2,6,10,14-Tetramethylpentadecane Derivatives, Their Preparation Methods and Applications

Abstract

This invention discloses a 2,6,10,14-tetramethylpentadecane derivative compound, its preparation method, and its applications. The structure of the compound is shown in formula (I). Compared with natural 2,6,10,14-tetramethylpentadecane, the compound has significantly enhanced binding ability to the malonyl / acetyltransferase functional domain (MAT) of fatty acid synthase (FASN), significantly enhanced ability to inhibit fatty acid synthesis, significantly enhanced therapeutic effect on diseases related to FASN abnormalities, significantly enhanced activation of anti-tumor immunity, and significantly enhanced inhibition of tumor growth. It can be effectively used in the preparation of drugs that inhibit fatty acid synthesis, drugs that treat fatty acid synthesis abnormalities, cancer treatment drugs, and immunotherapy drugs.

Description

Technical Field

[0001] This invention relates to a compound, and more particularly to a 2,6,10,14-tetramethylpentadecane derivative compound, its preparation method, and its applications. Background Technology

[0002] Existing research has extensively demonstrated that abnormal fatty acid synthesis can lead to various diseases, including cancer, obesity, diabetes, cardiovascular disease, and fatty liver. Fatty acid synthase (FASN) is a key enzyme in fatty acid synthesis, making the development of FASN inhibitors a key area of ​​biomedical development. FASN is the only protein in the human genome capable of reductively resynthesizing long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate. FASN is abnormally activated in various diseases such as cancer, obesity, diabetes, cardiovascular disease, and fatty liver, primarily producing 16-carbon saturated fatty acid palmitate. In mammals, FASN is highly conserved, mainly composed of two polypeptides with identical functions. Each polypeptide includes seven catalytic domains, such as β-ketoacyl synthase (KS), malonyl / acetyltransferase (MAT), dehydrogenase (DH), enoyl reductase (ER), β-ketoacyl reductase (KR), acyl carrier protein (ACP), and thioesterase (TE). A series of inhibitors have been developed targeting several domains of FASN, such as those inhibiting the KS domain (epiphycin, C75, EGCG, and other naturally occurring polyphenols); those inhibiting the ER domain (triclosan); and those inhibiting the TE domain (orlistat). However, the MAT domain of FASN is also a key subunit involved in de novo fatty acid synthesis, and currently there are no specific inhibitors targeting it. Summary of the Invention

[0003] Objective of the Invention: To address the problems of existing technologies, this invention provides a 2,6,10,14-tetramethylpentadecane derivative compound or a pharmaceutically acceptable salt thereof that targets MAT to inhibit fatty acid synthesis. This invention also provides a method for preparing this compound and its application in treating related diseases.

[0004] Technical solution: The 2,6,10,14-tetramethylpentadecane derivative compound or its pharmaceutically acceptable salt described in this invention, the structure of which is shown in formula (I):

[0005]

[0006] The group A is selected from any one of the following structures:

[0007]

[0008]

[0009] In a preferred embodiment of the present invention, the structure of the compound is shown in any of the following:

[0010]

[0011] The present invention also provides a method for preparing the above-mentioned compound or a pharmaceutically acceptable salt thereof, comprising the following steps: dissolving reactant 1 in an organic solvent, and reacting it with reactant 2 in the presence of a catalyst to obtain a compound as shown in formula (I); wherein reactant 1 is 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate; and wherein reactant 2 is selected from 8-chloro-6-hydroxyquinoline, 6-hydroxy-3,4-dihydroquinolineone, ethyl bromoacetate, 6-hydroxyquinoline, 5-hydroxy-3,4-dihydroquinolineone, 7-hydroxy-3,4-dihydroquinolineone, or acetaminophen.

[0012] As a specific embodiment of the present invention, the compound of the present invention is prepared by the following method:

[0013] (1) Synthesis of Compound 1:

[0014]

[0015] (2) Synthesis of Compound 2:

[0016]

[0017] (3) Synthesis of Compound 3:

[0018]

[0019] (4) Synthesis of Compound 4:

[0020]

[0021] (5) Synthesis of Compound 5:

[0022]

[0023] (6) Synthesis of Compound 6:

[0024]

[0025] (7) Synthesis of Compound 7:

[0026]

[0027] In a preferred embodiment of the present invention, the organic solvent is selected from tetrahydrofuran or N,N-dimethylformamide.

[0028] In a preferred embodiment of the present invention, the catalyst is a combination of diethyl azodicarbonate (DEAD) and triphenylphosphine (PPh3) or K2CO3.

[0029] In a preferred embodiment of the present invention, the reaction temperature is 10℃~100℃.

[0030] In a preferred embodiment of the present invention, the reaction temperature is room temperature or 90-100°C.

[0031] In a preferred embodiment of the present invention, the reaction time is 4 to 24 hours.

[0032] In a preferred embodiment of the present invention, the reaction time is 4 hours or 10 to 12 hours.

[0033] In a preferred embodiment of the present invention, the molar ratio of reactant 1 to reactant 2 is 0.6-1:1-0.6.

[0034] The present invention also provides pharmaceutical compositions comprising the above-described compounds or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0035] The present invention further provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for inhibiting fatty acid synthesis, drugs for treating abnormal fatty acid synthesis, cancer treatment drugs, and immunotherapy drugs.

[0036] Specifically, the applications described in this invention include: the application of the compound in targeting MAT to inhibit FASN activity, and the application of the compound in treating diseases caused by FASN abnormalities.

