs use the SG3932 fragment, such as AstraZeneca's B7-H4 targeted ADC AZD8205 (which is in clinical trials) which uses SG3932.

[0007] Structural analysis shows that the connection between the amino group of the toxin moiety and the alanine in the linker chain in SG3932 requires a condensation process. However, the applicant found that conventional condensation methods, such as those using condensation catalysis, could not yield the target structure. Alternatively, preparing the active ester first and then performing condensation also failed to produce the target structure.

[0008] The applicant disclosed the following compound 1 in WO2025167967A1, which has high linkage stability and enhanced solubility, allowing for efficient coupling of hydrophobic drugs and effective intracellular drug delivery. Summary of the Invention

[0009] To solve the above-mentioned technical problems, the present invention provides an intermediate compound represented by the formula IM2'.

[0010] Where R is selected from amino protecting groups; m is an integer from 2 to 16.

[0011] According to an embodiment of the present invention, the amino protecting group is selected from alkoxycarbonyl or acyl protecting groups, such as Alloc (allyloxycarbonyl), Cbz (benzyloxycarbonyl), Fmoc (fluoromethoxycarbonyl), Boc (tert-butyloxycarbonyl) or Teoc (trimethylsilylethoxycarbonyl).

[0012] According to an embodiment of the present invention, m is 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0013] In some embodiments of the present invention, R is selected from Alloc, Cbz, Fmoc, Boc, for example Alloc; m is 6, 7, 8, or 9.

[0014] The present invention also provides a method for preparing the intermediate compound represented by formula IM2' as described above, comprising:

[0015] Method 1. Direct condensation

[0016] Compound IM1' reacts with SM1 in the presence of a phosphite catalyst to give the intermediate compound shown in formula IM2'.

[0017] or,

[0018] Method 2. Friedland quinoline reaction

[0019] Compound IM4' reacts with SM4 via a Friedland quinoline reaction to yield the intermediate compound shown in formula IM2'.

[0020] Where m and R are defined as described above.

[0021] As an exemplary implementation, in method 1, R is selected from Alloc, Cbz, or Fmoc, for example, Alloc.

[0022] According to an embodiment of the present invention, in method 1, the phosphite is selected from at least one of the following compounds: triphenyl phosphite, tri-o-methylphenyl phosphite.

[0023] According to an embodiment of the present invention, in method 1, the molar ratio of the compounds IM1', SM1 to the catalyst phosphite is 1:(1-10):(1-10), for example 1:(1-5):(1-4.5), such as 1:(1.1-3):(1.1-2.5).

[0024] According to an embodiment of the present invention, in method 1, the reaction temperature is 60-200°C, for example 80-150°C; and the reaction time is 1-48h, for example 10-20h.

[0025] As an exemplary implementation, in method 2, R is selected from Alloc, Cbz, or Fmoc.

[0026] According to an embodiment of the present invention, in method 2, the Friedland quinoline reaction is carried out in the presence of catalyst TMSCl, hydrochloric acid, and PPTS (pyridinium p-toluenesulfonate).

[0027] According to an embodiment of the present invention, in method 2, a dehydrating agent, such as a molecular sieve, is also added to the Friedland quinoline reaction.

[0028] According to an embodiment of the present invention, in method 2, the reaction temperature is 40-100°C, for example 50-80°C; and the reaction time is 1-48h, for example 10-20h.

[0029] According to an embodiment of the present invention, in method 2, the molar ratio of compounds IM4', SM4 to catalysts (such as TMSCl, hydrochloric acid, PPTS) is 1:(1-5):(1-10), for example 1:(1-3):(1-8), such as 1:(1.1-1.5):(1.1-5).

[0030] According to an embodiment of the present invention, method 2 further includes a method for preparing compound IM4', comprising the following steps:

[0031] Compound IM1' reacts with compound SM3 to give compound IM4';

[0032] Where m and R are defined as described above.

[0033] According to an embodiment of the invention, the reaction of compound IM1' with compound SM3 is carried out in the presence of a condensing agent, such as EEDQ.

[0034] According to an embodiment of the present invention, the reaction temperature of compound IM1' and compound SM3 is room temperature; the reaction time is 1-12 h.

[0035] According to an embodiment of the present invention, the molar ratio of compound IM1', compound SM3 and condensing agent is 1:(1-5):(1-10), for example 1:(1-3):(1-8).

[0036] According to an embodiment of the present invention, method 2 includes the following steps:

[0037] Compound IM1' reacts with compound SM3 to give compound IM4'; compound IM4' reacts with SM4 via a Friedland quinoline reaction to give the intermediate compound shown in formula IM2'.

[0038] Where m and R are defined as described above.

