An active oxygen-responsive thione-aldehyde linker, and a preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses an active oxygen response thione aldehyde linker as well as a preparation method and application thereof. The thione aldehyde linker structure is a thione aldehyde structure with a p-hydroxyphenyl as a center. The thione aldehyde carbon atom is connected with two -CH2CH2COOH groups through a sulfur atom. The preparation method of the active oxygen response thione aldehyde linker comprises the following steps: dissolving 3-mercapto propionic acid and p-hydroxybenzaldehyde in ethyl acetate, adding trifluoroacetic acid as a catalyst, stirring and reacting at room temperature, and after the reaction is completed, the thione aldehyde linker is obtained through separation and purification. The thione aldehyde linker can respond to the increased ROS level in a tumor microenvironment, is broken under a high ROS condition, and thus realizes the release of a drug molecule connected therewith, which is beneficial to realizing the specific release of the drug at a tumor site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a reactive oxygen species-responsive thioketone aldehyde linker, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) achieve targeted drug delivery to tumor cells by covalently linking highly specific monoclonal antibodies with highly effective small-molecule cytotoxic drugs through linkers. This significantly reduces the systemic toxicity side effects of traditional chemotherapy while improving therapeutic efficacy, and has become an important research direction in the field of cancer treatment.

[0003] In antibody-drug conjugates (ADCs), the linker is a crucial component, and its stability and cleavage directly affect the safety and efficacy of the ADC. An ideal linker should remain stable during blood circulation and efficiently release the carried active drug upon reaching the target site to achieve precision treatment.

[0004] A significant characteristic of the tumor microenvironment (TME) is that its reactive oxygen species (ROS) levels are typically higher than those of normal tissues. Based on this characteristic, developing linkers responsive to ROS holds promise for achieving specific drug release at the tumor site, thereby further improving the targeting and therapeutic efficacy of ADCs.

[0005] Currently, although there have been some research reports on ROS-responsive linkers, there is still a need to develop novel linkers with novel structures, high response efficiency, and simple preparation processes to meet the higher requirements for safety, efficacy, and manufacturability in antibody-drug conjugate development. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a reactive oxygen species-responsive thioketone aldehyde linker, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A reactive oxygen species-responsive thioketone aldehyde linker having the following structure:

[0008] The structure includes a thioketaldehyde structure centered on a p-hydroxyphenyl group, wherein the carbon atoms of the thioketaldehyde are each connected to two -CH2CH2COOH groups via sulfur atoms.

[0009] The above-mentioned method for preparing reactive oxygen species-responsive thioketone aldehyde linkers includes the following steps: 3-Mercaptopropionic acid and p-hydroxybenzaldehyde were dissolved in ethyl acetate, and trifluoroacetic acid was added as a catalyst. The mixture was stirred at room temperature and the reaction was completed. After separation and purification, the thioketone aldehyde linker was obtained.

[0010] Preferably, the reaction process is monitored by thin-layer chromatography.

[0011] Preferably, the specific method for separation and purification is as follows: after the reaction is completed, the reaction solvent is removed to obtain a crude product; the crude product is washed with an organic solvent and further washed with water to remove residual reagents and catalysts; the washed product is dried to obtain the thioketaldehyde linker, wherein the organic solvent is preferably dichloromethane.

[0012] An antibody-drug conjugate comprising an antibody, a small molecule drug, and a reactive oxygen species-responsive thiophene aldehyde linker, as described above, linking the antibody to the small molecule drug.

[0013] Preferably, the antibody is a PD-L1 monoclonal antibody.

[0014] Preferably, the small molecule drug is tranylcypromine (Tran).

[0015] The above-mentioned method for preparing antibody-drug conjugates includes the following steps: Step 1: React the above-mentioned thiophene aldehyde linker with a small molecule drug under the action of a condensing agent to obtain a drug-linker intermediate; Step 2: The drug-linker intermediate is coupled with the antibody to obtain the antibody-drug conjugate.

