Small-molecule inhibitor based on lactic dehydrogenase targeted design and application of small-molecule inhibitor

Abstract

The invention discloses a small-molecule inhibitor based on lactic dehydrogenase targeted design and application thereof, and belongs to the field of biological medicine, the small-molecule inhibitor is a benzenesulfonamide compound or pharmaceutically acceptable salt thereof, and the structure of the small-molecule inhibitor is shown in the specification. The lactic dehydrogenase inhibitor is suitable for treating and / or preventing diseases mediated by lactic dehydrogenase.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a small molecule inhibitor based on lactate dehydrogenase targeting design. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, involving multiple etiologies. However, liver inflammation caused by different etiologies shares the same pathological process: the transformation of liver inflammation into cancer. In a state of chronic inflammation, the inflammatory microenvironment remodels cellular energy metabolism, promotes abnormal activation of the glycolysis pathway, leading to a large accumulation of lactate. This, in turn, drives the transformation of normal hepatocytes into malignant phenotypes through mechanisms such as histone lactation modification. Lactate dehydrogenase (LDH), a key enzyme in the final stage of glycolysis, has its A subtype (LDHA) significantly expressed in HCC tissues and is closely related to tumor proliferation, invasion, metastasis, and poor patient prognosis.

[0003] The catalytic redox reaction of LDHA is a precisely coordinated process. The enzyme functions as a tetramer, with each subunit binding the coenzyme NADH. NADH binding induces a conformational change in the enzyme, creating a specific pocket for the substrate pyruvate. This mechanism involves the transfer of hydride from the C4 of the NADH nicotinamide ring to the carbonyl carbon of pyruvate. This transfer is facilitated by the active site residues in the stable transition state, resulting in the reduction of pyruvate to lactate and the oxidation of NADH to NAD. + This reaction is crucial because NAD+ is essential under conditions of high glycolysis or impaired mitochondrial function. + The regeneration of LDHA is crucial for the continuation of glycolysis. If LDHA is inactive, glycolysis will be hindered by NAD+. + The depletion of oxidative phosphorylation leads to the exhaustion of the body's energy reserves. Therefore, when oxidative phosphorylation is restricted, the primary physiological role of LDHA is to maintain the production of ATP from glycolysis. In the case of hepatocellular carcinoma, this fundamental metabolic pathway becomes a major driver of the disease. The inflammatory environment induces hypoxia and redox states in the liver, promoting the transcriptional upregulation of LDHA through hypoxia-inducible factors (HIFs) and other stress response pathways. Overexpression of LDHA leads to metabolic reorganization processes, often described as the "Warburg effect," resulting in excessive lactate production. Lactate accumulation has many detrimental consequences, such as promoting intracellular acidification, thereby promoting apoptosis and creating an environment conducive to inflammation and fibrosis. Secondly, high lactate levels have been shown to directly activate hepatic stellate cells, the primary fibrotic cells in the liver, thereby accelerating the development of liver fibrosis, a key step in the progression of cirrhosis.

[0004] Therefore, targeting and inhibiting LDHA is an attractive target, and blocking lactate accumulation has become an important strategy to prevent liver inflammation and cancer transformation. Currently, there are no FDA-approved LDHA inhibitors on the market. Reported LDHA inhibitors generally exhibit non-specificity and poor cell penetration, such as oxalate (Oxamate), which has off-target effects; and phenol (Gossypol) and its derivative FX11; and have excessively high hepatic clearance rates. Summary of the Invention

[0005] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides a small molecule inhibitor based on lactate dehydrogenase targeting design. This small molecule inhibitor is suitable for treating and / or preventing conditions or diseases mediated by lactate dehydrogenase. This invention provides small molecule compounds that inhibit lactate dehydrogenase activity, which, as small molecule inhibitors targeting protein receptors, can be used to treat, for example, hepatocellular carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, and skin cancer.

[0006] Another object of the present invention is to provide the pharmaceutical use of the small molecule inhibitor.

[0007] Technical solution: To achieve the above objective, the present invention provides a small molecule inhibitor based on lactate dehydrogenase targeting design. The small molecule inhibitor is a benzenesulfonamide compound or a pharmaceutically acceptable salt thereof, and its structure is shown in Formula I below:

[0008]

[0009] Wherein, R1 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, deuterated C1-C6 alkyl, hydroxyl, carboxyl, amino, halogen, cyano, nitro, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted phenyl, 3-8 membered saturated or partially unsaturated monocyclic carbon ring, 4-8 membered saturated or partially unsaturated monocyclic heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur, wherein the heterocycle is a heterocyclic alkane ring or a heterocyclic alkene ring, and the above substitution is that one or more hydrogen atoms on the group are substituted by any one or more substituents: halogen, alkoxy with 1-3 carbons, trifluoromethyl, trifluoromethoxy, methylthio or trifluoromethylthio.

[0010] The small molecule inhibitor is selected from any one of the following:

[0011] .

[0012] The present invention relates to the application of small molecule inhibitors based on lactate dehydrogenase targeting in the preparation of lactate dehydrogenase inhibitors.

[0013] The small molecule inhibitor has binding activity with lactate dehydrogenase, and inhibits lactate production by binding with lactate dehydrogenase.

