2-hydroxysuccinic acid compound, pharmaceutical composition and application thereof

Abstract

The application discloses a 2-hydroxy succinic acid compound, a pharmaceutical composition and application thereof, and belongs to the field of medicine. The compound is a compound with a structure of formula I or II or an isomer, a pharmaceutically acceptable salt or a mixture thereof. The compound and the pharmaceutical composition can effectively inhibit the citric acid transport activity of HEK293 cells, are used for treating metabolic diseases and / or cardiovascular diseases, can exert a pharmaceutical effect at a molecular level, and have more excellent treatment effects, and most preferably can reach a nanomolar concentration level. In addition, the compound preparation method is simple and easy to operate.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound and a pharmaceutical composition and application thereof, in particular to a 2-hydroxy succinic acid compound and a pharmaceutical composition and application thereof. BACKGROUND

[0002] Cardiovascular disease is the first cause of death in the world, and dyslipidemia caused by high cholesterol is one of the major risks leading to the death of cardiovascular disease. Statins are the first-line drugs for hyperlipidemia, but 15% of patients have clinical defects such as statin intolerance or poor lipid-lowering effect of statins. Therefore, it is of great significance to develop safe and efficient drugs for the treatment of hyperlipidemia.

[0003] Cytoplasmic citrate is a key precursor and regulator of de novo fatty acid synthesis, and is considered to be an important intermediate connecting glucose metabolism and lipid metabolism. The concentration of citrate in the cytoplasm directly affects the rate of fat synthesis. There are mainly three sources of citrate in the cytoplasm: (1) the cytoplasmic citrate transporter (NaCT) encoded by SLC13A5 gene takes up citrate in the plasma into the cytoplasm. (2) The mitochondrial inner membrane citrate transporter (PMCT) encoded by SLC25A1 gene transports excess citrate in the tricarboxylic acid cycle to the cytoplasm. (3) Alpha-ketoglutarate (α-KG) generated by glutamine metabolism enters the tricarboxylic acid cycle to generate citrate. The citrate from the three sources all need to be decomposed by ATP-citrate lyase (ACLY) to generate acetyl-CoA and oxaloacetate, and then acetyl-CoA is used as the starting material to perform the de novo lipogenesis pathway (DNL). Therefore, inhibiting the key transporter of citrate uptake and the joint enzyme of citrate catabolism is an important target for the treatment of lipid metabolism diseases.

[0004] The citrate transporter encoded by SLC13A5 gene is also known as plasma membrane citrate transporter / sodium-dependent citrate transporter (NaCT). This carrier protein is located on the cell membrane and is responsible for the uptake of citrate in the plasma into the cytoplasm. SLC13A5 gene is highly expressed in the liver of mammals and has a certain distribution in the kidney, testis and brain. The expression level of SLC13A5 gene in patients with obesity, non-alcoholic fatty liver disease, diabetes and the like is significantly up-regulated. Mice with SLC13A5 gene knockout can avoid the occurrence and development of related metabolic diseases induced by high-fat diet, and NaCT has become an ideal target for regulating energy metabolism and lipid metabolism.

[0005] Currently, there is no NaCT inhibitor for treating hyperlipidemia successfully on the market, only Pfizer has reported PF-06649298 and PF-06761281, and BMS has reported BI-01383298, PF-06761281 can increase the concentration of citric acid in plasma and urine, so the safety needs to be further investigated, and the drug property needs to be further improved. SUMMARY

[0006] The present application aims to provide a 2-hydroxysuccinic acid compound for inhibiting NaCT, which can effectively solve the problems of insufficient NaCT inhibition activity and poor drug property of existing compounds; another object of the present application is to provide a pharmaceutical composition taking the 2-hydroxysuccinic acid compound as an active ingredient; another object of the present application is to provide an application of the 2-hydroxysuccinic acid compound in the preparation of a drug for treating diseases related to NaCT-dependent citrate transporter. + Extracellular citrate uptake inhibitors

[0007] Technical scheme: the 2-hydroxysuccinic acid compound provided by the present application is a compound having formula I or II or an isomer, a pharmaceutically acceptable salt or a mixture thereof:

[0008]

[0009] R1 is hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, pyrazolyl or phenyl;

[0010] R2 is 1-4 hydrogen R 2a substituted 6-10 membered aryl, 5-10 membered heteroaryl or tetrahydroquinolinyl, benzothiazolyl, benzoxazolyl;

[0011] R 2a is hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy;

[0012] L and M are selected from -CH2-, -NH- or -O-;

[0013] The heteroatom in the 5-10 membered heteroaryl is N, O or S, and the number of heteroatoms is 1-4.

[0014] Preferably, in the structure:

[0015] R1 is hydrogen, fluorine, cyano, methyl, ethyl, propyl, isopropyl, trifluoromethyl, isopropyl, methoxy, ethoxy, isopropoxy, pyrazolyl or phenyl;

[0016] R2 is 1-4 hydrogen R2a substituted phenyl, pyrazolyl, pyridyl or tetrahydroquinolinyl, benzothiazolyl, benzoxazolyl;

[0017] R 2a is hydrogen, fluorine, cyano, methyl, ethyl, isopropyl, tert-butyl, trifluoromethyl, dioxane, methoxy, ethoxy or isopropoxy.

[0018] Preferably, the 2-hydroxysuccinic acid compound is selected from any one of the following compounds:

[0019]

[0020]

[0021]

[0022] Preferably, the pharmaceutically acceptable salt is a salt of the above-mentioned compound with an acid or a base, the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid, the base is an inorganic base containing an alkali metal cation, an alkaline earth metal cation or an ammonium cation salt.

[0023] The above-mentioned inhibitors and pharmaceutically acceptable carriers form a pharmaceutical composition, which is prepared into a common pharmaceutical preparation, such as a tablet, a capsule, a syrup, a suspension or an injection, and the preparation can be added with a flavoring agent, a sweetener, a liquid / solid filler, a diluent and other commonly used pharmaceutical adjuvants.

[0024] The above-mentioned inhibitors and their pharmaceutical compositions can be prepared as drugs for treating diseases related to Na + dependent citrate transporter, in particular for treating hyperlipidemia, diabetes, non-alcoholic steatohepatitis or cancer.