[0039] The present invention also provides the compound IM4' as described above.

[0040] The present invention also provides a method for preparing the compound IM4' as described above, comprising the following steps:

[0041] Compound IM1' reacts with compound SM3 to give IM4';

[0042] Where m and R are defined as described above.

[0043] According to an embodiment of the invention, the reaction of compound IM1' with compound SM3 is carried out in the presence of a condensing agent, such as EEDQ.

[0044] According to an embodiment of the present invention, the reaction temperature of compound IM1' and compound SM3 is room temperature; the reaction time is 1-12 h.

[0045] According to an embodiment of the present invention, the molar ratio of compound IM1', compound SM3 and condensing agent is 1:(1-5):(1-10), for example 1:(1-3):(1-8).

[0046] The present invention also provides the use of the compound IM4' as described above as an intermediate for preparing the intermediate compound shown in formula IM2'.

[0047] The present invention also provides the compound IM1' as described above:

[0048] Where m and R are defined as described above.

[0049] According to an embodiment of the present invention, in compound IM1', m is 7 and R is allyloxycarbonyl, that is, compound IM1' is the following compound IM1:

[0050] The present invention also provides a method for preparing the compound IM1' as described above, comprising:

[0051] Method 1a. Compound A1' reacts in the presence of DCC and NHS to give compound A2'; compound A2' reacts with compound A3 to give compound IM1';

[0052] Alternatively, method 2a. Compound A1' reacts with compound A4 to give compound A5'; compound A5' undergoes a deprotection reaction to give compound IM1';

[0053] Where m and R are defined as described above.

[0054] This invention also provides the use of compound IM1' or IM1 in the intermediate compound of formula IM2' or compound IM4'.

[0055] This invention also provides the use of the intermediate compound of formula IM2' in the preparation of the intermediate compound of formula IM3' or antibody-drug conjugates.

[0056] Where m is an integer between 2 and 16.

[0057] This invention also provides intermediate compounds represented by formula IM3' as described above:

[0058] Where m is an integer between 2 and 16.

[0059] According to an embodiment of the present invention, m in the intermediate compound represented by formula IM3' is 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0060] In some embodiments of the present invention, the intermediate compound represented by formula IM3' is selected from the following compound IM3:

[0061] The present invention also provides a method for preparing an intermediate compound as shown in formula IM3' above, comprising: subjecting the intermediate compound as shown in formula IM2' above to a deamination protecting group reaction.

[0062] The present invention also provides the use of the intermediate compound represented by formula IM3' or the intermediate compound represented by formula IM2' as described above in the preparation of antibody-drug conjugates.

[0063] According to an embodiment of the present invention, the antibody-drug conjugate is selected from the following compound 1:

[0064] The present invention also provides a method for preparing compound 1 as described above, comprising the following steps:

[0065] b1) The intermediate compound shown in formula IM2' as described above is subjected to a deamination protecting group reaction to prepare the intermediate compound shown in formula IM3; at this time, m in the intermediate compound shown in formula IM2' is 7;

[0066] Compound 1 was prepared by reacting the intermediate compound shown in formula IM3 with compound SM2;

[0067] According to an embodiment of the present invention, the reaction between the intermediate compound represented by formula IM3 and compound SM2 is carried out in the presence of an organic base, for example, in the presence of N,N-diisopropylethylamine.

[0068] According to an embodiment of the present invention, the intermediate compound represented by formula IM3 reacts with compound SM2 at 0°C to 10°C.

[0069] According to an embodiment of the present invention, the preparation method of compound 1 further includes the step of preparing the intermediate compound shown in IM2' using methods 1 and 2 as described above.

[0070] According to an embodiment of the present invention, the preparation method of compound 1 further includes the step of preparing compound IM1' by means of methods 1a and 2a as described above.

[0071] In some more specific embodiments of the present invention, the preparation method of compound 1 includes: first preparing compound IM1' using method 1a and / or 2a, then using compound IM1' to prepare intermediate compound IM2' as shown in method 1 above, then deprotecting it to prepare intermediate compound IM3, and then reacting intermediate compound IM3 with compound SM2 to prepare compound 1; at this time, m is 7 in both compound IM1' and intermediate compound IM2'.

[0072] In some more specific embodiments of the present invention, the preparation method of compound 1 includes: first preparing compound IM1' using method 1a and / or 2a, then using compound IM1' to prepare intermediate compound IM2' as shown in method 2 above, then deprotecting intermediate compound IM2' to prepare intermediate compound IM3, and then reacting intermediate compound IM3 with compound SM2 to prepare compound 1; at this time, m is 7 in both compound IM1' and intermediate compound IM2'. Beneficial effects

[0073] First, the preparation methods 1a and 2a of formula IM1' provided by the present invention can obtain formula IM1' in high yield, and the product obtained by method 1a has higher yield and purity.