[0014] The present invention relates to the application of small molecule inhibitors based on lactate dehydrogenase targeting in the preparation of drugs for the treatment and / or prevention of lactate dehydrogenase-mediated diseases.

[0015] The diseases mediated by lactate dehydrogenase can include diseases of tumor cell metabolism and diseases of liver cell metabolism.

[0016] The diseases mentioned include liver cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, and skin cancer.

[0017] The present invention relates to a pharmaceutical composition for treating and / or preventing lactate dehydrogenase-mediated diseases, comprising the small molecule inhibitor as an active ingredient and pharmaceutically acceptable excipients.

[0018] The pharmaceutical composition is preferably in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

[0019] Furthermore, the present invention also provides a method for preparing the above-mentioned small molecule inhibitor, which can be obtained through the following reaction route:

[0020]

[0021] Specifically, the synthesis method of intermediate A is as follows:

[0022]

[0023] Add benzenesulfonamide (10 mmol, 2.02 g), ethyl acetate (20 mL), triethylamine (25 mmol, 3.48 mL), and DMAP (0.05 mmol, 6.1 mg) to a two-necked round-bottom flask. Seal the flask with a diaphragm and protect it with a nitrogen balloon. Over 15 minutes, add 8 mL of a mixture of acyl chloride (11 mmol) with an R1 group and toluene via a syringe. React at room temperature for 1 h. Extract with ethyl acetate and water, collect the organic layer, extract with brine (30 mL), dry the organic layer on magnesium sulfate, evaporate to dryness, then purify by column chromatography (eluent: methanol: dichloromethane = 1:50) and dry to obtain intermediate A.

[0024] Synthesis method of intermediate B:

[0025]

[0026] Intermediate A (10 mmol) was placed in a single-necked flask, and 20 ml of methanol was added as a solvent. 10 mg of 10% Pd / C was added as a catalyst. After purging with hydrogen, the reaction was carried out overnight at room temperature. Once the starting material spot had completely disappeared on a TLC plate, the reaction solution was filtered through diatomaceous earth to remove Pd / C. The solvent was then evaporated under reduced pressure to obtain intermediate B.

[0027] Synthesis method of intermediate C:

[0028]

[0029] At room temperature, 1-bromo-2-naphthol (10.0 mmol, 2.23 g) was dissolved in N,N-dimethylformamide (100 mL), followed by the addition of K₂CO₃ (25.0 mmol, 3.46 g) and methyl 2-bromoacetate (38.0 mmol, 3.63 mL). The reaction mixture was heated to 65 °C and reacted for 24 hours. The reactants were then added to EtOAc:H₂O (1:1, 300 mL). The organic layer was separated and washed with H₂O (3 × 50 mL) and brine (50 mL), then dried (MgSO₄), filtered, and concentrated under reduced pressure to obtain intermediate C.

[0030] Synthesis method of intermediate D:

[0031]

[0032] The intermediate C obtained above was dissolved again in MeOH and THF (volume ratio 1:1, 20 mL), and 1 M LiOH (5.0 equivalents, 23.95 g) was added. The reaction mixture was heated to 105°C under microwave irradiation for 15 minutes. The reaction mixture was cooled in an ice bath, and 1 N HCl was added until a clear precipitate appeared. After filtration and washing with H2O (10 mL), product D was obtained.

[0033] Synthesis method of final product E

[0034]

[0035] Intermediate B (0.39 mmol) and HATU (0.39 mmol) containing R1 were added sequentially to a solution of compound D (0.36 mmol) and TEA (Et3N, triethylamine) (0.54 mmol) in N,N-dimethylformamide (3 mL). After reacting at room temperature for 16 hours, the reactants were added to EtOAc and H2O (1:1, 50 mL, v / v). The organic layer was separated and washed with water (2 × 15 mL) and brine (10 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by reversed-phase liquid chromatography (30-75% acetonitrile:water, containing 0.1% trifluoroacetic acid) to give the final product E.

