A CPT1A protein degradation targeted chimera and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a protein degradation-targeting chimera for CPT1A, its preparation method, and application. (S,R,S)-AHPC-PEG4-NH2 HCl, etomoxir (sodium salt), and DIPEA are added to an organic solvent, followed by HATU, and the reaction is stirred at 20-25°C. After completion of the reaction, the chimera of formula (I) is obtained by purification. A new PROTAC capable of targeted degradation of the CPT1A protein was developed by linking the CPT1A inhibitor etomoxir (sodium salt) to a conjugate of the E3 ubiquitin ligase VHL ligand and a linker. This PROTAC can bind to CPT1A and trigger its effective degradation, significantly downregulating CPT1A levels, and has important application prospects in tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a CPT1A protein degradation targeted chimera and a preparation method and application thereof. Background Art

[0002] Abnormal metabolic reprogramming of tumor cells is a key characteristic of their survival and proliferation. Abnormal activation of fatty acid metabolism has been widely reported in various malignancies, particularly the mitochondrial fatty acid β-oxidation pathway, which supports sustained tumor growth by providing energy and biosynthetic precursors. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, catalyzing the transmembrane transport of long-chain fatty acids and playing a central role in maintaining lipid metabolic homeostasis.

[0003] Recent studies have found that CPT1A is significantly overexpressed in various tumors (such as liver cancer, breast cancer, colorectal cancer, and glioma). It not only enhances fatty acid oxidation to increase energy supply and support rapid tumor growth, but also downregulates acyl-CoA synthetase long-chain family member 4 (ACSL4) to reduce the content of phospholipid polyunsaturated fatty acids, thereby inhibiting cell ferroptosis and reducing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, regulating CPT1A has become an important target for improving the efficacy of tumor treatment.

[0004] Among the tumor treatment strategies targeting CPT1A, existing methods mainly include knocking down CPT1A expression, delivering siRNA to silence CPT1A, or using small molecule compounds to inhibit its activity. For example: (1) By specifically knocking down CPT1A expression, the fatty acid oxidation and growth ability of ovarian cancer cells can be significantly inhibited (Sawyer BT, Qamar L, Yamamoto TM, et al. Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression. Mol Cancer Res. 2020;18(7):1088-1098.), but it can only partially reduce the expression level of CPT1A and it is difficult to achieve complete elimination; there is also a risk of off-target effects, which may interfere with other metabolic pathways; (2) Delivery of CPT1A siRNA through iRGD-modified exosomes can reverse the sensitivity of resistant colon cancer cells to oxaliplatin (Lin D, ZhangH, Liu R, et al. iRGD-modified exosomes effectively deliver CPT1A siRNA to colon cancer cells, reversing oxaliplatin resistance by regulating fatty acidoxidation. Mol Oncol. 2021;15(12):3430-3446.), this strategy has poor in vivo stability, is easily degraded by nucleases, has limited delivery efficiency, and lacks sufficient tumor tissue targeting; (3) Through the CPT1A inhibitor (2R)-2-[6-(4-chlorophenoxy)hexyl] oxirane sodium formate, namely etomoxir (sodium salt), targeting CPT1A-mediated fatty acid oxidation, thereby enhancing the therapeutic effect of liver cancer (Ren M, Xu H, Xia H, Tang Q, Bi F. Simultaneously targeting SOAT1 and CPT1A ameliorates hepatocellular carcinoma by disrupting lipid homeostasis. Cell Death Discov. 2021;7(1):125.), it only has a reversible inhibitory effect on CPT1A enzyme activity and cannot block its non-enzyme function.

[0005] Currently, there is no strategy for degrading the key metabolic protein CPT1A. Therefore, developing a novel protein degradation targeting chimera (PROTAC) compound that can efficiently degrade CPT1A is expected to overcome the limitations of traditional treatments and significantly improve the clinical efficacy of tumor treatment. Summary of the Invention

[0006] In response to the above-mentioned problems existing in the prior art, the present invention provides a CPT1A protein degradation targeted chimera and its preparation method and application, which can target and degrade CPT1A and can be used to treat diseases related to CPT1A activity and expression.

[0007] To achieve the above-mentioned purpose, the technical solution provided by the present invention is as follows:

[0008] In a first aspect, the present application provides a CPT1A protein degradation targeted chimera, the chimera having the following structural formula:

[0009] ,

[0010] (Ⅰ).

