Protac compounds targeting lactate dehydrogenase and methods of making and using the same

Abstract

This invention provides PROTAC compounds targeting lactate dehydrogenase, their preparation methods, and applications, belonging to the field of biomedical technology. The compounds provided by this invention can efficiently degrade LDHA protein in pancreatic cancer cells and significantly inhibit the proliferation of pancreatic cancer Panc-1 cells at the cellular level. Several compounds exhibit excellent degradation and anti-proliferative activities, possessing the potential for further development into anti-pancreatic cancer drugs. This invention enriches the library of LDH-targeting PROTAC molecules, providing new candidate compounds for targeted therapy of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to PROTAC compounds that target lactate dehydrogenase, their preparation methods, and applications. Background Technology

[0002] Lactate dehydrogenase (LDH) is a key rate-limiting enzyme in the glycolysis pathway, catalyzing the reversible conversion between pyruvate and lactate, accompanied by the interconversion of NAD⁺ and NADH. In normal cells, LDH mainly participates in the homeostasis regulation of energy metabolism. However, in various malignant tumor cells, even with sufficient oxygen, cells preferentially obtain energy through the glycolysis pathway, a phenomenon known as the Warburg effect. This metabolic reprogramming leads to the production of large amounts of lactate, which not only provides ATP and biosynthetic precursors for the rapid proliferation of tumor cells but also promotes invasion and metastasis and inhibits anti-tumor immune responses by acidifying the tumor microenvironment.

[0003] LDH is a tetrameric protein encoded by the LDHA and LDHB genes. LDHA is significantly overexpressed in various tumors, including pancreatic cancer, lung cancer, colorectal cancer, and breast cancer, and is closely associated with poor patient prognosis and chemotherapy resistance. Therefore, LDH is considered an important metabolic target for anti-tumor drug development.

[0004] Currently reported small molecule inhibitors of LDH are mostly pyrazol- or thiazolyl-substituted compounds. While some compounds have shown certain enzyme inhibitory activity, they generally suffer from poor target selectivity, insufficient in vivo metabolic stability, and incomplete inhibitory effects, making it difficult to meet clinical treatment needs. Furthermore, traditional small molecule inhibitors can only temporarily block enzyme activity and cannot eliminate the LDH protein itself, thus making it difficult to completely block its mediated non-enzymatic carcinogenic function.

[0005] However, there is an urgent clinical need for safe, efficient, and highly selective LDH degradation drugs. Summary of the Invention

[0006] The purpose of this invention is to provide PROTAC compounds that target lactate dehydrogenase, their preparation methods, and applications, thereby solving the technical problems of existing small molecule LDH inhibitors, such as poor selectivity, inability to completely degrade target proteins, and limited efficacy against pancreatic cancer.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a compound represented by general formula (I), the structure of which is as follows: Wherein, R is -C(O)-NH-LX; L is selected from: -(CH2) n -or-(CH2CH2O) m-CH2CH2-, where n is an integer from 2 to 12 and m is an integer from 1 to 4; X is either a CRBN ligand or a VHL ligand.

[0008] Preferably, X is selected from the group represented by formula A or the group represented by formula B: Formula A; Formula B.

[0009] Preferably, the compound is selected from any one of the following compounds: .

[0010] The present invention also provides a method for preparing the above-mentioned compound, comprising the following steps: The compound shown in Formula V was subjected to an amide condensation reaction with HOOC-LX in the presence of a condensing agent to obtain the compound. The compound represented by formula V is: In HOOC-LX, the definitions of L and X are as described above.

[0011] Preferably, the conditions for the amide condensation reaction include: HATU as the condensing agent, DMSO as the solvent, room temperature as the reaction temperature, and 4 hours as the reaction time.

[0012] Preferably, the synthetic route of the compound provided by the present invention is as follows:

[0013] The present invention also provides pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs of the above-mentioned compounds.

[0014] The present invention also provides a pharmaceutical composition comprising the above-described compound and a pharmaceutically acceptable carrier or excipient.

[0015] Preferably, the dosage form of the pharmaceutical composition is an oral preparation, an injection, or a topical preparation.

[0016] The present invention also provides the use of the above-described compounds or pharmaceutical compositions in the preparation of medicaments for the treatment of pancreatic cancer.

[0017] The present invention also provides the use of the above-mentioned compounds in the preparation of reagents for degrading lactate dehydrogenase protein.