[0036] Lactate dehydrogenase plays a crucial and multidimensional role in the complex pathological process of hepatocellular carcinoma. This invention relates to a small molecule inhibitor targeting lactate dehydrogenase. The invention successfully designed and synthesized a class of small molecule inhibitors of lactate dehydrogenase, which are benzenesulfonamide compounds or their pharmaceutically acceptable salts. These inhibitors can be used to treat lactate dehydrogenase-mediated diseases, and can be used to treat diseases related to tumor cell and liver cell metabolism, such as hepatocellular carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, and skin cancer. Surface plasmon resonance (SPR) experiments demonstrated that the small molecule inhibitor has a strong affinity for the target protein, and enzyme activity experiments showed that the small molecule inhibitor has high activity. It can be used to treat multiple stages of liver disease, including chronic inflammation, fibrosis, cirrhosis, and eventual malignant transformation, as well as to block the transformation from inflammation to cancer. This invention also relates to pharmaceutical compositions containing the small molecule inhibitor of this invention, providing structural optimization directions for the development of novel lactate dehydrogenase inhibitors.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] (1) This invention provides a class of novel small molecule inhibitors with a clear structure-activity relationship. These small molecule inhibitor compounds exhibit high-affinity inhibitory activity by precisely targeting the catalytic active site of lactate dehydrogenase A (LDHA) or the NADH coenzyme binding pocket, and can block the LDHA-mediated final glycolysis reaction. At the molecular level, these small molecule inhibitor compounds interfere with the characteristic aerobic glycolysis (Warburg effect) of tumor cells by competitively inhibiting the conversion of pyruvate to lactate, leading to a reduction in intracellular lactate accumulation and an NADH / NAD ratio. +Redox imbalance and energy metabolism crisis. At the cellular level, this metabolic intervention can significantly disrupt the redox homeostasis of tumor cells, triggering severe metabolic stress, which in turn activates mitochondrial apoptosis pathways (such as the release of cytochrome c and activation of caspase cascades) or death receptor pathways, ultimately significantly promoting programmed apoptosis in tumor cells. Furthermore, by inhibiting the production and functional activity of LDHA, these small molecule inhibitory compounds can also block lactate-mediated immune escape and angiogenesis in the tumor microenvironment, thereby inducing tumor cell apoptosis and inhibiting its invasion and metastasis from multiple dimensions, demonstrating excellent antitumor activity and promising clinical application prospects.

[0039] (2) The small molecule inhibitors of the present invention are easy to prepare and inexpensive, and have simple structure, ingenious design, cheap and readily available raw materials, safe and environmentally friendly synthesis process, and easy to scale up production. Detailed Implementation

[0040] The present invention will now be specifically illustrated through embodiments. In this invention, the embodiments described below are for better explanation and are not intended to limit the scope of the invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0042] Example 1

[0043] Synthesis method of intermediate A:

[0044]

[0045] Add benzenesulfonamide (10 mmol, 2.02 g), ethyl acetate (20 mL), triethylamine (25 mmol, 3.48 mL), and DMAP (0.05 mmol, 6.1 mg) to a two-necked round-bottom flask. Seal the flask with a diaphragm and protect it with a nitrogen balloon. Over 15 minutes, add 8 mL of a mixture of acyl chloride (11 mmol) with an R1 group and toluene via a syringe. React at room temperature for 1 h. Extract with ethyl acetate and water, collect the organic layer, extract with brine (30 mL), dry the organic layer on magnesium sulfate, evaporate to dryness, then purify by column chromatography (eluent: methanol: dichloromethane = 1:50) and dry to obtain intermediate A.

[0046] Synthesis method of intermediate B:

[0047]

[0048] Intermediate A (10 mmol) was placed in a single-necked flask, and 20 mL of methanol was added as a solvent. 10 mg of 10% Pd / C was added as a catalyst. After purging with hydrogen, the reaction was carried out overnight at room temperature. Once the starting material spot had completely disappeared on a TLC plate, the reaction solution was filtered through diatomaceous earth to remove Pd / C. The solvent was then evaporated under reduced pressure to obtain intermediate B.

[0049] Synthesis method of intermediate C:

[0050]

[0051] At room temperature, 1-bromo-2-naphthol (10.0 mmol, 2.23 g) was dissolved in N,N-dimethylformamide (100 mL), followed by the addition of K₂CO₃ (25.0 mmol, 3.46 g) and methyl 2-bromoacetate (38.0 mmol, 3.63 mL). The reaction mixture was heated to 65 °C and reacted for 24 hours. The reactants were then added to EtOAc and H₂O (1:1, 300 mL, v / v). The organic layer was separated and washed with H₂O (3 × 50 mL) and brine (50 mL), dried (MgSO₄), filtered, and concentrated under reduced pressure to obtain intermediate C.

[0052] Synthesis method of intermediate D:

[0053]

[0054] Intermediate C was dissolved in MeOH and THF (volume ratio 1:1, 20 mL), and 1 M LiOH (5.0 equivalents, 23.95 g) was added. The reaction mixture was heated to 105°C under microwave irradiation for 15 minutes. The reaction mixture was cooled in an ice bath, and 1 N HCl was added until a distinct precipitate appeared. After filtration and washing with H2O (10 mL), product D was given.

[0055] Synthesis method of final product E:

[0056]

[0057] Intermediate B (0.39 mmol) and HATU (0.39 mmol) with R1 were added sequentially to a solution of compound D (0.36 mmol) and TEA (Et3N, triethylamine) (0.54 mmol) in N,N-dimethylformamide (3 mL). After reacting at room temperature for 16 hours, the reactants were added to EtOAc and H2O (1:1, 50 mL). The organic layer was separated and washed with water (2 × 15 mL) and brine (10 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by reversed-phase liquid chromatography (30–75% acetonitrile:water, containing 0.1% trifluoroacetic acid) to give the final product E.

[0058] The compounds obtained through the above reaction are as follows:

[0059] By replacing the R1 acyl chloride in the synthesis of intermediate A with acetyl chloride, the parent compound was obtained: N-(4-(N-acetylsulfonyl)phenyl)-2-((1-bromonaphth-2-yl)oxy)acetamide.