[0011] In a second aspect, the present application provides a method for preparing the CPT1A protein degradation targeted chimera described in the first aspect, comprising the following steps:

[0012] (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is reacted with sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxiranecarboxylate to obtain the chimera of formula (I).

[0013] Optionally, the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate to (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is 1:(1.1-1.5).

[0014] Optionally, the reaction is carried out in an organic solvent, using 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensation agent and N,N-diisopropylethylamine as a catalyst.

[0015] Optionally, after the reaction is completed, the reaction solution is purified, specifically comprising:

[0016] The reaction solution was diluted with ethyl acetate, washed with water and saturated sodium chloride aqueous solution, extracted, dried with sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness to obtain a concentrate;

[0017] The concentrate was dissolved in dichloromethane and separated by silica gel column chromatography, eluting with dichloromethane and a dichloromethane / methanol mixture as eluents, respectively. Thin layer chromatography was used for tracking detection, using dichloromethane:methanol in a volume ratio of 10:1 as the developing solvent. The components with an Rf value of 0.25-0.4 were collected, rotary evaporated until no liquid flowed out, and then vacuum dried to obtain the chimera described in formula (I).

[0018] Optionally, the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate to N,N-diisopropylethylamine is 1:(1.5-5); and / or the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:(1-3).

[0019] Optionally, the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate and (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is The amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl] oxirane formate to N,N-diisopropylethylamine is 1:3; the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl] oxirane formate to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.5.

[0020] Optionally, the organic solvent is selected from N,N-dimethylformamide, and the volume of the organic solvent is 0.1-10 mL / mmol based on the amount of sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate.

[0021] Optionally, the reaction temperature is 15-30° C., and the reaction time is 6-48 h.

[0022] In a third aspect, the present application provides a use of the protein degradation targeting chimera of CPT1A described in the first aspect in the preparation of an anti-tumor drug.

[0023] In a fourth aspect, the present application provides a use of the CPT1A protein degradation targeting chimera described in the first aspect in the preparation of a drug targeted for degradation of carnitine palmitoyltransferase 1A.

[0024] Compared with the prior art, this application has at least the following beneficial effects:

[0025] This invention develops a novel PROTAC that targets and degrades the CPT1A protein by linking the CPT1A inhibitor (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane sodium carboxylate (ethomoxir sodium salt) and (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride, a conjugate of the E3 ubiquitin ligase VHL ligand and a linker. This PROTAC binds to CPT1A and triggers its effective degradation, significantly downregulating CPT1A levels and holding great promise for application in cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation route of the protein degradation targeting chimera of CPT1A in the examples of this application;

[0027] Figure 2 This is a 1H NMR spectrum of the protein degradation targeting chimera of CPT1A in the examples of this application;

[0028] Figure 3 This is the 13C NMR spectrum of the protein degradation targeting chimera of CPT1A in the examples of this application;

[0029] Figure 4 This is a mass spectrum of the protein degradation targeting chimera of CPT1A in the examples of this application;

[0030] Figure 5 This is a high performance liquid chromatogram of the protein degradation targeting chimera of CPT1A in the examples of this application;

[0031] Figure 6 The cell survival rate of the protein degradation targeting chimera of CPT1A at different concentrations in mouse breast cancer cell lines (normal 4T1 cells and radiation-resistant 4T1 cells) in the examples of this application;

[0032] Figure 7 The immunoblot images and semi-quantitative analysis results of the degradation of CPT1A protein by the CPT1A protein degradation targeting chimera (10 μM) in mouse breast cancer cell lines (normal 4T1 cells and radiation-resistant 4T1 cells) in the examples of this application are shown. DETAILED DESCRIPTION

[0033] The following examples are merely examples of embodiments of the present invention and do not constitute any limitation to the present invention. It will be understood by those skilled in the art that modifications without departing from the spirit and scope of the present invention fall within the scope of protection of the present invention. In order to avoid repeated and cumbersome descriptions, the well-known structures and functions will not be described in detail. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products, and the raw materials and reagents are obtained from commercial channels with a purity of 95% or more. The test and calculation methods used are all conventional methods in the art.