[0074] Secondly, the antibody-drug conjugate prepared using the intermediate compound of formula IM2' provided by this invention as an intermediate exhibits good biological activity and stability. (Firstly, the antibody-drug conjugate prepared using the intermediate compound of formula IM2' through subsequent reactions shows significant inhibitory activity against the proliferation of HER2-positive cells SK-BR-3 and NCI-N87; simultaneously, it exhibits weak inhibitory activity against the proliferation of HER2-negative cells MDA-MB-468, demonstrating good selectivity. Secondly, the obtained antibody-drug conjugate shows a low release rate of free toxins in plasma, exhibiting significantly better plasma stability than DS8201. Finally, the obtained antibody-drug conjugate also possesses superior tumor suppressor activity compared to DS8201.) Furthermore, its preparation method, compared to conventional condensation methods or methods that first prepare the active ester and then perform condensation, avoids the generation of racemic products, resulting in a high yield of the target structure.

[0075] The method for preparing the intermediate compound shown in formula IM2' of this invention provides the possibility for the subsequent preparation of antibody-drug conjugates from the intermediate compound shown in formula IM2' or formula IM3'. Attached Figure Description

[0076] Figure 1 is the HPLC spectrum of the product from the first step of Example 1.

[0077] Figure 2 shows the mass spectrum of the reaction product from the first step of Example 1.

[0078] Figure 3 is the HPLC spectrum of the reaction product from the second step of Example 1.

[0079] Figure 4 shows the mass spectrum of the reaction product from the second step of Example 1.

[0080] Figure 5 shows the HPLC spectrum of the product from Example 3.

[0081] Figure 6 shows the mass spectrum of the product from Example 3.

[0082] Figure 7 shows the HPLC chromatogram of reaction product 13 in Table 1.

[0083] Figure 8 is the HPLC spectrum of the product from the first step of the reaction in Example 5.

[0084] Figure 9 shows the mass spectrum of the reaction product from the first step in Example 5.

[0085] Figure 10 is the HPLC spectrum of the product from the second step of the reaction in Example 5.

[0086] Figure 11 shows the mass spectrum of the reaction product in the second step of Example 5.

[0087] Figure 12 shows the HPLC chromatogram of the product from Example 6.

[0088] Figure 13 shows the mass spectrum of the product from Example 6.

[0089] Figure 14 shows the test results of the free toxin release rate of ADC-2 in Test Example 2.

[0090] Figure 15 shows the test results of the free toxin release rate of DS8201 in Test Example 2. Detailed Implementation

[0091] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0092] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0093] In this application, the Su group refers to N-succinimide group, and TCEP refers to tris(2-carbonylethyl) phosphate hydrochloride.

[0094] Synthesis of intermediate IM1:

[0095] Example 1

[0096] Under nitrogen protection, 35 g of compound A1, 11.5 g of NHS, 16.5 g of DCC, and 280 mL of DCM were added to a 500 mL reaction flask. The mixture was stirred at 25 °C for 2–3 h, and the reaction was confirmed to be complete by LC-MS. 200 mL of water was added to the reaction flask to wash the reaction solution. The solution was filtered to remove insoluble matter, and the filtrate was allowed to stand before separation. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 45 °C to dryness, yielding 45.4 g of crude product (95% purity), which was used directly in the next step. LC-MS m / z (ESI): 623.27 (M+1). Its HPLC spectrum is shown in Figure 1, and its mass spectrum is shown in Figure 2.

[0097] Under nitrogen protection, 43 g of compound A2, 15.6 g of compound A3, 26.8 g of DIEA, and 400 mL of DCM were added to a 1 L reaction flask. The mixture was stirred overnight at 25 °C, and the reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated to dryness under reduced pressure at 45 °C to obtain 80 g of crude product, which was then purified to obtain 24.6 g of product (purity 98%). LC-MS m / z (ESI): 696.41 (M+1). Its HPLC spectrum is shown in Figure 3, and its mass spectrum is shown in Figure 4.

[0098] Example 2

[0099] Under nitrogen protection, 2.9 g of compound A12, 11.2 g of compound A4, 34 g of PyBOP, 8.8 g of NMM, and 250 mL of DCM were added to a 500 mL reaction flask. The mixture was stirred overnight at 25 °C, and the reaction was confirmed to be complete by LC-MS. Subsequently, 120 mL of TFA was added to the reaction mixture, and the mixture was stirred at 25 °C for 3-4 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated under reduced pressure at 45 °C to obtain 55.3 g of crude product. After purification, 19.7 g of product (purity 98%) was obtained. LC-MS m / z (ESI): 696.41 (M+1).