[0018] The beneficial effects of this invention are: This invention provides a novel class of LDH-targeting PROTAC compounds. Cellular activity evaluation confirmed that these compounds can effectively degrade lactate dehydrogenase protein in human pancreatic cancer cells, while significantly inhibiting the proliferation of pancreatic cancer cells. They exhibit excellent effects in both target protein degradation and tumor cell growth inhibition, with some compounds showing particularly outstanding degradation and anti-proliferative activities. These results demonstrate that the compounds of this invention can exert anti-pancreatic cancer effects at both the protein degradation and cell proliferation inhibition levels, possessing a clear pharmacodynamic basis. This invention not only provides a novel degradation tool molecule for lactate dehydrogenase, an important metabolic target, enriching the application of PROTAC technology in the field of metabolic targets, but also provides novel candidate drugs with independent intellectual property rights for the treatment of pancreatic cancer. Based on the aforementioned excellent cellular activity, the compounds of this invention can be further developed into various pharmaceutical formulations for the preparation of anti-pancreatic cancer drugs, possessing broad prospects for clinical application and significant socio-economic value. Attached Figure Description

[0019] Figure 1 The image shows the results of Western blotting for detecting LDHA protein levels. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example This embodiment provides a method for preparing the compound: Step (1) 6-(4-bromophenyl)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one (compound 2) and synthesis Add a magnetic stir bar to a dry 50 mL round-bottom flask, then add methyl 5-amino-5-(4-bromophenyl)-3-oxo-5-(thiophen-3-yl)valerate (476 mg, 1.0 mmol) and anhydrous potassium carbonate (276 mg, 2.0 mmol), followed by 10 mL of anhydrous methanol. Stir thoroughly to dissolve / disperse the solids. Install a reflux condenser and place the flask in an oil bath. Heat to 80 °C (methanol reflux temperature) and stir for 2 hours. Monitor the reaction progress using TLC (developing solvent: petroleum ether / ethyl acetate = 2:1, v / v) and observe under a UV lamp at 254 nm. Stop the reaction when the starting material spot completely disappears and the product spot clearly appears. After the reaction is complete, remove the flask from the oil bath and allow it to cool naturally to room temperature. Concentrate under reduced pressure to remove most of the methanol, yielding a pale yellow oily residue. Add 20 mL of deionized water to the residue, stir well, and slowly adjust the pH of the solution to 5.0-6.0 with 1 M hydrochloric acid. The aqueous phase was extracted with ethyl acetate (3 × 20 mL), and all organic phases were combined. The organic phase was washed with 20 mL of saturated brine, and the layers were separated. The organic layer was dried over anhydrous sodium sulfate for 30 minutes. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3:1, v / v). The target product fraction was collected, concentrated, and dried under vacuum to give a white solid 6-(4-bromophenyl)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one, in 81% yield. 1 HNMR (400MHz, DMSO-) d 6)δ10.66(s,1H),9.21(s,1H),7.95(s,1H),7.60–7.45(m,6H),7.36–7.27(m,4H),7.26–7.20(m,2H),7.09(dt, J =4.1, 1.8 Hz, 2H), 4.78 (d, J =1.4Hz, 1H), 3.15–2.93(m, 4H). Step (2) Synthesis of 6-(4-bromophenyl)-3-((2-chlorophenyl)thio)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one (compound 3) Compound 2 (6-(4-bromophenyl)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one, 1.0 eq) was dissolved in anhydrous acetonitrile (MeCN), and 1,2-bis(2-chlorophenyl) disulfide (1.2 eq) was added. The reaction mixture was placed in an oil bath and heated to 80 °C, and stirred under reflux for 2 hours. After complete conversion of the starting material was monitored by TLC, the mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50:1, v / v) to give the target product compound 3 (6-(4-bromophenyl)-3-((2-chlorophenyl)thio)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one), in 72% yield. 1 HNMR (400MHz, Methanol-) d 4) δ7.55(d, J =8.5Hz,2H),7.52(dd, J =5.1, 3.0 Hz, 1H), 7.37 (d, J =8.6Hz,2H),7.31(dd, J =3.0, 1.4Hz, 1H), 7.21(dd, J =8.0, 1.2Hz, 1H), 7.15(dt, J =5.0, 1.0 Hz, 1H), 6.94 (td, J =7.7, 1.4 Hz, 1H), 6.76 (td, J =7.7, 1.2 Hz, 1H), 5.93 (d, J =7.9Hz, 1H), 3.47(d, J =3.0Hz, 2H). Step (3) Synthesis of 4-(4-(5-((2-chlorophenyl)thio)-4-hydroxy-6-oxo-2-(thiophen-3-yl)-1,2,3,6-tetrahydropyridin-2-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester (compound 4) Under nitrogen protection, compounds 3 (6-(4-bromophenyl)-3-((2-chlorophenyl)thio)-4-hydroxy-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one, 1.0 eq), 1-Boc-piperazine (1.5 eq), sodium tert-butoxide (t-BuONa, 2.0 eq), Brettphos ligand (0.05 eq), and tris(dibenzylideneacetone)palladium (Pd2(dba)3, 0.03 eq) were added sequentially to a dry reaction flask, followed by complete dissolution with anhydrous 1,4-dioxane. The reaction mixture was then placed in an oil bath and heated to 120°C, and stirred under reflux for 16 hours. After complete conversion of the starting material by TLC, the mixture was cooled to room temperature and filtered through diatomaceous earth to remove insoluble palladium salts. The filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2:1, v / v) to give the target product compound 4 (4-(4-(5-((2-chlorophenyl)thio)-4-hydroxy-6-oxo-2-(thiophen-3-yl)-1,2,3,6-tetrahydropyridin-2-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester), in 58% yield.