[0034] Ordinary 4T1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0035] Radiation-resistant 4T1 cells: A radiation-resistant 4T1 cell model was obtained by repeatedly administering a certain dose of radiation (4, 4, 4, 6, 6, 8, 8, 10, 10 Gy) to ordinary 4T1 cells for a total radiation dose of 60 Gy.

[0036] Example 1: Preparation of CPT1A protein degradation-targeted chimeras

[0037] like Figure 1As shown, (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (S,R,S)-AHPC-PEG4-NH2 HCl (66.66 mg, 100.41 μmol), sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxiranecarboxylate (sodium etomoxir) (25 mg, 78.11 μmol) and N,N-diisopropylethylamine (DIPEA) (32.45 mg, 251.04 μmol) were added to N,N-dimethylformamide (DMF) (67.66 mg, 100.41 μmol) in sequence. The mixture was stirred evenly, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (47.73 mg, 125.52 µmol) was added. The mixture was stirred at room temperature for 18 hours; diluted with ethyl acetate, washed twice with water and saturated sodium chloride aqueous solution, extracted, dried over sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness to obtain a concentrate; the concentrate was dissolved in dichloromethane and separated by silica gel column chromatography, eluting with dichloromethane and a dichloromethane / methanol mixture (volume ratio 20:1) as eluents, and detected by thin layer chromatography with dichloromethane:methanol (volume ratio 10:1) as the developing solvent. Fractions with Rf values ​​of 0.25-0.4 were collected, rotary evaporated until no liquid emanated, and then dried in vacuo at 25°C to obtain an off-white oily product, which is the protein degradation targeted chimera of CPT1A, with a molecular weight of about 87. mg (yield 55%, purity> 95%); CPT1A protein degradation targeting chimera (abbreviated as CP) 1 H NMR spectroscopy, 13 C NMR spectrum, mass spectrum and HPLC chromatography are shown in Figures 2 to 5 .

[0038] 1H NMR (400 MHz, Chloroform-d6) δ 8.68 (s, 1H), 7.39 – 7.33 (m, 5H),7.25 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 6.82 – 6.75 (m, 3H), 4.74 (t, J =7.9 Hz, 1H), 4.60 – 4.47 (m, 3H), 4.34 (dd, J = 14.9, 5.3 Hz, 1H), 4.10 (d, J= 11.5 Hz, 1H), 4.02 (d, J = 4.9 Hz, 2H), 3.90 (t, J = 6.5 Hz, 2H), 3.72 –3.54 (m, 16H), 3.50 (d, J = 5.2 Hz, 2H), 3.40 (q, J = 5.4 Hz, 2H), 2.83 –2.75 (m, 2H), 2.52 (s, 3H), 2.38 – 2.30 (m, 1H), 2.17 – 2.08 (m, 1H), 1.78 –1.72 (m, 2H), 1.43 (s, 2H), 1.25 (d, J = 5.5 Hz, 4H), 0.95 (s, 9H).

[0039] 1 H NMR (400 MHz, Chloroform-d6) δ171.49, 170.67, 170.51, 170.27,157.72, 150.31, 148.52, 138.14, 131.60, 131.00, 129.54, 129.27, 128.16,125.32, 115.77, 71.11, 70.58 (d, J = 6.6 Hz), 70.44, 70.27, 70.16, 69.63,68.18, 59.43, 58.35, 57.15, 56.66, 53.06, 43.27, 38.70, 35.78, 34.86, 30.74,29.70, 29.20, 29.04, 26.40, 25.81, 24.52, 16.06.

[0040] TOF-MS: m / z 944.4254 [M+H]⁺.

[0041] HPLC analysis was performed using an Ultimate XB-C18 column (4.5 x 250 mm, 5 µm) with a mobile phase of methanol / water (95:5, volume ratio). The detection wavelength was set at 254 nm. The injection volume was 10 µL, the flow rate was 1.0 mL / min, and the column temperature was 25°C. Under these conditions, the target compound had a retention time of 4.148 min, and its purity, calculated by area normalization, exceeded 95%.

[0042] Example 2: Cytotoxicity Evaluation of CPT1A Protein Degradation Targeting Chimera

[0043] Ordinary 4T1 cells and radiation-resistant 4T1 cells were cultured at 8 × 10 3 The cells were seeded at an initial density of 100 μM in a 96-well culture plate. After overnight incubation, different concentrations of CP compounds (1, 5, 10, 15, 20, and 25 μM, from Example 1) were added and incubated for 24 h. The cell proliferation and toxicity detection kit (CCK-8 method) was then used to measure the absorbance using a microplate reader, and the cell survival rate and IC were calculated. 50 The experimental results are as follows. Figure 6 shown.