[0060]

[0061] 1 H NMR (400 MHz, Chloroform-d) δ 10.50 (s, 1H), 7.95 – 7.88 (m, 2H), 7.10 (d, J = 8.6 Hz, 1H), 7.05 – 6.96 (m, 2H), 6.92 – 6.83 (m, 2H), 5.31 (s, 3H), 4.06 – 3.97 (m, 2H), 3.84 (d, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.39 (td, J= 11.7, 2.1 Hz, 2H), 2.21 (dqd, J = 11.1, 7.1, 3.4 Hz, 1H), 1.70 – 1.61 (m, 2H), 1.59 – 1.44 (m, 2H); MS (ESI) m / z Anal. Calcd for C 24 H 23 N4O5 (436.17), found:435.10 [M - H] - .

[0062] Replacing the R1 acyl chloride in intermediate A with 2-fluorobenzoyl chloride yields compound 1: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-2-fluorobenzoamide)

[0063]

[0064] 1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 10.76 (s, 1H), 8.12 (d, J= 8.5 Hz, 1H), 7.99 (dd, J-15.9, 8.6 Hz, 3H), 7.92 (d, J-8.6 Hz, 3H), 7.66-7.55 (m, 3H), 7.47 (d, J=8.5 Hz, 2H), 7.28 (dt, J=15.1, 8.4 Hz, 2H), 5.09 (s, 2H); MS (ESI) m / z Anal. Calcd for C 25 H 18 BrFN2O5S (556.01), found: 557.00 [M + H] + .

[0065] Replacing the R1 acyl chloride in intermediate A with 3-fluorobenzoyl chloride yields compound 2: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-3-fluorobenzoamide)

[0066]

[0067] 1 H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.32 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.88 – 7.79 (m, 6H), 7.65 (d, J = 8.6 Hz, 2H), 7.64 – 7.52 (m,1H), 7.51 – 7.44 (m, 2H), 7.29 (t, J = 8.7 Hz, 2H), 4.96 (s, 2H); MS (ESI) m / zAnal. Calcd for C 25 H 18 BrFN2O5S (556.01), found: 557.00 [M + H] + .

[0068] Replacing the R1 acyl chloride in intermediate A with 4-fluorobenzoyl chloride yields compound 3: N-(4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-4-fluorobenzoamide)

[0069]

[0070] 1 H NMR (400 MHz, DMSO-d6) δ 12.52 (s, 1H), 10.67 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 8.00 – 7.91 (m, 6H), 7.86 (d, J = 8.6 Hz, 2H), 7.68 – 7.61 (m,1H), 7.51 – 7.42 (m, 2H), 7.32 (t, J = 8.7 Hz, 2H), 5.04 (s, 2H); MS (ESI) m / zAnal. Calcd for C 25 H 18 BrFN2O5S (556.01), found: 557.21 [M + H] + .

[0071] Replacing the R1 acyl chloride in the synthesis of intermediate A with 4-methylbenzoyl chloride yields compound 4: N-(4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-4-methylbenzamide

[0072]

[0073] 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.68 (s, 1H), 8.12 (d, J= 8.6 Hz, 1H), 7.96 (td, J = 12.1, 10.5, 6.6 Hz, 4H), 7.86 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 7.9 Hz, 2H), 7.65 (t, J = 7.7 Hz, 1H), 7.52 – 7.42 (m, 2H), 7.29 (d, J = 7.9 Hz, 2H), 5.05 (s, 2H), 2.35 (s, 3H); MS (ESI) m / z Anal. Calcdfor C 26 H 21 BrN2O5S (553.43), found: 554.03 [M + H] + .

[0074] Replacing the R1 acyl chloride in the synthesis of intermediate A with 4-(trifluoromethyl)benzoyl chloride yields compound 5: N-(4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-4-(trifluoromethyl)benzamide

[0075]

[0076] 1 H NMR (400 MHz, DMSO-d6) δ 12.82 (s, 1H), 10.71 (s, 1H), 8.13 (d, J = 8.7 Hz, 1H), 7.99 (d, J = 9.2 Hz, 2H), 7.96 (d, J = 7.8 Hz, 2H), 7.89 (d, J= 8.7 Hz, 2H), 7.78 (t, J = 7.7 Hz, 1H), 7.73 (t, J = 8.6 Hz, 2H), 7.66 (t, J= 8.1 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.48 (t, J = 7.2 Hz, 2H), 6.63 (d, J= 8.4 Hz, 1H), 5.07 (s, 2H); MS (ESI) m / z Anal. Calcd for C 26 H 18 BrF3N2O5S (606.01), found: 607.04 [M + H] + .

[0077] Replacing the R1 acyl chloride in the synthesis of intermediate A with 2-(trifluoromethyl)benzoyl chloride yields compound 6: N-(4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-2-(trifluoromethyl)benzamide

[0078]

[0079] 1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H), 10.67 (s, 1H), 8.13 (dd, J= 8.7, 1.1 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 8.8 Hz, 3H), 7.88 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.4 Hz, 1H), 7.74 – 7.69 (m, 1H), 7.69 –7.63 (m, 2H), 7.61 – 7.54 (m, 1H), 7.49 (dd, J = 8.0, 6.1 Hz, 2H), 5.07 (s, 2H); MS (ESI) m / z Anal. Calcd for C 26 H 18 BrF3N2O5S (606.01), found: 607.04 [M + H] + .