[0025] Advantages: Compared with the prior art, the present application has the following remarkable advantages: (1) the compounds and their pharmaceutical compositions can effectively inhibit the activity of NaCT encoded by SLC13A5 gene, effectively inhibit the uptake of citrate by HEK293T cells, and the IC50 value of cell level inhibition is less than 60 nM; (2) the compounds and their pharmaceutical compositions are widely applicable, can be prepared as drugs for treating metabolic diseases such as hyperlipidemia, can exert drug efficacy at the molecular level, and the treatment effect is more excellent, and the optimal can reach the level of nanomolar concentration; (3) the compound preparation method is simple and easy to operate. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a graph showing the effect of compound LA-33 on lipid accumulation in AML12 cells;

[0027] Figure 2 Figure for the effect of compound LA-33 on SLC13A5, ACLY of AML12 cells;

[0028] Figure 3 Figure for the effect of compound LA-33 on lipid accumulation of mouse primary hepatocytes;

[0029] Figure 4 Figure for the effect of compound LA-33 on SLC13A5, ACLY of mouse primary hepatocytes;

[0030] Figure 5 Figure for the effect of compound LA-33 on the plasma lipid level of mice induced by starvation;

[0031] Figure 6 Figure for the effect of compound LA-33 on the lipid accumulation of mouse liver induced by starvation;

[0032] Figure 7 Figure for the effect of compound LA-33 on the mRNA related to the lipid accumulation of mouse liver induced by starvation. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below in combination with examples.

[0034] Example 1

[0035] Synthesis of LA-1:

[0036]

[0037] Step 1: Copper iodide (0.2 g, 1.05 mmol) was added to 60 ml THF, and triethylamine (1.13 g, 44.32 mmol) was added. The reaction solution changed from off-white turbidity to light gray slightly clear, and was replaced with nitrogen three times. Then, p-bromophenylacetylene (4 g, 22.09 mmol) and oxalyl chloride monoethyl ester (6 g, 43.94 mmol) were slowly added dropwise under ice bath. The reaction solution changed from light yellow turbidity to tan turbidity. The reaction was stopped after the reaction solution was reacted at room temperature for 16 h. 60 ml saturated sodium bicarbonate solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate three times, and the organic layer was combined and washed with saturated brine twice. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by reduced pressure concentration. Column chromatography was performed using PE:EA=60:1 to obtain 4 g of light yellow oil. 1 HNMR (300 MHz, DMSO-d6) δ 7.62-7.53 (m, 4H), 4.37 (s, 2H), 1.34 (s, 3H). HR-MS (ESI): Calculated for C 12 H 10BrO3[M+H] + : 280.9813, found 280.9803.

[0038] Step 2: Dissolve ethyl acetate (5.01 g, 56.9 mmol) in THF, cool to -78 °C, dropwise add lithium bis(trimethylsilyl)amide (56.9 ml, 56.9 mmol) reaction solution to keep -78 °C for 30 min, then dissolve 1-1 in THF and dropwise add to the reaction solution. Keep -78 °C for 1.5 h, TLC monitoring reaction complete. Stop the reaction, quench the reaction with saturated ammonium chloride, EA extraction, combined organic layer, dried over anhydrous sodium sulfate. PE:EA = 15:1 column chromatography to get 8 g of yellow oil. 1 H NMR (300 MHz, Chloroform-d) δ 7.52 - 7.42 (m, 2H), 7.37 - 7.27 (m, 2H), 4.39 (qd, J = 7.1, 1.3 Hz, 2H), 4.22 - 4.15 (m, 2H), 3.28 (d, J = 16.5 Hz, 1H), 3.14 (d, J = 16.5 Hz, 1H), 1.38 (t, J = 7.1 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H). HR-MS (ESI): Calculated for C 16 H 18 BrO5[M+H] + : 369.0338, found 369.0366.

[0039] Step 3: Dissolve 1-2 (0.50 g, 1.35 mmol) and 2-methoxypyridine-4-boronic acid pinacol ester (0.36 g, 1.62 mmol) in 7 ml solvent (dioxane: water = 6:1), add anhydrous sodium carbonate (0.43 g, 4.06 mmol), nitrogen replacement three times, then add Pd(dppf)Cl2(0.10 g, 0.14 mmol), nitrogen replacement three times, then react at 95 °C for 5 h, TLC detection reaction complete. The reaction solution is cooled to room temperature, then suction filtration, the filtrate is concentrated, add ethyl acetate, wash with water twice, saturated brine once, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure, use PE:EA = 10:1 column chromatography to get 200 mg of light yellow oil.

[0040] 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 5.0 Hz, 1H), 7.80 - 7.70 (m, 2H), 7.61 - 7.51 (m, 2H), 7.51 (dd, J = 5.1, 1.0 Hz, 1H), 7.08 (d, J = 1.0 Hz, 1H), 4.31 (s, 2H), 4.10 (s, 2H), 3.89 (s, 3H), 2.82 (d, J = 0.4 Hz, 2H), 1.25 (d, J = 15.6 Hz, 6H). HR-MS (ESI): Calcd for C 22 H 24 NO6[M+H] + :398.1604, found 398.1600.

[0041] Step 4: Intermediate 1-3 was dissolved in 30 ml anhydrous ethanol, 5 ml Raney nickel catalyst was added, hydrogen was replaced three times and the reaction was carried out overnight. After TLC detection of complete reaction, the reaction liquid was filtered, the filtrate was concentrated, and the colorless oil 120 mg was obtained by silica gel column purification. 1 H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 5.0 Hz, 1H), 7.80 - 7.70 (m, 2H), 7.61 - 7.51 (m, 2H), 7.51 (dd, J = 5.1, 1.0 Hz, 1H), 7.08 (d, J = 1.0 Hz, 1H), 4.31 (s, 2H), 4.10 (s, 2H), 3.89 (s, 3H), 2.82 (d, J = 0.4 Hz, 2H), 1.25 (d, J = 15.6 Hz, 6H). HR-MS (ESI): Calcd for C 22 H 28 NO6[M+H] + :402.1917, found 402.1933.