[0016] Preferably, the reaction temperature in step 1 is 4°C and the reaction time is 2 hours; the reaction time in step 2 is 12 hours.

[0017] Preferably, the condensing agent is EDC and NHS.

[0018] A pharmaceutical composition comprising an antibody-drug conjugate as described above and a pharmaceutically acceptable carrier.

[0019] The above-mentioned antibody-drug conjugates are used in the preparation of drugs for treating cancer.

[0020] The above-mentioned pharmaceutical composition is used in the preparation of a drug for treating cancer.

[0021] The beneficial effects of this invention are as follows: This invention provides a novel reactive oxygen species-responsive thioketone aldehyde linker with a novel structure that can respond to elevated ROS levels in the tumor microenvironment and break under high ROS conditions, thereby releasing the drug molecules linked to it, which is beneficial for achieving specific drug release at the tumor site.

[0022] The preparation method of the thioketone aldehyde linker described in this invention is simple, the reaction conditions are mild, the raw materials used are readily available, the reaction can be completed under acidic catalytic conditions, the process steps are relatively simple, and it has good operability and scale-up potential.

[0023] The antibody-drug conjugate (ADC) constructed using the thiophene aldehyde linker has been experimentally demonstrated to have good structural stability, remaining stable under simulated blood environment conditions, and exhibiting significant responsive drug release characteristics in the presence of ROS, indicating that this linker is suitable for constructing ROS-responsive ADCs.

[0024] The ADC constructed from the thiophene aldehyde linker has a high drug loading capacity. Experimental results show that each antibody molecule can effectively bind more than 10 small molecule drugs, exhibiting a high drug-antibody ratio (DAR value), demonstrating the advantage of this linker in improving the drug loading capacity of the ADC.

[0025] Due to the responsiveness of the thioketone aldehyde linker to ROS in the tumor microenvironment, the constructed ADC can achieve specific drug release under tumor microenvironment conditions, thereby helping to achieve precise drug delivery and reduce side effects. It is suitable for cancer treatment and has good application prospects. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the synthetic route of TK and TK-Tran intermediate in an embodiment of the present invention; Figure 2 The HPLC chromatograms of TK, Tran, and TK-Tran are shown in the embodiments of the present invention. Figure 3 The following are the ultraviolet absorption spectra of TK, Tran, and TK-Tran in the embodiments of the present invention; Figure 4 This is a mass spectrometry analysis diagram of TK-Tran in an embodiment of the present invention; Figure 5 The 1H NMR spectrum of the T-TK-P ADC in this embodiment of the invention (…) 1 H NMR spectrum; Figure 6 The ultraviolet absorption spectra of PDL1, Tran, TK, T-TK-P(50) and T-TK-P(100) in the embodiments of the present invention are shown. Figure 7 This is a drug release curve of T-TK-P ADC in the presence of reactive oxygen species in an embodiment of the present invention; Figure 8 This is a physical diagram of the TK linker of the present invention. Detailed Implementation

[0027] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] This invention provides a reactive oxygen species-responsive thioketone-aldehyde linker, characterized in that the thioketone-aldehyde linker has the following structure:

[0029] The structure includes a thioketaldehyde structure centered on a p-hydroxyphenyl group, wherein the carbon atoms of the thioketaldehyde are each connected to two -CH2CH2COOH groups via sulfur atoms.

[0030] The present invention also provides a method for preparing the above-mentioned reactive oxygen species-responsive thioketone aldehyde linker, comprising the following steps: 3-Mercaptopropionic acid and p-hydroxybenzaldehyde were dissolved in ethyl acetate, and trifluoroacetic acid was added as a catalyst. The mixture was stirred at room temperature and the reaction was completed. After separation and purification, the thioketone aldehyde linker was obtained.

[0031] The present invention also provides an antibody-drug conjugate comprising an antibody, a small molecule drug, and a reactive oxygen species-responsive thiophene aldehyde linker as described in claim 1, connecting the antibody and the small molecule drug, wherein the antibody is a PD-L1 monoclonal antibody and the small molecule drug is transphenylcyclopropylamine.