[0080] Replacing the R1 acyl chloride in the synthesis of intermediate A with 2-thiophene carboxyl chloride yields compound 7: N-((4-(2-((1-bromonaphth-2-yl)oxy)acetamyl)phenyl)sulfonyl)thiophene-3-carboxamide)

[0081]

[0082] 1 H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.12 (dd, J = 8.6, 1.0 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.81 – 7.73 (m, 2H), 7.70 – 7.62 (m, 3H), 7.61 (s, 1H), 7.52 – 7.46 (m, 2H), 7.46 – 7.42 (m, 1H), 7.34 (dd, J = 3.6, 1.3 Hz, 1H), 6.96 (dd, J = 5.0, 3.6 Hz, 1H), 5.01 (s, 2H); MS (ESI) m / z Anal. Calcd for C 23 H 17 BrN2O5S2 (543.98), found: 544.86 [M + H]+ .

[0083] Replacing the R1 acyl chloride in intermediate A with 2-naphthoyl chloride yields compound 8: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-2-naphthamide)

[0084]

[0085] 1 H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.51-8.44 (m, 1H), 8.14-8.10 (m, 1H), 8.03-7.96 (m, 3H), 7.93 (d, J = 8.2 Hz, 1H), 7.91-7.86 (m, 1H), 7.85-7.79 (m, 3H), 7.68-7.62 (m, 3H), 7.54-7.43 (m, 4H), 5.01 (s, 2H); MS (ESI) m / z Anal. Calcd for C 29 H 21 BrN2O5S (589.46), found: 590.03 [M + H] + .

[0086] Replacing the R1 acyl chloride in the synthesis of intermediate A with 2-furanoyl chloride yields compound 9: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)furan-3-carboxamide)

[0087]

[0088] 1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.16 (t, J = 1.7 Hz, 1H), 8.10 (dd, J = 7.6, 1.4 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1W 7.84 – 7.79 (m, 2H), 7.75 – 7.70 (m, 3H), 7.55 (td, J = 7.2, 1.3 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.12 (d, J = 8.2 Hz, 1H), 7.06 (t, J = 1.6 Hz, 1H), 4.76 (s, 2H). (MS) ESI m / zAnal. Calcd for C 23 H 17 BrN2O6S(529.36), found: 528.00 [M - H] - .

[0089] Replacing the R1 acyl chloride in the synthesis of intermediate A with 2,5-difluorobenzoyl chloride yields compound 10: N-((4-(2-(((1-bromonaphthyl-2-yl)oxy)acetamido)phenyl)sulfonyl)-2,5-difluorobenzoamide)

[0090]

[0091] 1 H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.97 (q, J = 8.9 Hz, 5H), 7.88 (d, J = 8.8 Hz, 2H), 7.67 (q, J = 8.2 Hz, 1H), 7.54 – 7.41 (m, 4H), 7.38 (td, J = 9.1, 4.3 Hz, 1H), 5.06 (s, 2H); MS (ESI) m / zAnal. Calcd for C 25 H 17 BrF2N2O5S (575.38), found: 576.00 [M + H] + .

[0092] Replacing the R1 acyl chloride in the synthesis of intermediate A with 4-(trifluoromethoxy)benzoyl chloride yields compound 11: N-((4-(2-((1-bromonaphthyl-2-yl)oxy)acetamido)phenyl)sulfonyl)-4-(trifluoromethoxy)benzoamide)

[0093]

[0094] 1 H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.11 (d, J = 8.5 Hz, 2H), 7.95 (dd, J = 20.3, 8.6 Hz, 3H), 7.68 – 7.50 (m, 6H), 7.47 (dd, J = 8.3, 5.5Hz, 3H), 5.04 (s, 2H); MS (ESI) m / z Anal. Calcd for C 26 H 18 BrF3N2O6S (623.40),found: 624.00 [M + H] + .

[0095] Replacing the R1 acyl chloride in intermediate A with cyclopropylformyl chloride yields compound 12: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)cyclopropanecarboxamide)

[0096]

[0097] 1 H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.64 (s, 1H), 8.13 (dd, J= 8.6, 1.0 Hz, 1H), 7.97 (dd, J= 17.4, 8.6 Hz, 2H), 7.90 – 7.81 (m, 4H), 7.66 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.52 – 7.45 (m, 2H), 5.05 (s, 2H), 1.68 (tt, J = 8.0, 4.5 Hz, 1H), 0.84 – 0.77 (m, 2H), 0.70 (dt, J = 4.4, 3.2 Hz, 2H); MS (ESI) m / z Anal. Calcd for C 22 H 19 BrN2O5S (503.37), found: 504.35 [M + H] + .