[0042] Step 5: Intermediate 1-4 was dissolved in 5 ml anhydrous ethanol, 1.5 ml 1N NaOH solution was added, and the reaction was carried out at room temperature overnight. After HPLC detection of complete reaction, the solvent was evaporated under reduced pressure, and the PH was adjusted to 2-3 using 1 mol / L hydrochloric acid. The water layer was extracted with ethyl acetate (3 x 5 ml), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain 70 mg of white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 5.0 Hz, 1H), 7.58 - 7.47 (m, 3H), 7.27 - 7.16 (m, 2H), 7.08 (d, J = 1.0 Hz, 1H), 5.14 (s, 1H), 3.88 (s, 3H), 2.82 - 2.66 (m, 2H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H). HR-MS (ESI): Calculated for C 18 H 20 NO6[M+H] + : 346.1291, found 346.1308.

[0043] Using similar procedures as in Example 1, the following compounds were prepared:

[0044]

[0045] 1 H NMR (300 MHz, DMSO-d6) δ 7.59 (d, J = 8.1 Hz, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.31 - 7.04 (m, 4H), 6.93 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 3.83 (s, 3H), 2.79 (t, J = 12.1 Hz, 2H), 2.57 (d, J = 15.7 Hz, 1H), 2.45 (dd, J = 13.5, 5.3 Hz, 1H), 2.01 - 1.83 (m, 2H). HR-MS (ESI): Calculated for C 19 H 21 O6[M+H] + : 345.1338, found 345.1372.

[0046]

[0047] 1 H NMR (300 MHz, DMSO-d6) δ 7.60 - 7.50 (m, 2H), 7.40 (dt, J = 7.4, 2.0 Hz, 1H), 7.35 - 7.16 (m, 4H), 6.96 (dt, J = 7.4, 2.0 Hz, 1H), 5.14 (s, 1H), 4.08 (s, 2H), 2.82 - 2.66 (m, 2H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H), 1.40 (s, 3H). HR-MS (ESI): Calculated for C 20 H 23O6[M+H] + : 359.1495, found 359.1507

[0048]

[0049] 1 H NMR (300 MHz, DMSO-d6) δ 7.26 (q, J = 8.2 Hz, 4H), 6.86 (dd, J = 7.4, 1.6 Hz, 1H), 6.76 (dd, J = 7.5, 1.7 Hz, 1H), 6.52 (t, J = 7.4 Hz, 1H), 3.16 (t, J = 5.5 Hz, 2H), 2.85 - 2.69 (m, 4H), 2.58 (d, J = 15.6 Hz, 1H), 2.45 (dd, J = 13.3, 5.4 Hz, 1H), 2.04 - 1.74 (m, 4H). HR-MS (ESI): Calculated for C 21 H 24 NO5[M+H] + : 370.1654, found 370.1660.

[0050]

[0051] 1 H NMR (300 MHz, DMSO-d6) δ 7.50 - 7.41 (m, 3H), 7.30 (d, J = 7.9 Hz, 2H), 6.37 (d, J = 1.9 Hz, 1H), 3.85 (s, 3H), 2.87 - 2.75 (m, 2H), 2.61 (s, 1H), 1.94 (qt, J = 14.0, 6.3 Hz, 2H). HR-MS (ESI): Calculated for C 16 H 19 N2O5[M+H] + : 319.1294, found 319.1300.

[0052]

[0053] 1H NMR (300 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.82 (s, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 4.15 (q, J = 7.3 Hz, 2H), 2.80 (d, J = 15.7 Hz, 1H), 2.73 - 2.64 (m, 1H), 2.59 (s, 1H), 2.40 (dd, J = 13.5, 5.5 Hz, 1H), 2.01 - 1.77 (m, 2H), 1.41 (t, J = 7.3 Hz, 3H). HR-MS (ESI): Calcd. for C 17 H 22 N2O5[M+H] + : 333.1450, found 333.1455.

[0054]

[0055] 1 H NMR (300 MHz, DMSO-d6) δ 7.70 (dd, J = 6.5, 1.0 Hz, 3H), 7.23 - 7.13 (m, 2H), 6.82 (s, 1H), 5.14 (s, 1H), 2.71 (d, J = 12.4 Hz, 1H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H). HR-MS (ESI): Calcd. for C 15 H 17 N2O5[M+H] + : 305.1137, found 305.1144.

[0056]

[0057] 1 H NMR (300 MHz, DMSO-d6) δ 7.80 - 7.70 (m, 2H), 7.63 (s, 1H), 7.28 - 7.18 (m, 2H), 6.80 (s, 1H), 5.14 (s, 1H), 4.52 (s, 1H), 2.71 (d, J = 12.4 Hz, 1H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H), 1.44 (d, J = 15.1 Hz, 5H). HR-MS (ESI): Calcd. for C 18 H 23 N2O5[M+H] + : 347.1607, found 347.1618.

[0058]

[0059] 1 H NMR (300 MHz, Methanol-d4) δ 7.98 (s, 1H), 7.58 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.28 (dd, J = 8.7, 1.6 Hz, 1H), 3.10 - 2.86 (m, 2H), 2.81 - 2.56 (m, 2H), 2.09 (dtd, J = 26.0, 13.5, 6.7 Hz, 2H). HR-MS (ESI): calculated for C 13 H 15 N2O5[M+H] + : 279.0981, found 279.0996.

[0060]

[0061] 1 H NMR (300 MHz, DMSO-d6) δ 7.60 - 7.50 (m, 2H), 7.42 - 7.30 (m, 2H), 7.27 - 7.16 (m, 2H), 6.89 (d, J = 7.5 Hz, 1H), 6.04 (d, J = 0.7 Hz, 2H), 5.14 (s, 1H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H). HR-MS (ESI): calculated for C 19 H 19 O7[M+H] + : 359.1131, found 359.1144.

[0062]

[0063] 1 H NMR (300 MHz, DMSO-d6) δ 7.50 (h, J = 4.6, 3.8 Hz, 4H), 7.39 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 2.81 (d, J = 15.6 Hz, 2H), 2.60 (s, 1H), 2.44 (d, J = 5.1 Hz, 1H), 1.94 (dq, J = 12.4, 6.9, 6.0 Hz, 2H). HR-MS (ESI): calculated for C 19 H 19 O7[M+H] + : 399.1055, found 399.1068.