[0022] Step (4) Synthesis of 3-((2-chlorophenyl)thio)-4-hydroxy-6-(4-(piperazin-1-yl)phenyl)-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one (compound 5) Compound 4 (1.0 eq) of 4-(4-(5-(((2-chlorophenyl)thio)-4-hydroxy-6-oxo-2-(thiophen-3-yl)-1,2,3,6-tetrahydropyridin-2-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester) was dissolved in anhydrous dichloromethane (DCM). Trifluoroacetic acid (TFA, 10 eq) was slowly added dropwise under ice bath conditions. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature (RT) for 4 hours. The reaction was monitored by TLC to ensure complete reaction of the starting material. After conversion, the solvent and excess trifluoroacetic acid were removed by vacuum concentration. Saturated sodium bicarbonate solution was added to the residue to adjust the pH to 8. The residue was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give the target product compound 5 (3-((2-chlorophenyl)thio)-4-hydroxy-6-(4-(piperazin-1-yl)phenyl)-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one) in 90% yield. 1 HNMR (400MHz, DMSO-) d 6)δ7.44(dd, J =5.0,2.9Hz,1H),7.34–7.22(m,3H),7.16(d, J =7.8Hz,1H),7.14–7.04(m,2H),6.86(d, J=8.4Hz,2H),6.80(t, J =7.6Hz, 1H), 6.68(t, J =7.7Hz, 1H), 6.12(d, J =7.9Hz,1H),3.24–3.17(m,4H),3.17–3.06(m,4H),3.06–2.93(m,2H). 13 CNMR(101MHz,DMSO)δ183.61,171.53,149.22,142.30,138.53,128.79,128.69,127.93,127.35,126.80,126.22,12 6.08,123.79,120.89,115.94,82.13,59.51,49.57,46.51,43.62,40.62,40.41,40.20,39.99,39.78,39.57,39.36. Step (5) Synthesis of the final product (compound 6-23) Compound 5 (3-((2-chlorophenyl)thio)-4-hydroxy-6-(4-(piperazin-1-yl)phenyl)-6-(thiophen-3-yl)-5,6-dihydropyridine-2(1H)-one) (1.0 eq) and its E3 ligand carboxylic acid derivative (1.2 eq) were dissolved in DMSO and reacted at room temperature with stirring for 4 hours. After the reaction was complete, water was added, the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane / methanol = 20:1) to give the final product in yields of 42%–60%.

[0023] The synthesis route diagram is as follows:

[0024] The product structure is as follows:

[0025] Compound 6 Structural characterization: ¹H NMR (400 MHz, DMSO-d⁶) δ 11.12 (s, ¹H), 7.61 (t, J = 7.8 Hz, 2H), 7.49 (dd, J = 5.0, 2.6 Hz, 1H), 7.33 – 7.24 (m, 3H), 7.20 (d, J = 7.9 Hz, 1H), 7.17 – 7.05 (m, 4H), 6.93 (d, J = 8.3 Hz, 2H), 6.86 (t, J = 7.6 Hz, 1H), 6.70 (t, J = 7.6 Hz, 1H), 6.02 (d, J = 7.9 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H), 4.26 (d, J = 4.6 Hz, 2H), 3.76 – 3.58 (m, 4H), 3.23 (d, J= 5.5 Hz, 2H), 3.20 – 3.08 (m, 4H), 2.90 (ddd, J = 17.9, 13.8, 5.4 Hz, 1H),2.65 – 2.52 (m, 2H), 2.04 (dd, J = 11.9, 6.1 Hz, 1H). MS (ESI+) 811.34 [M+H]+ Compound 7 Structural characterization: ¹H NMR (400 MHz, DMSO-d⁶) δ 11.12 (s, ¹H), 7.59 (t, J = 7.8 Hz, ¹H), 7.45 (dd, J = 5.0, 2.9 Hz, ¹H), 7.32 – 7.21 (m, 3H), 7.15 (t, J = 8.5 Hz, 2H), 7.12 – 6.98 (m, 3H), 6.87 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 7.3 Hz, 2H), 6.67 (t, J = 7.6 Hz, 1H), 6.08 (d, J = 7.9 Hz, 1H), 5.04 (dd, J = 12.7, 5.5 Hz, 1H), 3.59 – 3.55 (m, 4H),, 3.19 – 3.15 (m, 3H), 3.13 – 3.05 (m,4H), 3.03 – 2.95 (m, 2H), 2.94 – 2.80 (m, 1H), 2.72 (q, J = 6.6, 5.9 Hz, 2H), 2.58 (d, J = 18.4 Hz, 1H), 2.01 (dd, J = 14.7, 7.9 Hz, 1H). MS (ESI+) 825.36[M+H]+ Compound 8 Structural characterization: ¹H NMR (400 MHz, DMSO-d⁶) δ 11.12 (s, ¹H), 7.57 (d, J = 6.3 Hz, 2H), 7.48 (t, J = 3.9 Hz, 1H), 7.35 – 7.14 (m, 4H), 7.11 (d, J = 5.0 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.87 (dd, J = 16.9, 8.2 Hz, 3H), 6.69 (t, J = 7.1 Hz, 2H), 6.02 (d, J = 7.9 Hz, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 3.66– 3.54 (m, 4H), 3.34 (q, J = 6.9 Hz, 2H), 3.23 – 3.02 (m, 6H), 2.98 – 2.81(m, 1H), 2.66 – 2.53 (m, 2H), 2.46 (t, J = 7.1 Hz, 2H), 2.10 – 1.94 (m, 1H),1.83 (p, J = 6.9 Hz, 2H). MS (ESI+) 839.39 [M+H]+ Compound 9 Structural characterization: ¹H NMR (400 MHz, DMSO-d⁶) δ 11.14 (s, ¹H), 7.58 (t, J = 7.8 Hz, ¹H), 7.51 – 7.42 (m, ¹H), 7.37 – 7.21 (m, 3H), 7.11 (d, J = 8.4 Hz, 3H), 7.02 (d, J = 7.0 Hz, 1H), 6.98 – 6.83 (m, 3H), 6.78 (t, J = 7.4 Hz, 1H), 6.66 (t, J = 7.6 Hz, 1H), 6.57 (t, J = 6.0 Hz, 1H), 6.12 (d, J = 7.9 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 3.58 (d, J = 5.2 Hz, 4H), 3.33 (d, J = 5.9 Hz, 2H), 3.08 (dt, J = 18.9, 5.1 Hz, 4H), 2.98 – 2.83 (m, 3H), 2.64 – 2.52 (m,2H), 2.42 (d, J = 6.9 Hz, 2H), 2.01 (dd, J = 12.3, 6.1 Hz, 1H), 1.68 – 1.52(m, 4H). MS (ESI+) 853.42 [M+H]+ Compound 10 Structural characterization: ¹H NMR (400 MHz, DMSO-d⁶) δ 11.15 (s, ¹H), 7.57 (t, J = 7.8 Hz, ¹H), 7.46 (dd, J = 5.1, 2.9 Hz, ¹H), 7.33 – 7.21 (m, 3H), 7.15 – 7.06 (m, 3H), 7.03 – 6.97 (m, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.79 (t, J = 7.6 Hz, 1H), 6.67 (t, J = 7.7 Hz, 1H), 6.54 (t, J = 6.0 Hz, 1H), 6.11 (d, J = 7.9 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 3.62 – 3.54 (s, 4H), 3.30 (q, J = 6.7 Hz, 2H), 3.09 (d, J = 17.3 Hz, 4H), 2.96 (s, 2H), 2.93 – 2.82 (m, MS (ESI+) 867.44 [M+H]+ Compound 11 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 7.62 – 7.53 (m, 1H), 7.45(dd, J = 5.0, 2.8 Hz, 1H), 7.32 – 7.23 (m, 3H), 7.10 (dd, J = 9.9, 6.1 Hz,3H), 7.02 (d, J = 7.0 Hz, 1H), 6.91 – 6.82 (m, 3H), 6.78 (t, J = 7.4 Hz, 1H),6.66 (t, J = 7.6 Hz, 1H), 6.54 (q, J = 6.8, 6.0 Hz, 1H), 6.11 (d, J = 8.0 Hz,1H), 5.05 (dd, J = 12.9, 5.4 Hz, 1H), 3.63 – 3.54 (m, 4H), 3.29 (q, J = 6.7Hz, 2H), 3.14 – 3.04 (m, 4H), 2.89 (dt, J = 13.8, 9.0 Hz, 3H), 2.63 – 2.52(m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 2.08 – 1.96 (m, 1H), 1.66 – 1.44 (m, 5H),1.45 – 1.29 (m, 4H). MS (ESI+) 881.47 [M+H]+ Compound 12 1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 7.57 (dd, J = 8.5, 7.1 Hz,1H), 7.45 (dd, J = 5.0, 2.9 Hz, 1H), 7.32 – 7.22 (m, 3H), 7.15 – 7.05 (m,3H), 7.02 (d, J = 7.0 Hz, 1H), 6.96 – 6.84 (m, 3H), 6.78 (td, J = 7.5, 1.6Hz, 1H), 6.66 (t, J = 7.6 