[0100] Synthesis of intermediate compound IM2:

[0101] Comparative Example 1: Traditional Condensation Method

[0102] The condensation reaction was carried out using the condensing agents listed in Table 1 below:

[0103] The condensation conditions used and the results are shown in Table 1:

[0104] Table 1

[0105] In the table above, "*" indicates detection by HPLC. "N / A" indicates that trace amounts of the product are insufficient to determine whether alanine in IM2-3 is racemic.1 The HPLC chromatogram is shown in Figure 7.

[0106] The products in the "disordered reaction system" in the table above are trace amounts and have no value for separation.

[0107] Comparative Example 2: Conversion to an active ester followed by condensation:

[0108] Compound IM1-3, NHS, EDCI, and DCM were added to the reaction system, and the mixture was reacted at room temperature for 3 hours to obtain compound IM1-4. Compound IM1-4, SM1, and Et3N were reacted in DMF solution, but no target product was detected.

[0109] Comparative Example 3

[0110] 100 mg of intermediate compound IM1-2 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of triphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. The purity of the crude product was determined by LC-MS to be 40%, and it was further confirmed that 40% of the product was racemic.

[0111] Comparative Example 4

[0112] 100 mg of intermediate compound IM1-3 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of triphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. LC-MS showed that the purity of the crude product was 50%, and it was further confirmed that there was 30% racemic product in the product.

[0113] Example 3

[0114] 100 mg of intermediate compound IM1 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of triphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. The purity of the crude product was 89% as determined by LC-MS, and no racemic product was detected.

[0115] Dissolve the product obtained in the previous step in 5 ml of water and 5 ml of DCM, adjust the pH to 7-8 with sodium bicarbonate, extract with DCM, and then extract the aqueous phase again with 5 ml of DCM twice. Combine the organic phases, wash with 5 ml of water twice, dry, and evaporate the organic phase to dryness to obtain 147 mg of black oily intermediate IM2 for later use. MS m / z (ESI): 541.45 ((M+1) / 2). Its HPLC spectrum is shown in Figure 5, and its mass spectrum is shown in Figure 6.

[0116] Comparative Example 5

[0117] 100 mg of intermediate compound IM1 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of trimethyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. LC-MS showed that the purity of the crude product was 30%, and no racemic product was observed.

[0118] Example 4

[0119] 100 mg of intermediate compound IM1 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of tri-o-methylphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. LC-MS showed that the purity of the crude product was 80%, and no racemic product was observed.

[0120] Comparative Example 6

[0121] 100 mg of intermediate compound IM1 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of tri-o-methoxyphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. LC-MS showed that the purity of the crude product was 10%, and no racemic product was detected.

[0122] Comparative Example 7

[0123] 100 mg of intermediate compound (1 eq) IM1 and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 1.1 eq of tri-o-trifluoromethylphenyl phosphite was added. The mixture was heated to 100 °C and reacted for 16 hours. The purity of the crude product was 6% as determined by LC-MS, and no racemic product was detected.

[0124] Comparative Example 8

[0125] 100 mg of intermediate compound IM1 (1 eq) and 60 mg of SM1 were dissolved in 5 ml of N-methylpyrrolidone (NMP) solution, and 2 eq of Brønsted acid ionic liquid (BAIL) were added. The mixture was heated to 100 °C and reacted for 16 hours. LC-MS showed that the purity of the crude product was 10%, and no racemic product was observed. (This preparation method was carried out in accordance with the method described in the following literature: Lin Zhang, Jian Jiang, Linlin Li, Qian Chen, Ling Zhang, Hao Sun, and Chun Li, A CS Sustainable Chemistry & Engineering 2022 10(26), 8433-8442).

[0126] As can be seen from the yields and purities of the above examples and comparative products, the direct amidation reaction using triphenyl phosphite or tri-o-methylphenyl phosphite catalysts in this invention, compared to the condensation agents used in traditional condensation reactions, can directly yield the target product and avoid product racemization (the chiral carbon directly attached to the methyl group of alanine in the linker chain). Furthermore, in the preparation process of this application, when the amino protecting group is Alloc, the use of triphenyl phosphite or tri-o-methylphenyl phosphite as catalysis results in a high yield of the target chiral structure compared to other phosphites, as the Alloc protecting group does not racemize. In contrast, direct amidation reactions using other amino protecting groups and phosphites produce racemic products, resulting in a low yield of the target structure.