[0064]

[0065] 1 H NMR (300 MHz, DMSO-d6) δ 7.39 (d, J = 8.0 Hz, 2H), 7.30 (dd, J = 15.3, 7.6 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 7.15 - 6.97 (m, 2H), 3.76 (s, 3H), 2.87 (d, J = 19.9 Hz, 1H), 2.81 - 2.69 (m, 2H), 2.60 (s, 1H), 1.93 (qd, J = 14.0, 13.0, 5.9 Hz, 2H). HR-MS (ESI): Calcd. for C 19 H 21 O6[M+H] + : 345.1338, Found: 345.1372.

[0066]

[0067] 1 H NMR (300 MHz, Chloroform-d) δ 7.54 (d, J = 7.8 Hz, 2H), 7.37 (d, J = 7.9 Hz, 1H), 7.28 (d, J = 7.8 Hz, 2H), 7.22 - 7.10 (m, 2H), 6.91 (dd, J = 8.2, 2.5 Hz, 1H), 4.12 (q, J = 6.9 Hz, 2H), 3.16 (d, J = 17.0 Hz, 1H), 2.93 (q, J = 11.1 Hz, 2H), 2.68 (t, J = 12.3 Hz, 1H), 2.15 (t, J = 12.0 Hz, 2H), 1.48 (d, J = 13.9 Hz, 3H). HR-MS (ESI): Calcd. for C 20 H 23 O6[M+H] + : 359.1495, Found: 359.1508.

[0068]

[0069] 1H NMR (300 MHz, DMSO-d6) δ 7.61 (dd, J = 7.5, 2.0 Hz, 1H), 7.54 - 7.45 (m, 2H), 7.40 (td, J = 7.5, 2.0 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.11 (td, J = 7.5, 2.0 Hz, 1H), 6.95 (dd, J = 7.5, 2.0 Hz, 1H), 5.14 (s, 1H), 4.68 (s, 1H), 2.82 - 2.66 (m, 2H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H), 1.35 (d, J = 14.9 Hz, 5H). HR-MS (ESI): Calcd. for C 21 H 25 O6[M+H] + :373.1651, found 373.1673.

[0070]

[0071] 1 H NMR (300 MHz, DMSO-d6) δ 7.58 (d, J = 8.2 Hz, 2H), 7.35 (t, J = 7.9 Hz, 1H), 7.29 - 7.11 (m, 4H), 6.91 (dd, J = 8.2, 2.4 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 2.84 - 2.68 (m, 2H), 2.60 (s, 1H), 2.01 - 1.81 (m, 2H), 1.75 (p, J = 7.1 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). HR-MS (ESI): Calcd. for C 21 H 25 O6[M+H] + :373.1651, found 373.1679.

[0072]

[0073] 1H NMR (300 MHz, DMSO-d6) δ 7.84 (dd, J = 7.5, 2.0 Hz, 1H), 7.69 (td, J = 7.3, 2.0 Hz, 1H), 7.63 (dd, J = 7.5, 2.4 Hz, 1H), 7.58 - 7.45 (m, 3H), 7.25 - 7.14 (m, 2H), 5.14 (s, 1H), 2.82 - 2.66 (m, 2H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H). HR-MS (ESI): calculated for C 19 H 18 F3O5[M+H] + :383.1106, found 383.1116.

[0074]

[0075] 1 H NMR (300 MHz, DMSO-d6) δ 7.84 (dd, J = 7.5, 2.0 Hz, 1H), 7.69 (td, J = 7.3, 2.0 Hz, 1H), 7.63 (dd, J = 7.5, 2.4 Hz, 1H), 7.58 - 7.45 (m, 3H), 7.25 - 7.14 (m, 2H), 5.14 (s, 1H), 2.82 - 2.66 (m, 2H), 2.59 - 2.41 (m, 2H), 2.20 - 2.02 (m, 2H). HR-MS (ESI): calculated for C 19 H 18 F3O5[M+H] + :383.1106, found 383.1119.

[0076] Example 2

[0077] Synthesis of LA-18:

[0078]

[0079] Synthesis of Intermediate 2-2:

[0080] Cuprous iodide (0.34 g, 3.57 mmol) was added to 60 ml THF, triethylamine (7.22 g, 0.071 mol) was added, the reaction solution changed from off-white turbidity to light gray slightly clear, replaced with nitrogen three times, added (triethylsilyl) acetylene (5 g, 0.036 mol) and oxalyl chloride monoethyl ester (9.7 g, 0.076 mol) dropwise under ice bath, the reaction solution changed to light yellow turbidity, the reaction solution was stopped after 16 h reaction at room temperature. 60 ml saturated sodium bicarbonate solution was added to the reaction solution, extracted with ethyl acetate three times, the organic layer was combined, washed with saturated brine twice, dried with anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and column chromatography was performed using PE:EA=60:1 to obtain 9 g of light yellow oil. Yield 76%. 1 H NMR (300 MHz, DMSO-d6) δ 4.34 (s, 2H), 1.32 (s, 3H), 1.03 (s, 9H), 0.94 (s, 6H). HR-MS (ESI): Calculated for C 12 H 20 NaO3Si[M+Na] + : 263.1079, found 263.1088.

[0081] Synthesis of intermediate 2-3:

[0082] Ethyl acetate (1.12 g, 12.69 mmol) was dissolved in THF, cooled to -78°C, and LiHMDS (12.69 ml, 12.69 mmol) was added dropwise. The reaction solution was kept at -78°C for 30 min, and then intermediate 2b (2.70 g, 7.93 mmol) was dissolved in THF and added dropwise to the reaction solution. The reaction was kept at -78°C for 1.5 h, and TLC monitoring showed that the reaction was complete. The reaction was stopped, quenched with saturated ammonium chloride, extracted with EA, and the organic layer was combined and dried over anhydrous sodium sulfate. Column chromatography was performed using PE:EA=15:1 to obtain 1.3 g of yellow oil. Yield 34%. 1 H NMR (300 MHz, DMSO-d6) δ 4.10 (s, 2H), 3.69-3.53 (m, 2H), 2.65 (s, 2H), 1.22 (s, 3H), 1.14 (s, 3H), 1.03 (s, 9H), 0.87 (s, 6H). HR-MS (ESI): Calculated for C 16 H 30 NaO4Si[M+Na] + : 337.1811, found 337.1816.