[0098] Replacing the R1 acyl chloride in the synthesis of intermediate A with cyclobutylformyl chloride yields compound 13: N-((4-(2-(((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)cyclobutaneformamide)

[0099]

[0100] 1 H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 9.86 (s, 1H), 7.98 (dd, J= 7.4, 1.4 Hz, 1H), 7.56 (d, J= 8.2 Hz, 1H), 7.49 – 7.38 (m, 2H), 7.39 – 7.22 (m, 3H), 7.13 (td, J = 6.8, 1.4 Hz, 1H), 7.12 – 6.98 (m, 1H), 6.85 (d, 1H), 5.12 (s, 2H), 2.67 (p, J = 5.0 Hz, 1H), 2.13 – 2.04 (m, 2H), 2.04 – 1.96 (m, 2H), 1.93 – 1.83 (m, 1H), 1.83 – 1.76 (m, 1H).MS (ESI) m / z Anal. Calcd forC 23 H 21 BrN2O5S (517.39), found: 518.03 [M + H] + .

[0101] Replacing the R1 acyl chloride in the synthesis of intermediate A with trimellitic anhydride chloride yields compound 14: N-((4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamide)

[0102]

[0103] 1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 9.59 (s, 1H), 8.70 (d, J =1.9 Hz, 1H), 8.34 (dd, J = 8.2, 1.8 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.10 (dd, J = 7.6, 1.4 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.84 – 7.80 (m, 2H), 7.76 – 7.69 (m, 3H), 7.55 (td, J = 7.3, 1.4 Hz, 1H), 7.53 – 7.43 (m, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.76 (s, 2H).MS (ESI) m / z Anal. Calcd forC 27 H 17 BrN2O8S (609.40), found: 608.32 [M - H] - .

[0104] In the synthesis of intermediate A above, replacing R1 acyl chloride with benzoyl chloride yields compound 15: N-((4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)benzamide

[0105]

[0106] 1 H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.16 – 8.09 (m, 1H), 8.01 – 7.92 (m, 3H), 7.90 – 7.86 (m, 2H), 7.81 – 7.76 (m, 2H), 7.68 – 7.60 (m, 3H), 7.51 – 7.43 (m, 2H), 7.39 – 7.27 (m, 4H), 5.00 (s, 2H). MS (ESI) m / z Anal.C 25 H 19 BrN2O5S (538.02), found: 539.00[M + H] + .

[0107] In the synthesis of intermediate A above, replacing R1 acyl chloride with 3-tert-butylbenzoyl chloride yields compound 16, N-((4-(2-((1-bromonaphthyl-2-yl)oxy)acetamido)phenyl)sulfonyl)-3-(tert-butyl)benzamide

[0108]

[0109] 1 H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.96 (dd, J = 20.3, 8.6 Hz, 2H), 7.85 – 7.75 (m, 5H), 7.75 – 7.62 (m, 2H), 7.63 (d, J = 3.9 Hz, 1H), 7.52 – 7.42 (m, 2H), 7.37 – 7.29 (m, 2H), 5.01 (s, 2H). (MS) ESI m / z Anal. Calcd for C 29 H 27 BrN2O5S (595.51), found: 594.95 [M -H] - .

[0110] In the synthesis of intermediate A above, replacing R1 acyl chloride with 3,5-difluorobenzoyl chloride yields compound 17, N-((4-(2-((1-bromonaphth-2-yl)oxy)acetamido)phenyl)sulfonyl)-3,5-difluorobenzoamide.

[0111]

[0112] 1 H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 9.37 (s, 1H), 8.10 (dd, J =7.6, 1.4 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.85 – 7.79 (m, 3H), 7.76 – 7.69 (m, 4H), 7.55 (td, J = 7.3, 1.3 Hz, 1H), 7.52 – 7.46 (m, 4H), 7.15 – 7.07 (m, 3H), 4.76 (s, 2H). (MS) ESI m / z Anal. Calcd for C 25 H 17BrF2N2O5S (575.38), found:574.16 [M - H] - .

[0113] Example 2

[0114] Evaluation of lactate dehydrogenase activity inhibition capacity experiment

[0115] Detection Principle: The LDHA-catalyzed pyruvate reduction reaction is the "target reaction" for screening. The key to its detectability lies in the difference in physicochemical properties between the substrate NADH and the product NAD⁺. Substrate Binding: The active site of LDHA simultaneously binds to both pyruvate and NADH, forming an "LDHA-pyruvate-NADH" ternary complex; Hydrogen Transfer: The hydride anion of NADH (H⁺)... - The NADH is transferred to the carbonyl carbon of pyruvate, which is then reduced to lactic acid. Product release: Lactic acid and NAD⁺ dissociate from the active site of LDHA, and LDHA recovers its initial conformation and enters the next catalytic cycle. Throughout the reaction, the rate of NADH consumption is positively correlated with the enzyme activity of LDHA—the higher the enzyme activity, the more NADH is consumed per unit time, and the more NAD⁺ is generated.

[0116] Detection steps: 1. First, serially dilute the compound with DMSO; then dilute the DMSO solution 40 times with Assay buffer, and add 5 μL to the corresponding well (384 wells); 2. Dilute LDHA to 0.5 nM, add 10 μL to the corresponding well, and mix thoroughly; 3. Incubate at 25℃ for 15 min; 4. Mix: Dilute NADH to 250 μM with Assay Buffer, and simultaneously dilute sodium pyruvate to 500 μM, and mix thoroughly; set the microplate reader parameters: Exλ=340, Emλ=460, Flashes=20; Time Interval=30s, start reading the plate, and stop after 20 min.