[0127] Example 5

[0128] Intermediate SM3 (600 mg, 3.4 mmol) and intermediate compound IM1 (2.61 g, 3.75 mmol) were dissolved in 24 mL of DCM and 1.2 mL of methanol. EEDQ (842.01 mg, 3.40 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, dissolved in 20 mL of water and 20 mL of EA, and the pH was adjusted to 6-7. The aqueous phase was extracted again with 20 mL × 2 EA solutions. The organic phases were combined and evaporated to dryness to obtain a foamy solid. The solid was slurried at room temperature for 1 hour using 10V MTBE and filtered to obtain intermediate compound IM4, with a yield of 90% (purity 95%). MS m / z (ESI): 877.5 (M+23). Its HPLC chromatogram is shown in Figure 8, and its mass spectrum is shown in Figure 9.

[0129] Intermediate compounds IM4 (1.00 g) and SM4 (462.38 mg) were dissolved in 2 ml NMP and 10 ml toluene. Molecular sieves were added, followed by TMSCl (636.08 mg). The reaction mixture was heated to 50 °C and stirred for 16 h under nitrogen protection. The toluene was evaporated to dryness, dissolved in 20 ml water and 20 ml DCM, and the pH was adjusted to 7-8 with sodium bicarbonate. The mixture was extracted with DCM, and the aqueous phase was extracted again with 20 ml DCM twice. The organic phases were combined, washed with 20 ml water twice, dried, and evaporated to dryness to obtain 1.27 g of black oily intermediate compound IM2 (purity 85%). MS m / z (ESI): 541.45 ((M+1) / 2). Its HPLC spectrum is shown in Figure 10, and its mass spectrum is shown in Figure 11.

[0130] Example 6

[0131] 1.27 g of the black oily intermediate compound IM2 was dissolved in 10 ml of DCM, followed by the addition of 1.83 g of 1,3-dimethylbarbituric acid and 135.73 mg of tetrakis(triphenylphosphine)palladium. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction mixture was evaporated to dryness, and the intermediate compound IM3 was directly prepared with a yield of 95%. MS m / z (ESI): 499.46 ((M+1) / 2). Its HPLC spectrum is shown in Figure 12, and its mass spectrum is shown in Figure 13.

[0132] Example 7

[0133] The preparation method of SM2 is as follows:

[0134] In a 20L reactor, 750g of B1 and 4L of MeOH were added. The reactor temperature was adjusted to -5℃, and 117.8g of NaOMe and 4L of MeOH solution were slowly added dropwise while stirring at -5℃ for 1 hour. The methanol was concentrated, and the temperature was controlled at room temperature. 7.5L of water was added, and the mixture was stirred for 2 hours. After filtration and drying, 660g of B2 was obtained, with a yield of 89.7% and a purity of 95.0% as determined by HPLC.

[0135] In a 20L reactor, 1.27kg B2 and 6.5L THF were added. The reactor temperature was adjusted to 5℃. 600.9g NaSMe was added in batches, and the mixture was heated to 40℃ and stirred for 2 hours. The THF was concentrated while maintaining the temperature at room temperature. 12L of water was added, and the mixture was stirred for 1 hour. After filtration and drying, 10L of acetonitrile was added to dissolve the precipitate, and the solution was concentrated to 3L. 15L of water was slowly added dropwise, and the mixture was slurried and purified for 1 hour. After filtration and drying, 1.18kg B3 was obtained, with a yield of 88.3%. The purity was 98.2% as determined by HPLC, and LC-MS was 234.9 (M+1).

[0136] Add 600g B3, 450.7g methyl 5-hexyneate, 5 eq. triethylamine, 0.04 eq. tris(2-tolyl)phosphine, and 0.02 eq. Pd(dppf)Cl2·DCM to a 20L reactor, along with 6L isopropanol and 1.8L water. Heat to 80℃ and stir overnight. Cool to 25℃, filter through a diatomaceous earth sieve, and rinse the filter cake with 1.2L isopropanol. Transfer the filtered reaction solution to a reactor, add 1.8L of a 2.0 eq. LiOH·H2O aqueous solution dropwise, stir for 0.5h, filter through a diatomaceous earth sieve, and extract three times with 6L ethyl acetate. Collect the aqueous phase separately. Add 6L dichloromethane to the aqueous phase and stir with 4M... The pH was adjusted to 3-4 with HCl, and the mixture was allowed to stand and separated. The aqueous phase was extracted once with 3L of dichloromethane. The organic phases were combined, dried, concentrated, and 6L of methanol was added. After concentration, 6L of water was added, filtered, and dried to obtain 595g of B5, with a yield of 70.8% and a purity of 91.2%. LC-MS m / z (ESI): 267 (M+1).