Hz, 1H), 6.52 (t, J = 5.9 Hz, 1H), 6.11 (dd, J =8.0, 1.6 Hz, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 3.58 (q, J = 3.7 Hz, 4H),3.29 (q, J = 6.7 Hz, 2H), 3.08 (dt, J = 19.2, 5.1 Hz, 4H), 2.97 – 2.83 (m,3H), 2.64 – 2.52 (m, 2H), 2.33 (t, J = 7.4 Hz, 2H), 2.08 – 1.94 (m, 1H), 1.54(dt, J = 25.6, 6.9 Hz, 4H), 1.42 – 1.26 (d, J = 25.0 Hz, 6H). MS (ESI+)895.55[M+H]+ Compound 13 1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H),7.46 (d, J = 4.5 Hz, 1H), 7.32 – 7.20 (m, 3H), 7.15 – 7.07 (m, 3H), 7.03 (d,J = 7.0 Hz, 1H), 6.96 – 6.83 (m, 3H), 6.78 (t, J = 7.5 Hz, 1H), 6.67 (d, J =7.6 Hz, 1H), 6.58 (d, J = 5.8 Hz, 1H), 6.11 (d, J = 7.9 Hz, 1H), 5.05 (dd, J= 12.9, 5.3 Hz, 1H), 3.70 (t, J = 5.9 Hz, 2H), 3.66 – 3.54 (m, 8H), 3.14 –3.00 (m, 4H), 2.97 – 2.69 (m, 4H), 2.66 – 2.53 (m, 3H), 2.08 – 1.95 (m, 1H).MS (ESI+) 869.41[M+H]+ Compound 14 1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H),7.44 (dd, J = 4.9, 2.7 Hz, 1H), 7.33 – 7.22 (m, 3H), 7.17 – 7.06 (m, 3H),7.03 (d, J = 7.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 3H), 6.78 (t, J = 7.5 Hz, 1H),6.66 (t, J = 7.5 Hz, 1H), 6.60 (t, J = 5.8 Hz, 1H), 6.11 (d, J = 7.9 Hz, 1H),5.06 (dd, J = 12.9, 5.4 Hz, 1H), 3.69 – 3.49 (m, 12H), 3.44 (q, J = 6.3, 5.9Hz, 4H), 3.08 (dt, J = 16.7, 4.7 Hz, 4H), 2.97 – 2.84 (m, 2H), 2.62 – 2.52(m, 3H), 2.08 – 1.99 (m, 1H). MS (ESI+) 869.41[M+H]+ Compound 15 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.64 (t, J = 6.1 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.53 – 7.33 (m, 6H), 7.33 – 7.06 (m, 6H), 6.90 (d,J = 8.4 Hz, 2H), 6.82 (t, J = 7.6 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.06 (d,J = 7.9 Hz, 1H), 5.23 (s, 1H), 4.53 (d, J = 9.3 Hz, 1H), 4.47 – 4.32 (m, 3H),4.22 (dd, J = 15.9, 5.5 Hz, 1H), 3.69 – 3.56 (m, 6H), 3.17 – 3.02 (m, 6H),2.68 – 2.52 (m, 3H), 2.45 (s, 4H), 2.10 – 1.85 (m, 2H), 0.94 (s, 9H). MS (ESI+) 1010.69[M+H]+. Compound 16 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.61 (t, J = 6.1 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.49 – 7.33 (m, 5H), 7.28 (dd, J = 10.2, 5.6 Hz, 3H), 7.10 (dd, J = 10.6, 6.4 Hz, 2H), 6.92 – 6.84 (m, 3H), 6.78 (t, J = 7.5 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.13 (d, J = 7.9 Hz, 1H), 5.21 (s, 1H), 4.56 (d, J = 9.2 Hz, 1H), 4.48 – 4.31 (m, 3H), 4.22 (dd, J = 15.9, 5.4 Hz, 1H), 3.74 – 3.64 (m, 2H), 3.64 – 3.51 (m, 4H), 3.14 – 3.04 (m, 4H), 2.93 (s, 2H), 2.45 (s, 3H), 2.39 – 2.16 (m, 4H), 2.05 (td, J = 9.6, 7.6, 4.4 Hz, 1H), 1.91 (ddd, J = 12.9, 8.3, 4.6 Hz, 1H), 1.80 – 1.68 (m, 2H), 0.95 (s, 9H). MS (ESI+) 1024.62[M+H]+. Compound 17 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.61 (t, J = 6.1 Hz, 1H), 7.88 (d, J = 9.2 Hz, 1H), 7.48 – 7.34 (m, 5H), 7.33 – 7.21 (m, 3H), 7.10 (dd, J = 12.6, 6.4 Hz, 2H), 6.89 (d, J = 8.3 Hz, 3H), 6.78 (t, J = 7.5 Hz, 1H), 6.66 (t, J = 7.7 Hz, 1H), 6.11 (d, J = 7.8 Hz, 1H), 5.19 (s, 1H), 4.55 (d, J = 9.2 Hz, 1H), 4.48 – 4.31 (m, 3H), 4.22 (dd, J = 15.9, 5.5 Hz, 1H), 3.68 – 3.52 (m, 6H), 3.16 – 3.03 (m, 4H), 2.93 (s, 2H), 2.44 (s, 3H), 2.32 (dt, J = 20.9, 7.0 Hz, 3H), 2.14 (dd, J = 13.8, 6.8 Hz, 1H), 2.04 (t, J = 10.5 Hz, 1H), 1.90 (ddd, J = 13.1, 8.6, 4.5 Hz, 1H), 1.51 (dt, J = 14.5, 8.0 Hz, 4H), 0.94 (s, 9H). MS (ESI+) 1038.64[M+H]+. Compound 18 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.63 (t, J = 6.1 Hz, 1H),7.86 (d, J = 9.3 Hz, 1H), 7.50 – 7.33 (m, 5H), 7.33 – 7.20 (m, 3H), 7.11 (dd,J = 12.0, 6.4 Hz, 2H), 6.97 – 6.83 (m, 3H), 6.79 (t, J = 7.6 Hz, 1H), 6.66(t, J = 7.6 Hz, 1H), 6.10 (d, J = 7.9 Hz, 1H), 5.23 (s, 1H), 4.54 (d, J = 9.3Hz, 1H), 4.49 – 4.32 (m, 3H), 4.22 (dd, J = 15.8, 5.4 Hz, 1H), 3.73 – 3.54(m, 6H), 3.14 – 2.90 (m, 6H), 2.44 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.29 –2.09 (m, 2H), 2.06 – 