[0083] Synthesis of intermediate 2-4:

[0084] Intermediate 2c (1.3 g, 3.95 mmol) was dissolved in 15 ml of anhydrous ether, and tetrabutylammonium fluoride (5.94 ml, 5.94 mmol) was added dropwise with ice bath. The reaction was monitored by TLC for 1.5 h until completion. The reaction was quenched with 10 ml of saturated ammonium chloride solution, and the reaction was separated into layers. The aqueous layer was extracted with ether three times, and the organic layer was combined, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography to give a yellow oil, 0.63 g, 74% yield. 1 H NMR (300 MHz, DMSO-d6) δ 6.66 (s, 1H), 4.31 (s, 2H), 4.10 (s, 2H), 3.65 (s, 1H), 2.76 (d, J = 0.3 Hz, 3H), 1.25 (d, J = 15.6 Hz, 6H). HR-MS (ESI): Calculated for C 10 H 14 NaO5[M+Na] + : 237.0739, found 237.0749.

[0085] Synthesis of intermediate 2-5:

[0086] 4-(4-iodophenyl)morpholine (0.50 g, 1.73 mmol) was dissolved in 5 ml of tetrahydrofuran, and cuprous iodide (33 mg, 0.17 mmol), 0.7 ml of triethylamine were added. The reaction was replaced with nitrogen three times, and Pd(PPh3)4 (200 mg, 0.17 mmol) was added. The reaction was replaced with nitrogen again, and intermediate 2d (440 mg, 2.07 mmol) was added dropwise when the reaction temperature reached 60 °C. The reaction was monitored by TLC for 1.5 h until completion. The reaction was cooled to room temperature, filtered, and the filtrate was separated into layers. The aqueous layer was extracted with ethyl acetate, washed with saturated brine twice, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography to give a yellow solid, 0.64 g. 1 H NMR (300 MHz, DMSO-d6) δ 7.52 - 7.42 (m, 2H), 7.04 - 6.94 (m, 2H), 4.31 (s, 2H), 4.10 (s, 2H), 3.74 (d, J = 3.6 Hz, 4H), 3.18 (d, J = 1.1 Hz, 4H), 2.82 (d, J = 0.4 Hz, 2H), 1.25 (d, J = 15.6 Hz, 6H). HR-MS (ESI): Calculated for C 20 H 26 NO6[M+H] + : 376.1760, found 376.1772.

[0087] Synthesis of intermediate 2-6:

[0088] Intermediate 2-5 (0.60 g, 1.60 mmol) was dissolved in 30 ml of solvent (methanol:tetrahydrofuran = 6:1), 3 ml of Raney nickel was added, and the atmosphere was replaced with hydrogen three times. The reaction was allowed to react at room temperature overnight. After the reaction was completed as monitored by TLC, the reaction solution was filtered and the solvent was removed under reduced pressure to obtain 0.6 g of an off-white solid. 1 H NMR (300MHz, DMSO-d6) δ7.16–7.05(m,2H),6.87–6.77(m,2H),4.17(d,J=0.6Hz,2H),4.09(s,2H),3.74(d,J=3.6Hz,4H),3.19(d ,J=1.7Hz,4H),2.83–2.67(m,2H),2.59–2.40(m,2H),2.13(d,J=0.8Hz,2H),1.22(s,3H),1.14(s,3H).HR-MS(ESI):Calculated for C 20 H 30 NO6[M+H] + :380.2073,found 380.2079.

[0089] Synthesis of LA-18:

[0090] Intermediate 2-6 (0.50 g, 1.32 mmol) was dissolved in 8 ml of anhydrous ethanol, and 5 ml of 1 mol / L NaOH solution was added. The mixture was stirred at room temperature for 16 h, and the reaction was complete after TLC monitoring. The solvent was evaporated under reduced pressure, and the pH was adjusted to 2-3 by adding 1 mol / L HCl solution. The mixture was extracted three times with ethyl acetate (3 × 5 ml). The combined organic layers were washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to yield 300 mg of a white solid. 1 H NMR(300MHz,Deuterium Oxide)δ7.15(d,J=8.3Hz,2H),7.00–6.91(m,2H),3.79(dd,J=6.2,3.4Hz,4H),3.07–2.98(m,4H),2. 68–2.52(m,2H),2.41–2.21(m,2H),1.76(dtd,J=41.7,13.3,4.8Hz,2H).HR-MS(ESI):Calculatedfor C 16 H 22 NO6[M+H] + :324.1447,found 324.1459.

[0091] The following compounds were obtained by similar procedures to those in Example 2:

[0092]

[0093] 1 H NMR(300MHz,DMSO-d6)δ7.10(t,J=0.5Hz,4H),5.14(s,1H),2.82–2.63(m,4H),2.55–2.46(m,2H),2.20–2.02(m,2H),1.23(s,3H).HR-MS(ESI):Calculated for C 14 H 19 O5[M+H] + :267.1232,found 267.1246.

[0094]

[0095] 1 H NMR(300MHz,DMSO-d6)δ7.10(tdt,J=7.7,6.9,0.9Hz,4H),5.14(s,1H),2.90(t,J=0.9Hz,1H),2.74(d,J=5.5Hz,2H),2.59–2.42(m,2H),2.20–2.02(m,2H),1.25(d,J=15.1Hz,6H).HR-MS(ESI):Calculated for C 15 H 21 O5[M+H] + :281.1389,found 281.1366.

[0096]

[0097] 1 H NMR(300MHz,DMSO-d6)δ7.70–7.60(m,2H),7.49–7.39(m,2H),5.14(s,1H),2.82–2.66(m,2H),2.59–2.41(m,2H),2.20–2.02(m,2H).HR-MS(ESI):Calculated forC 13 H 14 F3O5[M+H] + :307.0793,found 307.0801.