[0037] The compounds in this application series possess sufficient functional groups and are therefore capable of reacting with any of a variety of inorganic bases, inorganic acids, and organic acids to form salts. Commonly used acids for forming acid addition salts are inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids, such as p-toluenesulfonic acid, methyl alkyl sulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, octanoates, heptanoates, propionates, oxalates, malonates, succinates, nitrites, sebacic acid salts, fumarates, maleates, butene-1,4-dicarboxylate, epoxy-1,6-dicarboxylate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrate, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, etc.

[0038] Base addition salts include salts derived from inorganic bases, such as ammonium or base or alkaline earth metal hydroxides, carbonates, bicarbonates, etc. Therefore, such bases used to prepare the salts of this invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc.

[0039] The novel compounds disclosed in this application represent a new class of FASN inhibitors that target the MAT functional domain. They exhibit excellent inhibitory activity against fatty acid synthesis in animal studies, and their application in treating human diseases caused by FASN abnormalities is demonstrated using tumors as an example. Therefore, the compounds of this invention can be used to treat human diseases caused by FASN abnormalities, including obesity, diabetes, fatty liver, and various cancers. They can be used alone or in combination with existing treatments to treat subjects with FASN abnormalities. When administered as a separate pharmaceutical composition, the compounds or the present invention and existing treatments can be administered simultaneously or at different times.

[0040] The amount of compound and existing treatment administered to the subject will depend on the type and severity of the disease or condition, as well as the subject's characteristics, such as general health, age, sex, weight, and drug tolerance. It will also depend on the type and severity of the disease. A skilled technician will be able to determine the appropriate application of the novel compound of this invention for the effective treatment of human diseases related to FASN abnormalities based on these and other factors.

[0041] The disclosed compounds may be administered via any suitable route, including, for example, oral administration or parenteral administration in capsules, tablets, or tablets. Parenteral administration may include, for example, systemic administration, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection. Depending on the type of cancer to be treated, the compounds may also be administered orally (e.g., by diet), topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation, or nasal drops), or rectally. Oral or parenteral administration is the preferred route of administration.

[0042] The disclosed compounds may be administered to a subject together with an acceptable drug carrier as part of a pharmaceutical composition for the treatment of an existing disease. The formulation of the compound to be administered will vary depending on the chosen route of administration (e.g., solution, emulsion, capsule). A suitable drug carrier may contain an inert component that does not interact with the compound.

[0043] Beneficial effects: This invention discloses a novel class of fatty acid synthesis inhibitors that bind to the MAT functional domain. The compounds designed in this invention or their pharmaceutically acceptable salts have significant inhibitory effects on FASN activity. For diseases caused by abnormal fatty acid synthesis, taking cancer as an example, they can significantly promote T cell infiltration in tumor tissue, significantly activate anti-tumor immune responses, and significantly inhibit tumor growth. They can be effectively used in the preparation of drugs that inhibit fatty acid synthesis, drugs that treat abnormal fatty acid synthesis, cancer treatment drugs, and immunotherapy drugs. Attached Figure Description

[0044] Figure 1 This is the NMR spectrum of compound 1;

[0045] Figure 2 This is the NMR spectrum of compound 2;

[0046] Figure 3 This is the NMR spectrum of compound 3;

[0047] Figure 4 This is the NMR spectrum of compound 4;

[0048] Figure 5 This is the NMR spectrum of compound 5;

[0049] Figure 6 This is the NMR spectrum of compound 6;

[0050] Figure 7 This is the NMR spectrum of compound 7;

[0051] Figure 8 The graph shows the binding affinity of 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3 to FASN-MAT.

[0052] Figure 9 This is a graph showing the inhibition of FASN activity by 2,6,10,14-tetramethylpentadecane.

[0053] Figure 10 This is a curve showing the inhibition of FASN activity by compound 2;

[0054] Figure 11 This is a curve showing the inhibition of FASN activity by compound 3;

[0055] Figure 12 The chart shows the effects of solvent and 2,6,10,14-tetramethylpentadecane on the inhibition of free fatty acid levels in mice.

[0056] Figure 13 The graph shows the inhibition of free fatty acid levels in mice by the solvent and compound 2, respectively.

[0057] Figure 14 The chart shows the inhibition of free fatty acid levels in mice by the solvent and compound 3, respectively.

[0058] Figure 15 The flow cytometry results show the effects of solvent and 2,6,10,14-tetramethylpentadecane on the promotion of invasive T cells in tumor tissue.

[0059] Figure 16 The flow cytometry results show the effects of solvent and compound 2 on the promotion of invasive T cells in tumor tissue.

[0060] Figure 17 The flow cytometry results show the effects of solvent and compound 3 on the promotion of invasive T cells in tumor tissue.

[0061] Figure 18 The flow cytometry results show the inhibition of invasive Treg cells in tumor tissue by the solvent and 2,6,10,14-tetramethylpentadecane, respectively.

[0062] Figure 19 The flow cytometry results show the inhibition of invasive Treg cells in tumor tissue by the solvent and compound 2, respectively.

[0063] Figure 20 These are flow cytometry results showing the inhibition of invasive Treg cells in tumor tissue by the solvent and compound 3, respectively.

[0064] Figure 21 The graph shows the inhibitory effects of the solvent and 2,6,10,14-tetramethylpentadecane on the growth of mouse mammary tumors.

[0065] Figure 22 This is a chart showing the inhibitory effects of the solvent and compound 2 on the growth of mouse mammary tumors.

[0066] Figure 23 The graph shows the inhibitory effects of the solvent and compound 3 on the growth of mouse mammary tumors. Detailed Implementation

[0067] The present application will now be described in detail with reference to specific embodiments.