[0117] Detection results: The inhibitory effect of the compounds on the activity of protein LDHA detected by fluorescence method is shown in Table 1.

[0118] Table 1. Inhibitory effect of compounds on protein LDHA by fluorescence assay

[0119]

[0120] The results in Table 1 show that ingredient 14 has a very good inhibitory effect on LDHA enzyme, IC50. 50 =1.9 μM, significantly superior to the commonly used experimental positive control compound FX-11, IC50 50 =22.3μM, CAS No. 213971-34-7.

[0121] Example 3

[0122] Surface plasmon resonance (SPR) experiments were used to verify the binding strength between the lactate dehydrogenase receptor and its target.

[0123] Detection Principle: Surface plasmon resonance (SPR) is a highly sensitive analytical technique based on optical principles, widely used in chemistry, biology, medicine, and materials science. In drug development and screening, it can rapidly screen the binding affinity of drug molecules to target proteins (such as receptors and enzymes) and optimize lead compounds. SPR experiments are performed on a subset of compounds from all available samples; the selected compounds cover the major substituents to ensure the representativeness of the results.

[0124] Detection steps: 1. LDHA protein fixation:

[0125] 1. Cleaning: Replace with a new CM5 chip (manufacturer: Cytiva; part number: BR-1005-30) and replace the running buffer. First, clean the new CM5 chip twice with a mixture of 400 μL of 50 mM NaOH and 4 μL of 5% SDS, then clean the CM5 chip once with 400 μL of 50 mM NaOH at a flow rate of 60 μL / min.

[0126] 2. Activation: Mix equal volumes of EDC and NHS and inject the mixture at a flow rate of 10 μL / min for 10 min to activate the Fc1 and Fc2 channels of the chip.

[0127] 3. Coupled protein: Dilute LDHA protein with 10 mM sodium acetate at pH 4.0 to a final concentration of 50 μg / mL, and then inject it multiple times into the Fc2 channel at a flow rate of 5 μL / min.

[0128] 4. Sealing: After coupling, seal the chip for 10 min with ethanolamine solution at a rate of 10 μL / min using channels Fc1 and Fc2.

[0129] II. Running buffer and sample preparation

[0130] 1. Configure running buffer and solvent calibration curves

[0131] (1) First, dilute 20×PBS with ultrapure water to 1×PBS, which contains 0.05% Tween 20. For later use, it is recorded as 1×PBST.

[0132] (2) The running buffer for small molecule samples is 1×PBST containing 5% DMSO.

[0133] Dilute 100 mL of 10×PBST with deionized water to 1 L to prepare 1×PBST. Add DMSO according to Table 2 to prepare 5% DMSO running buffer and 4% and 6% solvent correction stock solutions. Replace the original running buffer in the left tray of the system with 1×PBST containing 5% DMSO and insert the corresponding inlet tube.

[0134] Table 2 Concentration Table of PBST and DMSO

[0135]

[0136] 2. Preparation of small molecule inhibitor samples

[0137] (1) Dilute the 20 mM stock solution of the small molecule inhibitor compound of the present invention 20 times with 1×PBST buffer without DMSO to obtain 1000 μM small molecule inhibitor containing 5% DMSO in 1×PBST.

[0138] (2) The small molecule inhibitor compound was diluted to 100 μM with the prepared running buffer (1×PBST containing 5% DMSO) for single concentration detection, and 0 concentration was added.

[0139] 3. Inject running buffer and target molecules at gradient concentrations into the blank channel (the chip channel without LDHA fixation), and collect the blank signal for subsequent non-specific binding elimination. Inject running buffer into the LDHA-fixed channel, collect the baseline signal, and ensure the baseline is stable before sample detection.

[0140] 4. Set up the detection program: Association phase: Inject target molecules at gradient concentrations over 120 seconds at a flow rate of 30 μL / min (to accelerate binding and shorten experimental time); Dissociation phase: Stop target injection and continue injecting run buffer over 300 seconds at a flow rate of 30 μL / min, monitoring the dissociation of the target from the LDHA surface; Regeneration phase: Inject regeneration solution (e.g., 10 mM Glycine-HCl, pH 2.0) over 30 seconds at a flow rate of 30 μL / min to remove the target molecules bound to the chip surface, restoring LDHA binding activity and ensuring repeatability for the next round of detection. Inject target molecules sequentially from low to high concentrations, repeating the detection 2-3 times for each concentration to ensure data repeatability (coefficient of variation CV < 10%). After the experiment, rinse the chip channels with run buffer for 30 minutes and store the chip in a dry environment at 4°C for subsequent use.

[0141] 5. Data processing and fitting: Data preprocessing: Use the instrument's built-in software (Biacore InsightEvaluation) to subtract the blank channel signal and solvent background signal to obtain pure binding-dissociation curves.

[0142] 6. Dynamic model fitting: Select a suitable dynamic model based on the shape of the bonding curve.

[0143] Detection results: The results of the compounds binding with LDHA enzyme are shown in Table 3.