[0137] In a 20L reactor, 2.0 eq. of anhydrous potassium carbonate was added to 7.5L of water and stirred until dissolved. 900g of B5 was added and stirred until dissolved. 10L of water and 1.4 eq. of Oxone were added to the reactor and stirred until dissolved. The temperature was controlled to ≤40℃, and the prepared B5 aqueous solution was added dropwise, stirring for at least 1 hour after the addition was complete. 10L of DCM was added for extraction once, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated and then slurried with n-heptane. 745g of pale yellow B6 solid was obtained, with a purity of 96.3% and a yield of 81%. LC-MS m / z (ESI): 299 (M+1).

[0138] 1.25 eq. DCC was added to 2.1 L DCM and stirred until dissolved. 7 L DCM, 700 g B6, and 1.7 eq. NHS were added sequentially to a 20 L reactor. The reactor temperature was maintained at 25 °C. The prepared DCC solution in dichloromethane was slowly added dropwise, and the mixture was stirred for at least 2 hours. The mixture was filtered, and the filter cake was washed once with 1.4 L DCM. The external temperature was maintained at ≤30 °C. The mixture was concentrated under reduced pressure until no distillate was obtained, yielding an oily crude product. 7 L acetonitrile was added to the oily crude product, and the mixture was stirred for 10 minutes. The mixture was filtered, and the filtrate was concentrated under reduced pressure until no distillate was obtained. Methanol was added, and the mixture was filtered again to obtain 606 g of white solid SM2, with a purity of 97.7% and a yield of 53%. LC-MS m / z (ESI): 396 (M+1).

[0139] 5 g of compound IM3, 0.5 g of N,N-diisopropylethylamine, and 1.9 g of SM2 were dissolved in 100 mL of dichloromethane, and the reaction was stirred at 0 °C for 2 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain 4.5 g of white solid compound 1, MS m / z (ESI): 639.4 (M / 2+1). 1 ¹H NMR (400MHz, deuterated DMSO) δ 9.75 (s, 1H), 8.80 (s, 1H), 8.27 (d, J = 6.8Hz, 1H), 7.97–7.89 (m, 3H), 7.82 (d, J = 9.2Hz, 1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.43 (s, 2H), 5.26 (s, 2H), 4.57–4.50 (m, 1H), 4.27–4.24 (m, 1H), 4.08 (s, 3H), 3.60 (t, J = 6.8Hz, 1H) 6.8Hz, 2H), 3.50-3.48(m, 30H), 3.42-3.39(m, 5H), 3.22-3.14(m, 4H), 2.99-2.96(m, 2H), 2.47-2.35(m, 2H), 2.26 -2.22 (m, 2H), 2.06-1.95 (m, 3H), 1.93-1.84 (m, 2H), 1.82-1.74 (m, 2H), 1.40 (d, J=76.8Hz, 3H), 0.90-0.84 (m, 9H).

[0140] Preparation of ADC-2

[0141] Trastuzumab (16 mg) was placed in a 50 mL centrifuge tube, 50 mM PBS was added, and the pH was adjusted to 7.97 until the antibody concentration was 5 mg / mL. 0.2 M EDTA was added to bring the final EDTA concentration to 2 mM. TCEP (15 times the molar concentration) was added, and the mixture was incubated at 37°C for 2 h, continuously mixing. Compound 1 (DMA-prepared) solution was added under ice bath conditions at a drug-to-antibody molar ratio of 15:1. DMA was added at 10% of the total reaction volume, and the mixture was shaken and incubated at 22°C for 3 h, followed by incubation at 4°C for 18 h, continuously mixing. The reaction solution was purified using a Zeba desalting column, and the sample was concentrated using Amicon buffer, then the buffer was changed to a 30 mM His / Hac, pH 5.5 buffer system to obtain ADC-2 (12 mg). The average DAR value β of the ADC was calculated using the HIC method, which was 8.

[0142] Biological evaluation

[0143] Test Example 1: ADC Bioactivity Detection

[0144] 1. Test Objective

[0145] The purpose of this experiment was to detect the inhibitory activity of ADC compounds on the in vitro proliferation of HER2-expressing NCI-N87 cells, SK-BR-3 cells, and HER2-negative MDA-MB-468 cells. Cells were treated with different concentrations of the compounds in vitro, and after 5 days of culture, CTG was used for cell proliferation analysis. The Luminescent Cell Viability Assay detects cell proliferation based on IC50. 50 The value was used to evaluate the in vitro activity of the compound.

[0146] 2. Testing Methods

[0147] (1) On the first day, tumor cells were seeded on 96-well plates, with 5000 cells / 100μL of culture medium in each well, and 100μL of DPBS in each empty well at the edge. The plates were incubated overnight at 37°C.