[0068] I. Compound Synthesis

[0069] Example 1

[0070] Preparation of Compound 1

[0071]

[0072] Preparation of reactant 1 (2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate):

[0073]

[0074] In an ice bath, isophytol (3.6 g, 12 mmol), MgSO4 (7.2 g, 60 mmol, 5.0 equiv), and acetone (200 ml) were added to a three-necked flask, followed by the slow addition of KMnO4 (5.7 g, 36 mmol, 3.0 equiv). The reaction mixture was then stirred at room temperature for 12 hours, filtered, and washed with acetone. The solvent was evaporated under reduced pressure to give intermediate 1.

[0075] Under nitrogen protection, n-BuLi (1.1 mL, 2.8 mmol, 1.4 equiv) was added dropwise to methyltriphenylphosphine bromide (857 mg, 2.4 mmol, 1.2 equiv) in an ice bath, with THF (6.7 mL) as the solvent. The resulting mixture was stirred for 2 hours, and then intermediate 1 (537 mg, 2 mmol, 1.0 equiv) was added. The reaction mixture was then heated to room temperature and stirred for 12 hours, followed by quenching with a saturated aqueous NH4Cl solution. The organic layer was extracted with EtOAc (3 × 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product. After purification by column chromatography on silica gel (petroleum ether), intermediate 2 was obtained.

[0076] Under nitrogen protection, 9-BBN (293 mg, 2.4 mmol, 1.2 equiv) was added to a THF (6 mL) solution of intermediate 2 (533 mg, 2 mmol, 1.0 equiv). The reaction mixture was stirred at 66 °C for 6 hours, and then 30% H₂O₂ (2 mL) and 3M NaOH (20 mL) were added. The resulting mixture was stirred at room temperature for 2 hours. The organic layer was extracted with EtOAc (3 × 15 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (DCM / petroleum ether 1:10), intermediate 3 was obtained.

[0077] Under nitrogen protection, intermediate 3 (570 mg, 2 mmol) was added to a solution of DCM (20 mL, 0.1 M) with Et3N (607 mg, 6 mmol, 3.0 equiv), DMAP (12 mg, 5 mmol), and TsCl (418 mg, 2.2 mmol, 1.1 equiv). The reaction mixture was stirred at room temperature for 12 hours. The organic layer was extracted with EtOAc (3 × 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:20), reactant 1 (2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate) was obtained.

[0078] Under nitrogen protection, 8-chloro-6-hydroxyquinoline (43 mg, 0.24 mmol, 1.2 equiv), DEAD (70 mg, 0.4 mmol, 2.0 equiv), and PPh3 (105 mg, 0.4 mmol, 2.0 equiv) were added to 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (57 mg, 0.2 mmol) in THF (2 mL, 0.1 M). The reaction mixture was stirred at room temperature for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product. Purification by silica gel column chromatography (EtOAc / petroleum ether 1:10) yielded compound 1. The NMR structure is shown below. Figure 1 As shown. 1 HNMR(400MHz,Chloroform-d)δ8.87(dd,J=4.4,1.6Hz,1H),8.04(dd,J=8.4,1.6 Hz,1H),7.56(d,J=2.4Hz,1H),7.40(dd,J=8.0,4.0Hz,1H),7.00(d,J=2.4Hz,1H ),3.94–3.90(m,1H),3.85–3.82(m,1H),2.04–1.96(m,1H),1.44–1.33(m,2H),1 .29–1.19(m,4H),1.15–1.09(m,9H),1.07–1.05(m,9H),0.87–0.83(m,12H)ppm. 13C NMR(101MHz,Chloroform-d)δ156.9,148.3,140.5,135.2,134.2,130.2,122.6,122.1,105.3,73.7,39.3,37.4(2C), 37.3,37.2,33.8,33.7,33.1(2C),32.8,32.7,28.0,24.8,24.6,24.4,22.7,19.7(2C),17.1ppm.HRMS(ESI)m / z:[M+H] + Calcd for C 28 H 45 ClNO 446.3184; Found 446.31833.

[0079] Example 2

[0080] Preparation of compound 2

[0081]

[0082] Under nitrogen protection, in an ice bath, K₂CO₃ (124 mg, 0.9 mmol, 3.0 equiv) was added to 6-hydroxy-3,4-dihydroquinolinone (73 mg, 0.45 mmol, 1.5 equiv) in DMF (3 mL, 0.1 M). The reaction mixture was stirred at room temperature for 2 hours. Then, 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (131 mg, 0.3 mmol, synthesized as in Example 1) was added and stirred at 100 °C for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:2), compound 2 was obtained. The NMR structure is shown below. Figure 2 As shown. 1 H NMR(400MHz,Chloroform-d)δ9.08(s,1H),6.76–6.69(m,3H),3.78–3.74(m,1H),3.69–3.65(m,1H),2.94(t,J=6.8Hz,2H),2.63(t,J =7.6Hz,1H),1.93–1.88(m,1H),1.55–1.46(m,2H),1.39–1.20(m,13H),1.16–1.04(m,6H),1.01–1.00(m,3H),0.87–0.83(m,12H)ppm. 13C NMR(101MHz,Chloroform-d)δ171.7,155.3,130.6,124.9,116.2,114.4,113.0,73.6,39.3,37.4,37.3,37.2, 33.8,33.2,32.8,32.7,30.6,28.0,25.7,24.8,24.5,24.3,22.7,19.7,19.6,17.1ppm.HRMS(ESI)m / z:[M+Na] + Calcd forC 28 H 47 NNaO2 452.3499; Found 452.3497.