1.85 (m, 2H), 1.50 (h, J = 7.0 Hz, 4H), 1.37 – 1.25 (m,2H), 0.94 (s, 9H). MS (ESI+) 1052.66 [M+H]+. Compound 19 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.60 (t, J = 6.1 Hz, 1H),7.85 (d, J = 9.3 Hz, 1H), 7.50 – 7.34 (m, 5H), 7.32 – 7.22 (m, 3H), 7.15 (d,J = 7.7 Hz, 2H), 7.10 (d, J = 5.1 Hz, 1H), 6.89 (d, J = 8.4 Hz, 2H), 6.81 (t,J = 7.5 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.06 (d, J = 7.9 Hz, 1H), 5.18 (s,1H), 4.54 (d, J = 9.3 Hz, 1H), 4.51 – 4.30 (m, 3H), 4.22 (dd, J = 15.9, 5.4Hz, 1H), 3.69 – 3.63 (m, 2H), 3.60 – 3.56 (m, 4H), 3.14 – 3.02 (m, 6H), 2.44(s, 3H), 2.29 (dt, J = 27.7, 7.3 Hz, 3H), 2.19 – 1.86 (m, 3H), 1.48 (h, J =7.2 Hz, 4H), 1.31 – 1.24 (m, 4H), 0.94 (s, 9H). MS (ESI+) 1066.68 [M+H]+. Compound 20 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.60 (t, J = 6.1 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H), 7.72 (s, 1H), 7.50 (dd, J = 5.0, 2.8 Hz, 1H), 7.40 (q, J = 8.1 Hz, 4H), 7.33 – 7.17 (m, 4H), 7.12 (d, J = 5.1 Hz, 1H), 6.88 (dd, J = 18.2, 8.2 Hz, 3H), 6.70 (t, J = 7.6 Hz, 1H), 5.99 (d, J = 8.0 Hz, 1H), 4.55 (d, J = 9.3 Hz, 1H), 4.47 – 4.31 (m, 3H), 4.22 (dd, J = 15.9, 5.5 Hz, 1H), 3.66 (d, J = 4.8 Hz, 2H), 3.58 (d, J = 5.3 Hz, 4H), 3.23 (s, 2H), 3.15 – 3.06 (m, 4H), 2.44 (s, 3H), 2.30 (dt, J = 29.4, 7.2 Hz, 3H), 2.08 (ddd, J = 33.2, 11.8, 6.7 Hz, 2H), 1.96 – 1.83 (m, 1H), 1.50 (dd, J = 13.2, 6.6 Hz, 4H), 1.27 – 1.23 (m, 6H), 0.94 (s, 9H). MS (ESI+) 1080.62 [M+H]+. Compound 21 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.64 (t, J = 6.1 Hz, 1H),7.85 (d, J = 9.3 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.41 (p, J = 9.2, 7.9 Hz,4H), 7.33 – 7.18 (m, 3H), 7.13 (dd, J = 19.3, 6.4 Hz, 3H), 6.89 (d, J = 8.4Hz, 2H), 6.81 (t, J = 7.6 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.06 (d, J = 8.0Hz, 1H), 5.23 (s, 1H), 4.54 (d, J = 9.3 Hz, 1H), 4.48 – 4.32 (m, 3H), 4.22(dd, J = 15.9, 5.4 Hz, 1H), 3.69 – 3.63 (m, 2H), 3.58 (t, J = 4.7 Hz, 4H),3.14 – 3.04 (m, 6H), 2.45 (s, 3H), 2.29 (dt, J = 29.4, 7.2 Hz, 3H), 2.17 –2.00 (m, 2H), 1.95 – 1.84 (m, 1H), 1.49 (dd, J = 13.5, 6.9 Hz, 4H), 1.29 –1.26 (m, 8H), 0.94 (s, 9H). MS (ESI+) 1094.65 [M+H]+. Compound 22 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.59 (d, J = 6.1 Hz, 1H),7.93 (d, J = 9.3 Hz, 1H), 7.46 (t, J = 3.9 Hz, 1H), 7.40 (q, J = 8.1 Hz, 5H),7.27 (dd, J = 19.1, 5.6 Hz, 3H), 7.17 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 5.1Hz, 1H), 6.86 (dd, J = 21.2, 8.0 Hz, 3H), 6.69 (t, J = 7.6 Hz, 1H), 6.04 (d,J = 7.9 Hz, 1H), 5.17 (s, 1H), 4.55 (d, J = 9.3 Hz, 1H), 4.48 – 4.32 (m, 3H),4.22 (dd, J = 15.8, 5.5 Hz, 1H), 3.73 – 3.59 (m, 5H), 3.55 – 3.41 (m, 3H),3.17 – 3.04 (m, 8H), 2.69 – 2.52 (m, 3H), 2.44 (s, 3H), 2.41 – 2.31 (m, 1H),2.10 – 1.83 (m, 2H), 0.94 (s, 9H). MS (ESI+) 1054.64 [M+H]+. Compound 23 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.66 (q, J = 5.3, 4.7 Hz, 1H), 7.92 (dd, J = 9.5, 3.9 Hz, 1H), 7.50 – 7.21 (m, 10H), 7.16 (d, J = 7.8Hz, 1H), 7.11 (d, J = 5.1 Hz, 1H), 6.86 (dd, J = 31.6, 8.0 Hz, 3H), 6.68 (t,J = 7.6 Hz, 1H), 6.05 (d, J = 7.9 Hz, 1H), 4.56 (d, J = 9.3 Hz, 1H), 4.50 –4.31 (m, 3H), 4.22 (dd, J = 16.0, 5.4 Hz, 1H), 3.70 – 3.59 (m, 8H), 3.55 –3.41 (m, 6H), 3.17 – 3.05 (m, 6H), 2.66 – 2.53 (m, 3H), 2.44 (s, 3H), 2.40 –2.30 (m, 1H), 2.11 – 1.85 (m, 2H), 0.94 (s, 9H). MS (ESI+) 1098.69 [M+H]+. Based on the above compounds, the following tests were conducted: 1. Highly efficient degradation of LDH and blocking of glycolysis: The compound provided by this invention can simultaneously and efficiently degrade LDHA, with a DC50 level of nanomolar, completely clearing the target protein and completely blocking aerobic glycolysis of pancreatic cancer cells, thereby inhibiting tumor proliferation from the root.