[0144] Table 3. Surface Plasmon Resonance (SPR) Results

[0145]

[0146] As shown in Table 3, all small molecule inhibitor compounds in this invention have a strong affinity for lactate dehydrogenase protein.

[0147] Example 4

[0148] Lactate dehydrogenase dual-concentration enzyme activity inhibition experiment

[0149] Detection principle: By setting dual substrate concentrations (high / low) or dual enzyme concentrations (high / low), the inhibitor concentration is determined, and the rate change of the LDHA catalytic reaction is monitored. The type of action and inhibitory intensity (Ki value) of the inhibitor are derived by combining the equation. The core is to eliminate the interference of a single variable through the concentration gradient and accurately characterize the interaction mechanism between the inhibitor and LDHA.

[0150] Detection steps: (1) Calibrate the UV spectrophotometer (340nm), preheat the constant temperature water bath to 37℃, and prepare sterile consumables. Prepare reagents in an ice bath: 50mmol / L Tris-HCl buffer (pH 7.4), low (0.5×Km) / high (5×Km) concentration lactate substrate, 20mmol / L NAD + (Protect from light), 0.1 mg / mL LDHA enzyme solution, gradient concentration inhibitor (DMSO ≤ 0.1%) (2) Grouped sample addition: Blank group: 60 μL buffer + 20 μL substrate + 20 μL NAD + Mix well. Negative control group: 50 μL buffer + 20 μL substrate + 20 μL NAD ++10μL enzyme solution, mix well. Inhibitor group: 40μL buffer + 20μL substrate + 20μL NAD⁺ + 10μL enzyme solution + 10μL inhibitor compound, mix well. (3) Reaction and detection: Except for the enzyme solution, all systems are preheated at 37℃ for 10 minutes. Add enzyme to start the reaction, immediately place in a spectrophotometer, and continuously detect at 340nm for 10 minutes (record the value every 30 seconds), and simultaneously detect the blank group. (4) Data processing: Correct ΔA / min = ΔA / min of each group - ΔA / min of the blank group, calculate the initial rate V0. Inhibition rate (%) = [1-(V0 of inhibitor group / V0 of negative group)]×100%. Determine the inhibition mechanism: the inhibition rate of the low substrate group > the high substrate group is competitive; the two groups are close to each other and are non-competitive; both inhibit and the high substrate cannot cancel each other out and are mixed type, and then calculate the Ki value.

[0151] Test results: The results of the dual-concentration enzyme activity inhibition experiment of lactate dehydrogenase are shown in Table 4.

[0152] Table 4. LDHA dual-concentration inhibition rate test experiment

[0153]

[0154] As shown in Table 4, compounds 8, 11-15, and 17 of this invention exhibit good enzymatic activity at 10 μM and 100 μM, and can significantly inhibit lactate dehydrogenase. Compound 14 has 100% inhibitory effect on lactate dehydrogenase at a high concentration of 100 μM, and can also significantly inhibit lactate dehydrogenase activity at 10 μM.

[0155] In summary, the small molecule inhibitor designed and prepared in this invention has a significant inhibitory effect on lactate dehydrogenase. As a small molecule inhibitor targeting protein receptors, it can be used to treat lactate dehydrogenase-mediated diseases such as hepatocellular carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, and skin cancer.

Claims

1. A small molecule inhibitor based on lactate dehydrogenase targeting design, characterized in that, The small molecule inhibitor is a benzenesulfonamide compound or a pharmaceutically acceptable salt thereof, with the structure shown in Formula I below: ； Wherein, R1 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, deuterated C1-C6 alkyl, hydroxyl, carboxyl, amino, halogen, cyano, nitro, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted phenyl, 3-8 membered saturated or partially unsaturated monocyclic carbon ring, 4-8 membered saturated or partially unsaturated monocyclic heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur, wherein the heterocycle is a heterocyclic alkane ring or a heterocyclic alkene ring, and the above substitution is that one or more hydrogen atoms on the group are substituted by any one or more substituents: halogen, alkoxy with 1-3 carbons, trifluoromethyl, trifluoromethoxy, methylthio or trifluoromethylthio.

2. The small molecule inhibitor based on lactate dehydrogenase targeting design according to claim 1, characterized in that, The small molecule inhibitor is selected from any one of the following: 。 3. The application of a small molecule inhibitor based on lactate dehydrogenase targeting design as described in claim 1 or 2 in the preparation of lactate dehydrogenase inhibitors.

4. The application according to claim 3, characterized in that, The small molecule inhibitor inhibits lactate production by binding to lactate dehydrogenase.

5. The use of a small molecule inhibitor based on lactate dehydrogenase targeting as described in claim 1 or 2 in the preparation of a medicament for treating and / or preventing lactate dehydrogenase-mediated diseases.

6. The application according to claim 5, characterized in that, The lactate dehydrogenase-mediated diseases include those involving tumor cell metabolism and those involving liver cell metabolism.

7. The application according to claim 6, characterized in that, The diseases mentioned include liver cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, and skin cancer.

8. A pharmaceutical composition for treating and / or preventing lactate dehydrogenase-mediated diseases, comprising a small molecule inhibitor as described in claim 1 or 2 as an active ingredient and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

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