[0083] Example 3

[0084] Preparation of compound 3

[0085]

[0086] Under nitrogen protection and in an ice bath, NaH (60% dispersed in mineral oil, 24 mg, 0.6 mmol, 3.0 equiv) was added to compound 2 (89 mg, 0.2 mmol) in DMF (2 mL, 0.1 M). The reaction mixture was stirred at room temperature for 0.5 h. Then, ethyl bromoacetate (40 mg, 0.24 mmol, 1.2 equiv) was added and stirred for 4 h. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:3), compound 3 was obtained. The NMR structure is shown below. Figure 3 As shown. 1 H NMR(400MHz,Chloroform-d)δ6.75–6.64(m,3H),4.62(s,2H),4.20(q,J=7.2Hz,2H),3.79–3.65(m,2H),2.92–2.88(m,2H),2.70–2.66(m,2H) ,1.97–1.84(m,1H),1.56–1.45(m,2H),1.43–1.35(m,3H),1.34–1.18( m,14H),1.14–1.04(m,5H),1.01–0.99(m,3H),0.89–0.83(m,12H)ppm. 13CNMR(101MHz,Chloroform-d)δ170.2,168.7,155.2,132.9,127.6,115.1,114.6,112.5,73.5,61.4,44.5,39.3,37.4,37.3, 37.2(2C),33.7,33.1,32.7,31.4,27.9,25.6,24.7,24.4(2C),24.3,22.7,19.7,19.6,17.0,14.1ppm.HRMS(ESI)m / z:[M+H] + Calcd forC 32 H 54 NO4 516.4047; Found 516.4047.

[0087] Example 4

[0088] Preparation of compound 4

[0089]

[0090] Under nitrogen protection, K₂CO₃ (124 mg, 0.9 mmol, 3.0 equiv) was added to 6-hydroxyquinoline (65 mg, 0.45 mmol, 1.5 equiv) in an ice bath, with DMF (3 mL, 0.1 M) as the solvent. The reaction mixture was stirred at room temperature for 2 hours. Then, 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (131 mg, 0.3 mmol, synthesized as in Example 1) was added and stirred at 100 °C for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:3), compound 4 was obtained. The NMR structure is shown below. Figure 4 As shown. 1 H NMR(400MHz,Chloroform-d)δ8.76–8.74(m,1H),8.04–7.98(m,2H),7.39–7.32(m,2H),7.05(d,J=2.8Hz,1H),3.95–3.91(m,1H),3.86–3.82(m, 1H),2.05–1.99(m,1H),1.78–1.71(m,3H),1.56–1.48(m,2H),1.38–1.3 1(m,5H),1.29–1.20(m,12H),1.09–1.02(m,7H),0.87–0.83(m,12H)ppm. 13C NMR(101MHz,Chloroform-d)δ157.4,147.7,144.2,134.7,130.6,129.3,122.7,121.3,105.7,73.5,39.3,37.5, 37.4,37.3,37.2,33.9,33.1,32.7,32.8,28.0,24.8,24.5,24.4,22.7,19.7(2C),17.2ppm.HRMS(ESI)m / z:[M+H] + Calcdfor C 28 H 46 NO 412.3574; Found 412.3573.

[0091] Example 5

[0092] Preparation of compound 5

[0093]

[0094] Under nitrogen protection, in an ice bath, K₂CO₃ (124 mg, 0.9 mmol, 3.0 equiv) was added to 5-hydroxy-3,4-dihydroquinolinone (73 mg, 0.45 mmol, 1.5 equiv) in DMF (3 mL, 0.1 M). The reaction mixture was stirred at room temperature for 2 hours. Then, 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (131 mg, 0.3 mmol, synthesized as in Example 1) was added and stirred at 100 °C for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:2), compound 5 was obtained. The NMR structure is shown below. Figure 5 As shown. 1 H NMR(400MHz,Chloroform-d)δ9.12(brs,1H),7.09(t,J=8.0Hz,1H),6.55(d,J=8.8Hz,1H),6.47(d,J=8.0Hz,1H),3.85–3.72(m,2H),2.97(t . 13C NMR(101MHz,Chloroform-d)δ171.9,156.4,138.2,127.8,111.8,108.0,106.2,73.3,39.3,37.4(2C),37.3(2C) ,33.8,33.3,32.8,32.7,30.2,28.0,24.8,24.5,24.4,22.7(2C),19.7(2C),18.5,17.2ppm.HRMS(ESI)m / z:[M+H] + Calcd for C 28 H 48 NO2 430.3680; Found 430.3680.

[0095] Example 6

[0096] Preparation of compound 6

[0097]

[0098] Under nitrogen protection, in an ice bath, K₂CO₃ (124 mg, 0.9 mmol, 3.0 equiv) was added to 7-hydroxy-3,4-dihydroquinolinone (73 mg, 0.45 mmol, 1.5 equiv) in DMF (3 mL, 0.1 M). The reaction mixture was stirred at room temperature for 2 hours. Then, 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (131 mg, 0.3 mmol, synthesized as in Example 1) was added and stirred at 100 °C for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:3), compound 6 was obtained. The NMR structure is shown below. Figure 6 As shown. 1 H NMR(400MHz,Chloroform-d)δ8.28(br,1H),7.04(d,J=8.0Hz,1H),6.53–6.51( m,1H),6.34(s,1H),3.79–3.75(m,1H),3.70–3.66(m,2H),2.89(t,J=6.8Hz,2H) ,2.62(t,J=8.0Hz,2H),1.93–1.87(m,1H),1.55–1.47(m,2H),1.38–1.33(m,3H) ,1.33–1.20(m,11H),1.16–1.06(m,4H),1.01–1.00(m,3H),0.87–0.83(m,12H). 13C NMR(101MHz,Chloroform-d)δ171.8,159.0,138.0,128.6,115.5,108.7,102.2,73.5,39.35,37.5,37.4,37.3 (2C),33.8,33.2,32.8,32.7,31.1,28.0,24.8,24.6,24.5,24.4,22.7,19.7(2C),17.1.HRMS(ESI)m / z:[M+H] + Calcd for C 28 H 48 NO2 430.3680; Found 430.3679.