[0098]

[0099] 1H NMR (300 MHz, Chloroform-d) δ 7.26 - 7.09 (m, 4H), 5.14 (s, 1H), 2.77 (d, J = 12.4 Hz, 1H), 2.71 (d, J = 12.4 Hz, 1H), 2.59 - 2.46 (m, 2H), 2.46 (dt, J = 7.7, 1.0 Hz, 1H), 2.20 - 2.02 (m, 2H), 1.73 (dd, J = 16.9, 13.0 Hz, 4H), 1.59 (dd, J = 13.0, 10.1 Hz, 4H). HR-MS (ESI): calculated for C 17 H 23 O5[M+H] + : 307.1575, found 307.1554.

[0100]

[0101] 1 H NMR (300 MHz, Chloroform-d) δ 7.16 - 7.06 (m, 2H), 6.84 - 6.74 (m, 2H), 3.43 - 3.29 (m, 4H), 2.89 - 2.71 (m, 2H), 2.59 (dt, J = 12.4, 1.0 Hz, 1H), 2.51 (dt, J = 12.4, 1.0 Hz, 1H), 2.16 (d, J = 12.4 Hz, 1H), 2.08 - 1.98 (m, 3H). HR-MS (ESI): calculated for C 16 H 22 O5[M+H] + : 308.1498, found 308.1495.

[0102]

[0103] 1 H NMR (300 MHz, Chloroform-d) δ 7.26 - 7.09 (m, 4H), 2.89 - 2.71 (m, 2H), 2.61 - 2.42 (m, 3H), 2.19 (d, J = 12.3 Hz, 1H), 2.08 (s, 1H), 1.80 (d, J = 13.1 Hz, 2H), 1.70 (d, J = 13.0 Hz, 2H), 1.61 - 1.31 (m, 6H). HR-MS (ESI): calculated for C 18 H 25 O5[M+H] +:321.1702, found 321.1718.

[0104]

[0105] 1 H NMR (300 MHz, Chloroform-d) δ 7.17 - 7.06 (m, 2H), 6.84 - 6.74 (m, 2H), 3.19 (d, J = 13.0 Hz, 4H), 2.84 (d, J = 12.4 Hz, 1H), 2.76 (d, J = 12.4 Hz, 1H), 2.56 (dd, J = 12.0, 3.8 Hz, 7H), 2.29 (s, 3H), 2.16 (d, J = 12.4 Hz, 1H), 2.03 (d, J = 12.4 Hz, 1H). HR-MS (ESI): Calculated for C 16 H 23 N2O5[M+H] + :323.1607, found 323.1608.

[0106]

[0107] 1 H NMR (300 MHz, Chloroform-d) δ 7.17 - 7.06 (m, 2H), 6.84 - 6.74 (m, 2H), 3.19 (d, J = 13.0 Hz, 4H), 2.84 (d, J = 12.4 Hz, 1H), 2.76 (d, J = 12.4 Hz, 1H), 2.56 (dd, J = 12.0, 3.8 Hz, 7H), 2.29 (s, 3H), 2.16 (d, J = 12.4 Hz, 1H), 2.03 (d, J = 12.4 Hz, 1H). HR-MS (ESI): Calculated for C 17 H 25 N2O5[M+H] + :337.1763, found 337.1766.

[0108]

[0109] 1H NMR (300 MHz, Chloroform-d) δ 7.15 - 7.05 (m, 2H), 6.86 - 6.76 (m, 2H), 3.78 (s, 3H), 2.89 - 2.71 (m, 2H), 2.55 (qt, J = 12.4, 1.0 Hz, 2H), 2.16 (d, J = 12.4 Hz, 1H), 2.03 (d, J = 12.4 Hz, 1H). HR-MS (ESI): calculated for C 13 H 17 O6[M+H] + : 269.1025, found 269.1033.

[0110]

[0111]

[0112] 1 H NMR (300 MHz, Chloroform-d) δ 7.17 - 7.06 (m, 2H), 6.85 - 6.74 (m, 2H), 4.08 (s, 2H), 2.84 (d, J = 12.4 Hz, 1H), 2.76 (d, J = 12.4 Hz, 1H), 2.55 (qt, J = 12.4, 1.0 Hz, 2H), 2.16 (d, J = 12.4 Hz, 1H), 2.03 (d, J = 12.4 Hz, 1H), 1.42 (s, 3H). HR-MS (ESI): calculated for C 14 H 19 O6[M+H] + : 283.1182, found 283.1189.

[0113]

[0114] 1 H NMR (300 MHz, Chloroform-d) δ 7.15 (d, J = 7.4 Hz, 1H), 6.94 (dtt, J = 7.5, 2.0, 1.0 Hz, 1H), 6.88 - 6.76 (m, 2H), 3.81 (s, 3H), 2.89 - 2.71 (m, 2H), 2.57 (dt, J = 12.4, 1.0 Hz, 1H), 2.47 (dt, J = 12.4, 1.0 Hz, 1H), 2.16 (d, J = 12.4 Hz, 1H), 2.04 (d, J = 12.4 Hz, 1H). HR-MS (ESI): calculated for C 13 H17 O6[M+H] + :269.1025, found 269.1033.

[0115]

[0116] 1 H NMR (300 MHz, DMSO-d6) δ 7.18 (t, J = 7.4 Hz, 1H), 6.95 (dtt, J = 7.6, 2.0, 1.0 Hz, 1H), 6.88 - 6.75 (m, 2H), 4.07 (s, 2H), 2.82 - 2.66 (m, 2H), 2.56 (dt, J = 12.4, 1.0 Hz, 1H), 2.47 (dt, J = 12.3, 1.0 Hz, 1H), 2.10 (d, J = 12.4 Hz, 1H), 2.00 (d, J = 12.4 Hz, 1H), 1.39 (s, 3H). HR-MS (ESI): Calculated for C 14 H 19 O6[M+H] + :283.1182, found 283.1185.

[0117]

[0118] 1 H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 7.9 Hz, 1H), 8.03 (dd, J = 11.4, 8.0 Hz, 3H), 7.55 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.9 Hz, 2H), 2.81 (s, 1H), 2.76 (s, 1H), 2.58 (d, J = 15.5 Hz, 2H), 1.95 (dq, J = 12.4, 7.0, 6.2 Hz, 2H). HR-MS (ESI): Calculated for C 19 H 18 NO5S[M+H] + :372.0906, found 372.0916.