[0032] The present invention also provides a method for preparing the above-mentioned antibody-drug conjugate, comprising the following steps: Step 1: React the thioketal linker as described in claim 1 with a small molecule drug under the action of a condensing agent to obtain a drug-linker intermediate; Step 2: The drug-linker intermediate is coupled with the antibody to obtain the antibody-drug conjugate.

[0033] The condensing agents are EDC and NHS. The reaction temperature in step 1 is 4°C and the reaction time is 2 hours. The reaction time in step 2 is 12 hours.

[0034] The present invention further provides a pharmaceutical composition comprising the above-described antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0035] The present invention further provides the application of the above-mentioned antibody-drug conjugate in the preparation of drugs for treating cancer.

[0036] The present invention further provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating cancer.

[0037] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Synthesis of a reactive oxygen species (ROS) responsive thioketone aldehyde (TK) linker; A mixture of 3-mercaptopropionic acid (11.44 g, 108.02 mmol) and p-hydroxybenzaldehyde (6.0 g, 49.1 mmol) was dissolved in an appropriate amount of ethyl acetate, and a catalytic amount of trifluoroacetic acid was added. The reaction was stirred at room temperature, and the reaction progress was monitored by thin-layer chromatography. The reaction ended when p-hydroxybenzaldehyde was exhausted. The solvent was removed, and the crude product was washed several times with dichloromethane and cold water, followed by drying to obtain a white product, namely the ROS-responsive TK linker.

[0039] Example 2: Synthesis of a drug-linker intermediate (TK-Tran); TK (32.6 mg) obtained in Example 1 and tranylcypromine (Tran, 18.4 mg) were dissolved in 10 mL of deionized water. EDC (15.5 mg) was added and stirred for 10 min, followed by NHS (11.5 mg). The reaction was carried out at 4 °C for 2 hours. After the reaction was completed, the reaction solution was dialyzed and lyophilized to obtain the TK-Tran drug-linker intermediate. The obtained TK-Tran intermediate was characterized by high performance liquid chromatography (HPLC, peak elution time 4.73 min), ultraviolet absorption spectroscopy (maximum absorption range 260–300 nm), and mass spectrometry (a fragment ion peak at m / z 314.9 was observed, corresponding to a molecular weight of 431.06), verifying its successful synthesis.

[0040] Example 3: Synthesis of antibody-drug conjugate (T-TK-P); 10 mL of the TK-Tran solution obtained in Example 2 was taken and mixed with 50 mg or 100 mg of PD-L1 monoclonal antibody (adebecilimab), respectively. After stirring for 10 minutes, EDC / NHS buffer (corresponding amounts of 15.5 mg / 11.5 mg or 31 mg / 23 mg) was added, and the reaction was continued for 12 hours. Precipitation was generated during the reaction. After the reaction was completed, the conjugates were washed, centrifuged and lyophilized to obtain two antibody-drug conjugates, T-TK-P(50) and T-TK-P(100).

[0041] The obtained antibody-drug conjugates were characterized by proton nuclear magnetic resonance (NMR) spectroscopy (changes in the aromatic region hydrogen signal peak and carbon-based α-H signal peak), ultraviolet absorption spectroscopy (absorption at both 260 nm and 280 nm), and infrared spectroscopy (3288, 1635, and 1562 cm⁻¹). -1 (Increased signal peaks at locations such as [location name]) verify the successful synthesis of the antibody-drug conjugate.

[0042] The drug-antibody ratios (DAR values) of T-TK-P(50) and T-TK-P(100) were measured to be 13.8 and 10.2, respectively.

[0043] Test Example 1: Evaluation of ROS-responsive drug release characteristics of T-TK-P; T-TK-P (50) prepared in Example 3 was placed in a release medium containing 50 μM or 100 μM hydrogen peroxide (H2O2) and incubated at 37°C. Samples were taken at different time points, and the concentrations of released transphenylcyclopropylamine (Tran) and PD-L1 were determined by ultraviolet spectrophotometry.