[0099] Example 7

[0100] Preparation of compound 7

[0101]

[0102] Under nitrogen protection, in an ice bath, K₂CO₃ (124 mg, 0.9 mmol, 3.0 equiv) was added to acetaminophen (68 mg, 0.45 mmol, 1.5 equiv) in DMF (3 mL, 0.1 M). The reaction mixture was stirred at room temperature for 2 hours. Then, 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate (131 mg, 0.3 mmol, synthesized as in Example 1) was added and the mixture was stirred at 100 °C for 12 hours. The organic layer was extracted with EtOAc (3 × 5 mL), washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to give the crude product. After purification by silica gel column chromatography (EtOAc / petroleum ether 1:2), compound 7 was obtained. The NMR structure is shown below. Figure 7 As shown. 1 H NMR(400MHz,Chloroform-d)δ7.44(br,1H),7.36(d,J=9.6Hz,2H),6.83(d,J=8.8Hz,2H),3.79–3.76(m,1H),3.69–3.66(m,1H),2.13(s,3H) ,1.94–1.88(m,1H),1.55–1.45(m,2H),1.42–1.33(m,4H),1.32–1.21( m,9H),1.16–1.05(m,6H),1.01–0.99(m,3H),,0.89–0.83(m,12H)ppm. 1313C NMR (101 MHz, Chloroform-d) δ 168.3, 156.1, 130.7, 121.9, 114.7, 73.5, 39.3, 37.4, 37.3, 37.2, 33.8, 33.1, 32.8, 32.7, 27.9, 24.8, 24.4, 24.3 (2C), 22.7, 19.7 (2C), 17.1 ppm. HRMS (ESI) m / z: [M+H] + Calcd for C 27 H 48 NO2 418.3680; Found 418.3679.

[0103] Experimental Example

[0104] II. Experimental Materials

[0105] 2.1 Cell Lines

[0106] All the cells used in this invention are listed in the cell lines shown in Table 1:

[0107] Table 1 Cell Lines

[0108]

[0109] 2.2 Experimental Animals and Related Feeding Materials

[0110] The Balb / c mice used in this invention were purchased from Jiangsu Ailingfei Biotechnology Co., Ltd., license number: SCXK (Su) 2020-0009; all experimental mice were housed in the SPF (specific pathogen free) - level experimental room of the Animal Experiment Center of Nanjing Normal University. According to the feeding requirements, the feeding density of all experimental mice was less than or equal to 5 mice per cage, the room temperature was maintained within the range of 20 - 25 °C, the humidity was maintained at around 50%, and the automatic light control was (12 h light / 12 h dark). The feed and bedding for the mice were both purchased from the Qinglongshan Animal Breeding Farm in Jiangning District, Nanjing.

[0111] 2.3 Experimental Reagents

[0112] (1) All the reagents used for cell culture are listed in Table 2 Cell Culture Reagents

[0113] Table 2 Cell Culture Reagents

[0114]

[0115] (2) All the reagents used for biochemical experiments are listed in Table 3

[0116] Table 3 Biochemical Experiment Reagents

[0117]

[0118]

[0119] (3) All antibodies used in this invention are listed in Table 4.

[0120] Table 4 Antibodies

[0121]

[0122] 2.4 Experimental Apparatus

[0123] (1) Milli-Q ultrapure water system, Millipore Corporation, USA;

[0124] (2) UB-7PH meter, Nanjing Henglian Biotechnology Co., Ltd.;

[0125] (3) EASYPET3 automatic inhaler, Eppendorf GmbH, Germany;

[0126] (4) XD-202 microscope, Nanjing Jiangnan Yongxin Optical Co., Ltd.;

[0127] (5) SW-CJ-IF type ultra-clean workbench, Suzhou Antai Air Technology Co., Ltd., Suzhou Group;

[0128] (6) Benchtop refrigerated centrifuge, Eppendorf, Germany;

[0129] (7) BECKMAN Microfuge 16 benchtop centrifuge, BECKMAN Corporation, USA;

[0130] (8) VOTTEX-2 vortex oscillator, Nanjing Henglian Biotechnology Co., Ltd.;

[0131] (9) HWS12 electric thermostatic water bath, Shanghai Yiheng Technology Co., Ltd.;

[0132] (10) Cell incubator, Thermo Fisher Scientific;

[0133] (11) StepOnePlus TM Real-Time PCR instrument, Bio-Rad Laboratories, USA;

[0134] (12)BD FACSVerse TM Flow cytometer, BD Biosciences, USA;

[0135] (13) BCD-328EDPT refrigerator, Qingdao Haier Co., Ltd.;

[0136] (14) BCD-539WT Low Temperature Refrigerator, Qingdao Haier Co., Ltd.;

[0137] (15) DW-86L626 Ultra-low Temperature Freezer, Qingdao Haier Co., Ltd.;

[0138] (16) Vernier caliper, Nanjing SuCe Measurement Instruments;

[0139] (17)Monolith NT.115, Nanotemper Technologies.