[0026] Human pancreatic cancer cells PANC-1 were treated with compound 6-23 (2 μM) for 48 hours. Cells were collected, total protein was extracted, and LDHA protein levels were detected by Western blotting. The results are as follows: Figure 1 As shown in the figure. The results show that numbers 7, 9, 15, 20, 21, and 22 have better degradation effects on LDHA.

[0027] Human pancreatic cancer cells PANC-1 were treated with 7, 9, 15, 20, 21, and 22 mg of amino acids (0, 12 nM, 37 nM, 110 nM, 330 nM, and 1000 nM) for 48 hours, respectively. Cells were collected, total protein was extracted, and LDHA was detected by Western blotting. After gray-scale quantification, the results were normalized, and DC50 was calculated by fitting a dose-response curve. The results are shown in Table 1 below. Table 1. Degradation activity of compounds against LDHA protein in PANC-1 cells (DC50) compound DC50 compound <![CDATA[DC 50 ]]> 6 D 15 C 7 B 16 D 8 D 17 D 9 B 18 D 10 D 19 D 11 D 20 A 12 D 21 A 13 D 22 A 14 D 23 D A < 100 nM; 100 nM <B<300 nM;300 nM<C<1000 nM; D> 1000 nM 2. Inhibitory effect on the proliferation of pancreatic cancer cells Panc1 Log-phase human pancreatic cancer cells (Panc-1) were collected and analyzed at a concentration of 1×10⁻⁶. 3 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The original culture medium was discarded, and complete culture medium containing a series of concentration gradients of the test compounds was added to each well, with three replicates per group. A blank control group and a solvent control group were also included. After culturing for another 96 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 1–2 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The cell proliferation inhibition rate was calculated using the formula: Inhibition rate (%) = [1 − (OD value of drug-treated wells − OD value of blank wells) / (OD value of control wells − OD value of blank wells)] × 100%. The IC50 of the test compounds on Panc-1 cells was calculated using GraphPad Prism with the logarithm of compound concentration as the x-axis and the inhibition rate as the y-axis. 50 The values ​​and results are shown in Table 2 below: Table 2. Inhibitory activity of compounds on PANC-1 cell proliferation (IC50) 50 ) compound IC50 (μM) compound <![CDATA[IC 50 (μM)]]> 7 B 20 A 9 B 21 A 15 C 22 A A < 10 μM; 10 μM <B<30μM; 30μM<C<100μM; D> 100μM As demonstrated by the above embodiments, this invention provides a class of PROTAC compounds targeting lactate dehydrogenase. Compounds 7, 9, 15, 20, 21, and 22 can effectively degrade LDHA protein at the cellular level, with compounds 20, 21, and 22 exhibiting nanomolar-level degradation activity. Simultaneously, these compounds show significant inhibitory effects on the proliferation of human pancreatic cancer Panc-1 cells, with compounds 20, 21, and 22 exhibiting particularly prominent inhibitory activity. In summary, the compounds of this invention achieve highly efficient inhibition of pancreatic cancer cells by degrading LDH protein, possessing the potential to be developed into anti-pancreatic cancer drugs.