[0119]

[0120] 1H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 7.9 Hz, 1H), 8.01 (dd, J = 11.4, 8.0 Hz, 3H), 7.55 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.9 Hz, 2H), 2.81 (s, 1H), 2.76 (s, 1H), 2.58 (d, J = 15.5 Hz, 2H), 1.95 (dq, J = 12.4, 7.0, 6.2 Hz, 2H). HR-MS (ESI): Calcd. for C 19 H 18 NO6[M+H] + : 356.1155.

[0121]

[0122] 1 H NMR (300 MHz, DMSO-d6) δ 7.69 - 7.54 (m, 4H), 7.46 (t, J = 7.6 Hz, 2H), 7.36 (dd, J = 8.4, 6.2 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 2.82 (d, J = 15.7 Hz, 2H), 2.61 (s, 1H), 2.51 - 2.40 (m, 1H), 2.05 - 1.81 (m, 2H).. HR-MS (ESI): Calcd. for C 18 H 19 O5[M+H] + : 315.1239.

[0123]

[0124] 1 H NMR (300 MHz, DMSO-d6) δ 7.89 (d, J = 5.1 Hz, 3H), 7.50 (d, J = 5.1 Hz, 3H), 7.28 (d, J = 0.6 Hz, 1H), 2.78 - 2.54 (m, 5H), 2.11 (d, J = 12.4 Hz, 1H), 2.00 (d, J = 12.3 Hz, 1H). HR-MS (ESI): Calcd. for C 16 H 17 O5[M+H] + : 289.1077.

[0125] Example 3

[0126] The present application part of the compounds to high expression of SLC13A5 HEK-293T extracellular D4-citrate uptake inhibition experiment:

[0127] 1. Experimental method

[0128] (1) Cell recovery and culture: the HEK293T cells taken from liquid nitrogen were quickly placed in a 37°C water bath for about 1 min, then transferred to a centrifuge tube containing 5 mL of culture medium, 1100 rpm, centrifuged for 3 min, the supernatant was discarded, 2 mL of 10% FBS DMEM high glucose medium was added, 1 mL was inoculated into a cell culture dish containing 7 mL of medium, and the cell culture dish was placed in a 5% CO2, 37°C cell incubator, and the cells were grown as a monolayer. When the cells were about 90% confluent, the culture medium was discarded, washed twice with PBS preheated at 37°C, 2 mL of 0.25% trypsin was added and incubated at room temperature, gently shaken for 30 s, the trypsin was discarded, 2 mL of fresh medium was added to terminate the reaction, and the medium containing the cells was transferred to a sterile Ep tube and centrifuged at 800 rpm for 3 min. After centrifugation, the supernatant was discarded, 2 mL of 10% FBS DMEM high glucose medium was added, and the cells were inoculated into a cell culture dish and incubated in a 5% CO2, 37°C cell incubator.

[0129] (2) Transfection: the original culture medium was replaced with double-antibody-free serum-free high-glucose DMEM medium, and the SLC13A5 overexpression plasmid and Lipofectamine TM 2000 transfection reagent were diluted with Opti-MEM serum-free medium, respectively. The overexpression plasmid and Lipofectamine TM 2000 were added to a 24-well cell culture plate, gently shaken to mix, and incubated in a cell culture incubator.

[0130] (3) Uptake: 24 h after transfection, the original medium in 24-well plates was aspirated, 1 mL of sodium buffer was added to each well and washed three times, then 250 μL of sodium buffer containing different concentrations of compounds was added, and the plate was placed in a shaking incubator, the temperature was 37°C, and the pre-warming incubation time was 30 min; the sodium buffer containing the compound was aspirated, 1 mL of sodium buffer was added to wash three times; 250 μL of sodium buffer containing the above different concentrations of compound and 200 μM D4-citrate was added to each well, and the temperature was 37°C, and the uptake time was 30 min; the sodium buffer containing D4-citrate was discarded, 1 mL of choline buffer was added to each well to wash three times to terminate the uptake; after the reaction was completed, 200 μL of double distilled water was added to each well, and the plate was placed in a -80°C freezer for 30 min, then thawed at room temperature, and the freeze-thawing was repeated for 3 times, and the cells were fully broken by ultrasonic treatment for 10 min. The solution containing the broken cells was transferred to an Ep tube, and an internal standard acetonitrile was added, centrifuged at 12000 rpm for 10 min, and the supernatant was detected by LC-MS / MS to detect the intracellular D4-citrate concentration, and the HEK-293T-vector group was subtracted to calculate the net content.

[0131] 2. Data processing

[0132] (1) Calculation formula of citrate uptake experiment

[0133] % Inhibition = [1-(A_sample / A_max)]

[0134] Where: A_sample represents the content of D4-citrate in the sample, and A_max represents the content of D4-citrate in the blank.

[0135] (2) Fitting dose-effect curve: taking the log value of concentration as the X axis and the percentage inhibition rate as the Y axis, using the analysis software GraphPad Prism5 to fit the dose-effect curve of log(inhibitor) vs. response-Variable slope, and thus the IC 50 value of each compound to citrate uptake activity was obtained.

[0136] Calculation formula:

[0137] Y = Bottom + (Top-Bottom) / (1+10^((Log IC 50 -X)×Hill Slope)).

[0138] IC 50 The data are shown in Table 1.

[0139] Table 1. Inhibition activity of compounds on citrate uptake of HEK293 cells

[0140] No. Citrate uptake inhibitory activity No. Citrate uptake inhibitory activity LA-1 A LA-2 A LA-3 A LA-4 A LA-5 A LA-6 A LA-7 A LA-8 A LA-9 B LA-10 C LA-11 A LA-12 B LA-13 A LA-14 A LA-15 A LA-16 A LA-17 A LA-18 A LA-19 B LA-20 A LA-21 B LA-22 A LA-23 C LA-24 A LA-25 A LA-26 A LA-27 B LA-28 C LA-29 A LA-30 B LA-31 B LA-32 C LA-33 C LA-34 C

[0141] Note: A: <1 μM, B: 1-5 μM, C: >5 μM.

[0142] As shown in Table 1, all the tested compounds have good inhibitory effect on citrate transporter of HEK293 cells, and all the compounds have micromolar inhibitory rate on NaCT, in which the compound LA-33 has the best inhibitory rate on citrate uptake of HEK293T cells, and the IC 50 = 60 nM.