[0148] (2) On the second day, first aspirate 50 μL of the old culture medium per well; add ADC at different concentration gradients, with the initial concentration of ADC being 200 nM, diluted 5 times, resulting in 9 concentrations. The volume of drug added is 50 μL per well.

[0149] (3) On the sixth day, thaw CellTiter-Glo Buffer and CellTiter-Glo Substrate reagent at 4°C. Before use, aspirate 10 ml of Buffer and add it to Substrate, mix well, and equilibrate to room temperature.

[0150] (4) On the seventh day, equilibrate the 96-well plate at room temperature for 30 minutes, and add 100 μL of Cell-Titer-Glo to each well. Shake at room temperature in the dark for 5 minutes, incubate for 10 minutes, transfer 100 μL of the liquid in the well to the white plate, and then use a microplate reader to detect chemiluminescence.

[0151] 3. Data Analysis

[0152] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The inhibitory activity of the ADC compound on the in vitro proliferation of NCI-N87 cells, SK-BR-3 cells, and MDA-MB-468 cells was tested. The results are shown in Table 2 below.

[0153] Table 2

[0154] DS8201 is trastuzumab.

[0155] Conclusion: The antibody-drug conjugate of ADC-2 targeting HER2 has significant inhibitory activity on the proliferation of HER2-positive cells SK-BR-3 and NCI-N87; while it has weak inhibitory activity on the proliferation of HER2-negative cells MDA-MB-468; it has good selectivity.

[0156] Test Example 2: ADC Plasma Stability Experiment

[0157] The mice used in this experiment were CD-1 mice, the rats were SD rats, and the monkeys were cynomolgus monkeys.

[0158] (1) Free toxin release test and results

[0159] DS8201 samples and ADC-2 were added to sterile mouse plasma, sterile rat plasma, sterile human plasma, and sterile monkey plasma at a final concentration of 200 μg / mL, respectively, and incubated in a cell culture incubator at 37°C. The day of incubation was recorded as day 0. Samples were then taken out on days 1, 4, 7, 14, and 21 for the detection of free toxin content.

[0160] The free toxin release rate is shown in Figures 14-15. The results indicate that ADC-2 is quite stable in mouse, rat, human, and monkey plasma, with a maximum free toxin release rate not exceeding 0.2%, which is significantly better than the reference DS8201.

[0161] (2) ADC DAR value test and results

[0162] The DS8201 sample and ADC-2 were added to the above-mentioned human sterile plasma at a final concentration of 200 ug / mL and incubated in a cell culture incubator at 37°C. The day of incubation was recorded as day 0. Subsequently, samples were taken out on day 1, day 4, day 7, day 14 and day 21, and the changes in DAR value were detected.

[0163] The experimental results of ADC DAR value changes are shown in Table 3. The results show that the DAR value change of the conjugate formed by the small molecule linker of ADC-2 in human plasma is significantly smaller than that of DS8201, indicating better plasma stability, which further confirms the stability of the small molecule linker of the present invention.

[0164] Table 3: Plasma stability of ADC (DAR value changes)

[0165] Test Example 3: Efficacy Evaluation of NCI-N87 Tumor-Bearing Mice

[0166] 3.1 Experimental Objective

[0167] Using Balb / c nude mice as test animals, this study investigates whether tumor growth is inhibited, delayed, or cured, and evaluates the efficacy of the ADC in this application.

[0168] 3.2 Experimental Procedure

[0169] 3.2.1 Test Drug

[0170] Vehicle: PBS

[0171] Reference ADC (DS8201): 3 mg / kg

[0172] ADC-2: 3 mg / kg

[0173] 3.2.2 Preparation method: All were prepared by diluting with PBS.

[0174] 3.2.3 Test Methods

[0175] NCI-N87 cells were subcutaneously injected into the right rib area of ​​mice. After 7 days of tumor growth, the animals were randomly divided into three groups (n=6 per group, 2 treatment groups + 1 control group). The drugs were administered via tail vein injection once daily. Tumor volume (diameter) and body weight were measured twice weekly for four weeks. Data were recorded. Statistical analysis was performed using Excel 2023: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for inter-group differences were calculated using TTEST.

[0176] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0177] The antitumor efficacy of the compound was evaluated using TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0178] TGI(%) = [1-(Ti-T0) / (Ci-C0)]×100%, where Ti is the average tumor volume of a certain treatment group on a certain day, T0 is the average tumor volume of this treatment group at the beginning of administration; Ci is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the solvent control group at the beginning of administration.