[0140] 2.5 Experimental Consumables

[0141] (1) 10μL, 100μL, 1000μL pipette tips, 10μL, 100μL, 1000μL DNase- / RNase-free pipette tips, Jiangsu Haimen Jiawei Glassware Factory;

[0142] (2) 0.2 mL DNase- / RNase-free PCR tubes and 1.5 mL DNase- / RNase-free flat-cap centrifuge tubes, Axygen, USA;

[0143] (3) 1.5mL and 2mL ordinary centrifuge tubes, Jiangsu Haimen Jiawei Glassware Factory;

[0144] (4) 2mL externally rotated cell cryopreservation tube, Corning;

[0145] (5) 100mm and 60mm cell culture dishes and 96-well cell culture plates, Nanjing SORFA Biotechnology Co., Ltd.

[0146] (6) 10mL pipettes, 15mL and 50mL centrifuge tubes, Nanjing SORFA Biotechnology Co., Ltd.

[0147] (7) Disposable PE gloves, Jiangsu Haimen Jiawei Glassware Factory;

[0148] (8) Disposable powder-free latex gloves, Jiangsu Haimen Jiawei Glassware Factory;

[0149] (9) Capillary, Nanotemper Technologies.

[0150] III. Experimental Methods

[0151] 3.1 Cell Culture

[0152] 4T1 breast cancer cells were cultured in 1640 medium supplemented with 10% serum and 1% penicillin / streptomycin in a cell culture incubator at 37°C containing 5% CO2.

[0153] 3.2 Mouse tumor model and drug administration

[0154] A mouse model of tumor formation from transplanted homologous breast cancer cells (i.e., 4T1 cells): Six-week-old female Balb / c mice purchased commercially were tagged using the toe-clipping method. One million 4T1 cells were injected into the third mammary pad on the left side of each mouse. After tumor formation (approximately 50–100 mm in volume), 3 Tumors were treated with either compound 2 or compound 3 via intraperitoneal administration, starting with a single dose of 4 g / kg / animal of 2,6,10,14-tetramethylpentadecane or four doses (every two days) of 100 mg / kg / animal. A solvent treatment (basic 1640, compound-free) served as a control. Throughout the trial, animals were monitored daily for signs of toxicity, including weight loss.

[0155] 3.3 Evaluation of the activity of experimental compounds in mouse tumor models

[0156] After drug treatment, the bedding of each cage of mice was changed 1-2 times per week, and the food and drinking water were replenished 1-2 times per week. Starting from the injection of 4T1 cells, each mouse was palpated at each weighing to check for nodular palpable masses in the mammary glands, i.e., mammary tumors. The time when mammary tumors were first palpable was recorded, and the length and width of the tumors were recorded twice a week. The tumor volume was calculated using the formula: Tumor Volume = Length × Width × Height, and the tumor volume of each mouse at each time point was recorded. The length, width, and height of the tumors were measured using calipers. The tumor volume was recorded when it reached 2500 mm². 3 Euthanasia was performed, and blood and mammary tumor tissue from tumor-bearing mice were collected. Tumor tissue was washed once with PBS and then wiped clean with absorbent paper. All tumor tissue was wrapped in aluminum foil and labeled with the mouse number, treatment method, and date, and stored at -80°C. Mouse blood was allowed to stand at room temperature for 30 minutes, then centrifuged to collect the supernatant and mouse serum, and stored at -20°C. The anticancer effect of the novel compound was evaluated using a tumor growth inhibition assay. Flow cytometry was used to detect the infiltration of T cells and Treg cells in tumors by the novel compound, and the effect of the novel compound on FASN activity in mice was assessed using free fatty acid content.

[0157] 3.4 Detection of free fatty acids in mice

[0158] Follow the instructions in the kit package insert. In short, equilibrate all components to room temperature before use. Briefly centrifuge the tube before opening. Thaw the tube on ice during the assay. Prepare sufficient "working reagent" as per the instructions. Transfer 30 μL of serum sample to 420 μL of Working Reagent 1, vortex thoroughly for 10 min, centrifuge at 3000 rpm for 10 min, and add 50 μL of the supernatant to 200 μL of Working Reagent 3. Vortex thoroughly for 2 min, let stand for 15 min, and transfer 0.2 mL to a microvolume cuvette / 96-well plate. Measure the absorbance at 550 nm. Calculate the corresponding free fatty acid content using the OD value based on the standard curve.

[0159] 3.5 Protein Expression and Purification, and Microthermophoresis (MST)

[0160] The FASN-MAT domain was cloned into the pet-MBP-3C vector and transformed into *E. coli* BL21-CodonPlus (DE3) for protein expression and purification. When the bacterial density reached 0.6–0.8 OD600 nm, the protein was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and then grown at 20 °C for 16 h. After induction, the bacteria were collected and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 30 mM imidazole and lysed by sonication. The protein was purified using Ni-NTA beads and washed with wash buffer containing an additional 50 mM imidazole compared to the lysis buffer, followed by elution in elution buffer containing an additional 500 mM imidazole compared to the lysis buffer. The eluted protein was further purified by size exclusion purification using AKTA chromatography. Protein concentration was determined using the BCA protein assay. The binding affinity of the MAT protein to 2,6,10,14-tetramethyltetradecane, compound 2, or compound 3 was determined using Monolith NT.115 software. The His-Tag Labeling Kit RED-tris-NTA was used according to the manufacturer's procedure. 2nd The protein was fluorescently labeled with a generation dye, and the protein was adjusted to an appropriate concentration in PBST. Each assay was performed at room temperature, with the labeled protein mixed with the same volume of 16 different series of unlabeled compounds at varying concentrations. The samples were then loaded into capillaries and measured at 25°C using 60% LED power and moderate MST power. Data analysis was performed using MO.Affinity Analysis v.2.3 software, with Kd values ​​within a given range.