[0044] Test results are as follows Figure 7 As shown, under 100 μM H2O2 conditions, the cumulative release of Tran was close to 100% after 48 hours, and the release of PD-L1 was about 40%, indicating that the T-TK-P has significant ROS-responsive drug release characteristics in the presence of reactive oxygen species.

[0045] Test Example 2: Stability evaluation of T-TK-P; T-TK-P prepared in Example 3 was dispersed in BEME solution to simulate the blood environment, and its ultraviolet absorption spectrum was detected on day 1, day 3 and day 5 respectively.

[0046] Test results showed that the UV absorption spectrum of the T-TK-P did not change significantly during the observation period, indicating that the antibody-drug conjugate has good stability in a simulated blood environment.

[0047] In the above specific embodiments, through specific examples and test cases, the reactive oxygen species (ROS) responsive thioketaldehyde (TK) linker, its preparation method, and the antibody-drug conjugate (ADC) constructed based on the linker have been described in this invention.

[0048] Specifically, this invention prepares a well-defined ROS-responsive TK linker by reacting 3-mercaptopropionic acid with p-hydroxybenzaldehyde under acidic catalytic conditions. Based on this, the TK linker is then conjugated with small molecule drugs and antibodies, respectively, successfully constructing antibody-drug conjugates. The structure and composition of the linker and the antibody-drug conjugates are verified using relevant characterization methods.

[0049] Further test results showed that the constructed antibody-drug conjugate had good structural stability in a simulated blood environment and exhibited obvious responsive drug release characteristics in the presence of reactive oxygen species. At the same time, the antibody-drug conjugate had high drug loading capacity and could achieve effective conjugation of multiple small molecule drugs with a single antibody molecule.

[0050] In summary, this invention has verified the feasibility and effectiveness of the ROS-responsive TK linker and its constructed antibody-drug conjugate in terms of structural stability, ROS-responsive drug release, and application in tumor treatment through specific embodiments, indicating that it has good application prospects in the field of targeted cancer therapy.

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A reactive oxygen species-responsive thioketone-aldehyde linker, characterized in that, The thioketone aldehyde linker has the following structure: ； The structure includes a thioketaldehyde structure centered on a p-hydroxyphenyl group, wherein the carbon atoms of the thioketaldehyde are each connected to two -CH2CH2COOH groups via sulfur atoms.

2. The method for preparing the reactive oxygen species-responsive thioketone-aldehyde linker as described in claim 1, characterized in that, Includes the following steps: 3-Mercaptopropionic acid and p-hydroxybenzaldehyde were dissolved in ethyl acetate, and trifluoroacetic acid was added as a catalyst. The mixture was stirred at room temperature and the reaction was completed. After separation and purification, the thioketone aldehyde linker was obtained.

3. An antibody-drug conjugate, characterized in that, The invention comprises an antibody, a small molecule drug, and a reactive oxygen species-responsive thiophene aldehyde linker as described in claim 1, which links the antibody to the small molecule drug.

4. The antibody-drug conjugate according to claim 3, characterized in that, The antibody is a PD-L1 monoclonal antibody.

5. The antibody-drug conjugate according to claim 3, characterized in that, The small molecule drug is tranylcyclopropane.

6. The method for preparing the antibody-drug conjugate according to any one of claims 3-5, characterized in that, Includes the following steps: Step 1: React the thioketal linker as described in claim 1 with a small molecule drug under the action of a condensing agent to obtain a drug-linker intermediate; Step 2: The drug-linker intermediate is coupled with the antibody to obtain the antibody-drug conjugate.

7. The method for preparing the antibody-drug conjugate according to claim 6, characterized in that, The condensing agents are EDC and NHS.

8. A pharmaceutical composition, characterized in that, It comprises the antibody-drug conjugate as described in any one of claims 3-5 and a pharmaceutically acceptable carrier.

9. The use of the antibody-drug conjugate as described in any one of claims 3-5 in the preparation of a medicament for treating cancer.

10. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating cancer.

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