[0179] 3.3 Experimental Results and Conclusions

[0180] The in vivo tumor inhibition (TGI) effects of the test drugs on the NCI-N87 transplantation model are shown in Table 4. The results indicate that the ADC molecules of this application can significantly reduce tumor volume, exhibiting superior tumor inhibition effects compared to the reference ADC (DS8201).

[0181] Table 4: In vivo tumor suppression effect of ADC on NCI-N87 transplantation model

[0182] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The intermediate compound represented by formula IM2', in, R is selected as a white amino protecting group; m is an integer from 2 to 16; Preferably, the amino protecting group is selected from alkyloxycarbonyl or acyl protecting groups, such as alkyloxycarbonyl. c Cb z 、Fmo c Bo c Or Teo c One of them; Preferably, m The possible values ​​are 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

2. The method for preparing the intermediate compound according to claim 1, wherein, include: Method 1. Direct condensation Compound IMl' reacts with SMl in the presence of a phosphite catalyst to give the intermediate compound shown in formula IM2'. or, Method 2. Friedland quinoline reaction Compound IM4' reacts with SM4 via a Friedland quinoline reaction to yield the intermediate compound shown in formula IM2'. in, m R has the definition of claim 1; Preferably, in method 1, the molar ratio of compounds IMl', SMl to the catalyst phosphite is 1:(1-10):(1-10): Preferably, in method 1, the reaction temperature is 60-200°C; Preferably, in method 1, R is selected as Allo. c ; Preferably, in method 1, the phosphite is selected from at least one of the following compounds: triphenyl phosphite, tri-o-methylphenyl phosphite; Preferably, in method 2, R is selected as Allo. c Cb z or Fmo c ; Preferably, in method 2, the Friedland quinoline reaction is carried out in the presence of a catalyst; Preferably, in method 2, a dehydrating agent, such as a molecular sieve, is also added to the Friedland quinoline reaction; Preferably, in method 2, the reaction temperature is 40–1000°C; Preferably, in method 2, compounds IM4' and SM4 are reacted with a catalyst; Preferably, method 2 further includes a method for preparing compound IM4', comprising the following steps: Compound IMl' reacts with compound SM3 to give IM4'; in, m R has the definition of claim 1; Preferably, method 2 includes the following steps: Compound IM1' reacts with compound SM3 to give IM4'; compound IM4' reacts with SM4 via a Friedland quinoline reaction to give the intermediate compound shown in formula IM2'. in, m R has the definition of claim 1.

3. Compound IM4', R is selected as a white amino protecting group; m is an integer from 2 to 16.

4. The method for preparing the compound IM4' according to claim 3, wherein, Includes the following steps: Compound IMl' reacts with compound SM3 to give IM4'; in, m R has the definition of claim 3; Preferably, the reaction between compound IMl' and compound SM3 is carried out in the presence of a condensing agent, such as EEDQ.

5. Use of the compound IM4' of claim 3 as an intermediate in the preparation of the intermediate compound IM2' of claim 1.

6. Compound IMi': in, m R has the definition of claim 1; Preferably, in compound IMl', m is 7 and R is allyloxycarbonyl, that is, compound IMl' is the following compound IMl:

7. The method for preparing compound IMl' according to claim 6, wherein, include: Method 1 a Compound A1' reacts in the presence of DCC and NHS to give compound A2'; Compound A2' reacts with compound A3 to give compound IMe'; Alternatively, method 2 a Compound A1' reacts with compound A4 to give compound A5'; compound A5' undergoes a deprotection reaction to give compound IMe'; in, m R has the definition of claim 1.

8. The intermediate compound represented by formula IM3', in, m Integers between 2 and 16; Preferably, the intermediate compound represented by formula IM3' is selected from the following compound IM3:

9. Use of the intermediate compound of formula IM2' as claimed in claim 1 in the preparation of the intermediate compound of formula IM3' as claimed in claim 8; or, use of the intermediate compound of formula IM2' as claimed in claim 1 in the preparation of antibody-drug conjugates; Preferably, the antibody-drug conjugate is selected from the following compound 1:

10. A method for preparing compound 1, wherein, Includes the following steps: b1) The intermediate compound represented by formula IM2' as described in claim 1 is subjected to a deamination protecting group reaction to prepare the intermediate compound represented by formula IM3; in this case, m in the intermediate compound represented by formula IM2' is 7; Compound 1 was prepared by reacting the intermediate compound shown in formula IM3 with compound SM2; Preferably, the reaction between the intermediate compound represented by formula IM3 and compound SM2 is carried out in the presence of an organic base; Preferably, the method for preparing compound 1 further includes the step of preparing the intermediate compound represented by formula IM2' using method 1 or method 2 as described in claim 2.

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