[0161] 3.6 Flow cytometry

[0162] Mince tumor tissue of appropriate size in a 60mm dish, resuspend the tissue fragments in 4mL of freshly prepared tumor tissue digestion solution, and transfer to a 50mL centrifuge tube. Digest at 37°C and 180rpm for 2 hours. Pass the digestion solution through a 200-mesh sieve and transfer to a 2mL centrifuge tube. Centrifuge at 1000×g for 5 minutes at room temperature and discard the supernatant. Resuspend the pellet in 500μL of freshly prepared 5% FACS Buffer and collect in one tube. Centrifuge at 1000×g for 5 minutes at room temperature and discard the supernatant. Wash again with 500μL of 5% FACS Buffer and discard the supernatant. Resuspend the pellet in 500μL of 5% FACS Buffer, count the cells, and take 1×10⁻⁶ cells. 6 Dissolve tumor cells in 500 μL of 5% FACS Buffer (replace with a 1.5 mL centrifuge tube at this step). Prepare three extra tubes of cells for blank control and single-antibody control. Wrap the centrifuge tubes with aluminum foil. Add 5 μL of the corresponding antibody to each tube, with no antibody added to the blank tubes and only one antibody added to the single-antibody control tubes. Mix well and incubate on ice for 30 min. Centrifuge at 1000 x g for 5 min at room temperature, discard the supernatant, resuspend the pellet in 800 μL of 5% FACS Buffer, and place on ice. Preheat the flow cytometer, pass each sample through a 200-mesh sieve again, connect to the flow cytometer tube, and load the sample into the flow cytometer.

[0163] IV. Experimental Results

[0164] 4.1 NMR spectra of the compounds shown in this invention

[0165] All reactions involving air- or moisture-sensitive reagents were performed using standard Schlenk techniques in flame-dried glassware under a nitrogen atmosphere. Solvents were freshly distilled or commercially available ultra-dry grade solvents, stored in molecular sieves. Top. Refluxed onto dichloromethane (DCM)CaH2 and used as fresh distillation. Merck silica gel 60F254 plates were used for thin-layer chromatography (TLC) with UV (254 / 366 nm) or KMnO4 as staining agent. NM spectrometry was performed on a Bruker Avance 400 spectrometer in CDCl3 at 400 MHz (1H), 101 MHz (13C), and 376 MHz (19F), with tetramethylsilane as an internal standard. Chemical shifts (δ) are reported in parts per million (ppm). HRMS-ESI was performed on an AB Triple 5600 mass spectrometer. Figures 1-7 The NMR spectra correspond to compounds 1 through 7, respectively.

[0166] 4.2 Effects of 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3 on the in vitro binding affinity of the FASN-MAT domain

[0167] After purifying the FASN-MAT protein in vitro, the protein concentration was adjusted to 40 nM using PBST. The binding affinity of 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3 to the FASN-MAT domain was then detected using micro-thermophoresis (MST). The results are as follows: Figure 8 As shown in Table 5, compounds 2 and 3 exhibit stronger binding affinity to FASN-MAT compared to 2,6,10,14-tetramethylpentadecane, with compound 3 showing superior affinity to compound 2. Compound 2 demonstrates a nearly 40-fold increase in binding affinity to the MAT functional domain in FASN compared to its natural analog 2,6,10,14-tetramethylpentadecane, while compound 3 demonstrates a nearly 100-fold increase in binding affinity to the MAT functional domain in FASN compared to its natural analog 2,6,10,14-tetramethylpentadecane.

[0168] Table 5. Binding coefficients of compounds with MAT

[0169]

[0170]

[0171] 4.3 Inhibition of FASN hemienzyme activity by 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3

[0172] After treating 4T1 cells with different concentration gradients of 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3 for 48 hours, the cell pellets were collected, lysed, and the protein supernatant was obtained. The effect of different drug concentrations on FASN enzyme activity was assessed in vitro by measuring the inhibition of NADPH oxidation capacity in the protein supernatant, and the EC50 value was then calculated. Figures 9-11 As shown, compared to 2,6,10,14-tetramethylpentadecane, compounds 2 and 3 exhibit stronger inhibitory activity against FASN enzyme activity, with compound 3 being superior to compound 2. Several other compounds of this invention also possess inhibitory activity against FASN enzyme activity.

[0173] 4.4 2,6,10,14-Tetramethylpentadecane, compound 2, and compound 3 inhibit fatty acid synthesis in mice.

[0174] Mice were injected intraperitoneally with either 4 g / kg of 2,6,10,14-tetramethylpentadecane or four times (every two days) of 100 mg / kg of compound 2 or compound 3, using their respective solvents (basic 1640 medium containing no compounds) as controls. Mouse serum was then collected to detect the effects of the drugs on the free fatty acid content in the mice. Results are as follows: Figure 12-14As shown in Table 6, the serum free fatty acid content of mice treated with 2,6,10,14-tetramethylpentadecane was as follows: Figure 12 As shown, the serum free fatty acid content of mice treated with compound 2 compared to control mice was as follows: Figure 13 As shown, the serum free fatty acid content of mice treated with compound 3 compared to control mice was as follows: Figure 14 As shown. Compared with the solvent control, 2,6,10,14-tetramethylpentadecane, compounds 2 and 3 all significantly inhibited the content of free fatty acids in mice; compounds 2 and 3 showed more than 40 times greater ability to inhibit the synthesis of live fatty acids compared with their natural analogues 2,6,10,14-tetramethylpentadecane. Several other compounds of this invention can also inhibit the content of free fatty acids in mice.