[0044] The results showed that the IC value of compound CP in normal 4T1 cells and radiation-resistant 4T1 cells was 50 were 13.18 ± 0.11 µM and 13.79 ± 0.24 µM, respectively, indicating that CP had good killing activity against both cell lines.

[0045] Example 3: Evaluation of the degradation effect of CPT1A protein targeting chimera on CPT1A protein

[0046] Ordinary 4T1 cells and radiation-resistant 4T1 cells were cultured at 1×10 6 The initial density of cells was seeded in a 6-well culture plate. After incubation overnight, compound CP (10 μM, from Example 1) was added and incubated for another 24 h. After collecting the cells, the cells were lysed using a radioimmunoprecipitation assay lysis buffer (RIPA buffer) containing 1% phenylmethylsulfonyl fluoride (PMSF). After the total protein concentration was determined by bicinchoninic acid (BCA) protein assay, equal amounts of protein samples were loaded onto sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, blocking treatment, primary and secondary antibody incubation, and enhanced chemiluminescence (ECL) development were performed in sequence, and the images were analyzed using Image Lab software. The experimental results are shown in Figure 2. Figure 7 shown.

[0047] The results showed that, using β-actin as the internal reference, the CPT1A protein bands in ordinary 4T1 cells and radiation-resistant 4T1 cells (with high CPT1A expression) were significantly weakened compared with the control group after treatment with 10 μM compound CP, and the protein expression levels decreased by approximately 46% and 53%, respectively. Obviously, the CPT1A protein degradation targeting chimera can significantly degrade CPT1A protein under different CPT1A expression backgrounds.

[0048] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

[0049] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A CPT1A protein degradation targeting chimera, characterized in that: The chimera structural formula is as follows: （Ⅰ）。 2. A method for preparing the CPT1A protein degradation targeted chimera according to claim 1, characterized in that: The steps include: (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is reacted with sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxiranecarboxylate to obtain the chimera of formula (I).

3. The method for preparing a CPT1A protein degradation targeted chimera according to claim 2, characterized in that: The amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate and (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is 1:(1.1-1.5).

4. The method for preparing a CPT1A protein degradation targeted chimera according to claim 2, characterized in that: The reaction is carried out in an organic solvent, using 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensation agent and N,N-diisopropylethylamine as a catalyst.

5. The method for preparing a CPT1A protein degradation targeted chimera according to claim 2, characterized in that: After the reaction is completed, the reaction liquid is purified, specifically including: The reaction solution was diluted with ethyl acetate, washed with water and saturated sodium chloride aqueous solution, extracted, dried with sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness to obtain a concentrate; The concentrate was dissolved in dichloromethane and separated by silica gel column chromatography, eluting with dichloromethane and a dichloromethane / methanol mixture as eluents, respectively. Thin layer chromatography was used for tracking detection, using dichloromethane:methanol in a volume ratio of 10:1 as the developing solvent. The components with an Rf value of 0.25-0.4 were collected, rotary evaporated until no liquid flowed out, and then vacuum dried to obtain the chimera described in formula (I).

6. The method for preparing a CPT1A protein degradation targeted chimera according to claim 4, characterized in that: The amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate to N,N-diisopropylethylamine is 1:(1.5-5); and / or the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:(1-3).

7. The method for preparing a CPT1A protein degradation targeted chimera according to claim 4, characterized in that: The amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate and (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride is 1:

1. :1.2; the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl] oxirane formate and N,N-diisopropylethylamine is 1:3; the amount ratio of the sodium (2R)-2-[6-(4-chlorophenoxy)hexyl] oxirane formate and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.

5.

8. The method for preparing a CPT1A protein degradation targeted chimera according to claim 4, wherein: The organic solvent is selected from N,N-dimethylformamide, and the volume of the organic solvent is 0.1-10 mL / mmol based on the amount of sodium (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane formate.

9. Use of the CPT1A protein degradation targeted chimera according to claim 1 in the preparation of anti-breast cancer drugs.

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