[0143] Example 4

[0144] In vitro pharmacodynamic experiment of representative compound LA-33 of the present application on AML12 cells

[0145] Experimental procedure:

[0146] The AML12 cells were cultured in DMEM / F12 high glucose medium containing 10% FBS and placed in a cell incubator at 37°C containing 5% CO2. First, the cells were pre-protected with serum-free medium containing different concentrations of compounds for 30 min, and then stimulated with a mixture of PA and OA (designated as OPA, PA / OA, 1:4) at 300 μM for 24 hours to develop a lipid accumulation model. In order to simulate the physiological state of endogenous extracellular matrix, an aliquot of 200 μM citrate was supplemented to the culture medium. The AML12 cells were incubated with OPA for 24 hours, and different concentrations of compound LA-33 were added to the culture medium respectively 24 h later for evaluation of lipid accumulation and mRNA expression.

[0147] As shown in Table 1, all the tested compounds have good inhibitory effect on citrate transporter of HEK293 cells, and all the compounds have micromolar inhibitory rate on NaCT, in which the compound LA-33 has the best inhibitory rate on citrate uptake of HEK293T cells, and the IC Figure 1 As shown in Table 1, all the tested compounds have good inhibitory effect on citrate transporter of HEK293 cells, and all the compounds have micromolar inhibitory rate on NaCT, in which the compound LA-33 has the best inhibitory rate on citrate uptake of HEK293T cells, and the IC Figure 2 It is shown that ACLY is significantly down-regulated, and the compound LA-33 only inhibits the function of SLC13A5 without affecting its expression.

[0148] Example 5

[0149] In vitro pharmacodynamic experiment of representative compound LA-33 of the present application on mouse primary hepatocytes

[0150] Experimental procedure:

[0151] Mouse primary hepatocytes were cultured in DMEM high glucose medium containing 10% FBS and placed in a cell incubator at 37°C with 5% CO2. First, pre-protect with serum-free medium containing different concentrations of compounds for 30 min, then stimulate hepatocytes with a mixture of PA and OA (designated as OPA, PA / OA, 1:4) at 300 mM for 24 hours to develop a lipid accumulation model. To mimic the physiological state of the endogenous extracellular matrix, an aliquot of 200 mM citrate was supplemented to the culture medium. AML12 cells were incubated with OPA for 24 hours, and compound LA-33 with different concentrations was added to the culture medium respectively 24 hours later to evaluate lipid accumulation and mRNA expression.

[0152] As shown in Figure 3 , compound LA-33 effectively reduced the TC, TG content in OPA-stimulated AML cells. Figure 4 The results showed that ACLY was significantly down-regulated, and compound LA-33 only inhibited the function of SLC13A5 without affecting its expression, which showed the same results as the AML12 cell line.

[0153] Example 6

[0154] In vivo pharmacodynamic experiment of the representative compound LA-33

[0155] 1. Experimental procedure

[0156] C57BL / 6J mice were purchased from Jiangsu Ailinfeng Biotechnology Co., Ltd. After 1 week of adaptive feeding, they were randomly divided into four groups. The blank group was normally fed, and the other three groups were fasted for 48 h. After the end of fasting, the low-dose group and the high-dose group were injected intraperitoneally at a dose of 10 mg / kg and 30 mg / kg, respectively. 1.5 h later, the animals were anesthetized with intraperitoneal sodium pentobarbital (50 mg / kg) to collect blood samples or liver tissue. Free fatty acid kit was used to measure fatty acids (NEFA) in plasma and liver; triglyceride test kit was used to measure triglyceride (TG) content in plasma and liver; total cholesterol test kit was used to measure triglyceride (TC) content in plasma and liver; HDL-c and LDL-c in plasma were measured using low-density lipoprotein cholesterol test kit and high-density lipoprotein cholesterol test kit, respectively. Plasma samples were directly measured, and tissue samples were homogenized with 9 volumes of normal saline before measurement.

[0157] 2. Data processing:

[0158] Plasma sample calculation formula:

[0159] Cholesterol (TC) content (mmol / L) = (A 样本 -A 空白 ) / (A 标准 -A 空白)×C 标准

[0160] Triglyceride (TG) content (mmol / L) = (A 样本 -A 空白 ) / (A 标准 -A 空白 )×C 标准

[0161] NEFA content (mmol / L) = (ΔA 样本 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )×C 样本 ;

[0162] LDL-c and HDL-c content (mmol / L) = (ΔA sample - ΔA blank) / (ΔA standard - ΔA blank) × C sample;

[0163] like Figure 5 As shown, compound LA-33 can significantly reduce the levels of total cholesterol, total triglycerides and free fatty acids in mouse plasma in a dose-dependent manner, and at the same time significantly reduce the level of low-density lipoprotein in the plasma of starvation-induced mice at a dose of 30 mg / kg.

[0164] like Figure 6 As shown in the results, compound LA-33 can significantly reduce the levels of total cholesterol, total triglycerides and free fatty acids in the liver of mice in a dose-dependent manner.

[0165] like Figure 7 As shown in the results, compound LA-33 can significantly reduce the mRNA expression of ACLY and the mRNA expression of ACC1 and FASN in the DNL pathway in mouse liver, but the mRNA expression of HMGCR, a key enzyme in the TC synthesis pathway, has no significant change. This conclusion is consistent with the results of liver TC and TG determination.

Claims

1. A 2-hydroxysuccinic acid compound, characterized in that Any one of the following compounds:

2. The compound according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by the above-mentioned compound with an acid or a base, wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid, and the base is an inorganic base containing a basic metal cation, an alkaline earth metal cation or an ammonium cation salt.

3. A pharmaceutical composition containing the compound according to claim 1 or 2, characterized in that The pharmaceutical composition contains the compound as an active ingredient and a pharmaceutically acceptable carrier.

4. A compound according to claim 1 or 2 or a pharmaceutical composition according to claim 3 in the preparation of Na + Application of drugs in diseases related to citric acid transporter dependence.

5. The use according to claim 4, characterized in that The Na + Diseases associated with dependence on citrate transporters are metabolic diseases or cancer.

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