[0028] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound represented by general formula (I), characterized in that, The structure of the general formula (I) is as follows: Wherein, R is -C(O)-NH-LX; L is selected from: -(CH2) n -or-(CH2CH2O) m -CH2CH2-, where n is an integer from 2 to 12 and m is an integer from 1 to 4; X is either a CRBN ligand or a VHL ligand.

2. The compound according to claim 1, characterized in that, X is selected from the group shown in Formula A or the group shown in Formula B: Formula A; Formula B.

3. The compound according to claim 1 or 2, characterized in that, The compound is selected from any one of the following compounds: 。 4. The method for preparing the compound according to claim 3, characterized in that, Includes the following steps: The compound shown in Formula V was subjected to an amide condensation reaction with HOOC-LX in the presence of a condensing agent to obtain the compound. The compound represented by formula V is: In HOOC-LX, L is as defined in claim 1, and X is as defined in claim 2.

5. The preparation method according to claim 4, characterized in that, The conditions for the amide condensation reaction are as follows: the condensing agent is HATU, the solvent is DMSO, the reaction temperature is room temperature, and the reaction time is 4 hours.

6. A pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug of the compound of any one of claims 1 to 3.

7. A pharmaceutical composition, characterized in that, The compound comprising any one of claims 1 to 3, and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an oral preparation, an injection, or a topical preparation.

9. Use of the compound of any one of claims 1 to 3 or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating pancreatic cancer.

10. Use of the compound of any one of claims 1 to 3 in the preparation of a reagent for degrading lactate dehydrogenase protein.

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