[0175] Table 6 shows the dosages of compounds that significantly inhibit fatty acid synthesis in mice.

[0176] medicine A dose that significantly inhibits fatty acid synthesis in mice 2,6,10,14-Tetramethylpentadecane 4g / kg Compound 2 100mg / kg Compound 3 100mg / kg

[0177] 4.5 2,6,10,14-Tetramethylpentadecane, compound 2 and compound 3 promote the infiltrating of T cells in tumor tissue.

[0178] A homologous xenograft tumor model was established by injecting a certain number of 4T1 murine breast cancer cells into the fat pad of Balb / c mice. After tumor formation, mice were intraperitoneally injected with either 4 g / kg of 2,6,10,14-tetramethylpentadecane or four times (every two days) of compound 2 or compound 3, using their respective solvents (basic 1640 medium) as controls. Flow cytometry was used to detect the effects of different drugs on T cells in tumor tissue. The results are as follows: Figures 15-17 As shown in Table 7, the ability of 2,6,10,14-tetramethylpentadecane to activate antitumor immune activity relative to the control is as follows: Figure 15 As shown, compound 2's ability to activate antitumor immune activity relative to the control is as follows: Figure 16 As shown, compound 3's ability to activate antitumor immune activity relative to the control is as follows: Figure 17 As shown. Compared with the solvent control, 2,6,10,14-tetramethylpentadecane, compounds 2 and 3 all significantly increased the number of infiltrating T cells in tumor tissue. Compounds 2 and 3 showed more than 40-fold increased ability to activate antitumor immune activity compared with their natural analogues 2,6,10,14-tetramethylpentadecane. Several other compounds of this invention can also increase the number of infiltrating T cells in tumor tissue.

[0179] Table 7. The ability of compounds to activate antitumor immune activity

[0180] medicine A dose that significantly activates antitumor immune activity in mice 2,6,10,14-Tetramethylpentadecane 4g / kg Compound 2 100mg / kg Compound 3 100mg / kg

[0181] 4.6 2,6,10,14-Tetramethylpentadecane, compound 2 and compound 3 inhibit invasive Treg cells in tumor tissue.

[0182] A homologous xenograft tumor model was established by injecting a certain number of 4T1 murine breast cancer cells into the fat pad of Balb / c mice. After tumor formation, mice were intraperitoneally injected with either 4 g / kg of 2,6,10,14-tetramethylpentadecane or four times (every two days) of 100 mg / kg of compound 2 or compound 3, using their respective solvents (basic 1640 medium) as controls. Flow cytometry was used to detect the effects of different drugs on suppressor Treg cells in tumor tissue. Figures 18-20 As shown, compared with the solvent control, 2,6,10,14-tetramethylpentadecane, compound 2, and compound 3 all significantly reduced the number of invasive Treg cells in tumor tissue. Several other compounds of this invention can also reduce the number of invasive Treg cells in tumor tissue.

[0183] 4.7 Effects of 2,6,10,14-tetramethylpentadecane, compound 2 and compound 3 on the growth of mouse mammary tumors.

[0184] A certain number of 4T1 murine breast cancer cells were injected into the fat pad of Balb / c mice to construct a homologous xenograft model. After tumor formation, the mice were intraperitoneally injected with either 4 g / kg of 2,6,10,14-tetramethylpentadecane or four times (every two days) of compound 2 or compound 3, using their respective solvents (basic 1640 medium) as controls. Figures 21-23 As shown in Table 8, the ability of 2,6,10,14-tetramethylpentadecane to activate antitumor immune activity relative to the control is as follows: Figure 21 As shown, compound 2's ability to activate antitumor immune activity relative to the control is as follows: Figure 22 As shown, compound 3's ability to activate antitumor immune activity relative to the control is as follows: Figure 23 As shown. Compared with the solvent control, 2,6,10,14-tetramethylpentadecane, compounds 2 and 3 all significantly inhibited the growth of mouse mammary tumors. Compounds 2 and 3 showed more than 40-fold increased antitumor activity compared with their natural analogues 2,6,10,14-tetramethylpentadecane, with compound 3 showing the most significant effect. Several other compounds of this invention can also inhibit the growth of mouse mammary tumors.

[0185] Table 8. The tumor growth inhibitory ability of compounds

[0186] medicine Significantly inhibits tumors in mice at the dose 2,6,10,14-Tetramethylpentadecane 4g / kg, single dose Compound 2 100mg / kg, 4 times Compound 3 100mg / kg, 4 times .

Claims

1. A 2,6,10,14-tetramethylpentadecane derivative compound or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the compound is selected from any one of the following: 。 2. A process for the preparation of a compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The process includes the following steps: dissolving reactant 1 in an organic solvent, and reacting it with reactant 2 in the presence of a catalyst to obtain the compound shown; reactant 1 is 2,6,10,14-tetramethylpentadecanyl-4-methylbenzenesulfonate; reactant 2 is selected from 6-hydroxy-3,4-dihydroquinolineone.

3. The production method according to claim 2, characterized by, The organic solvent is selected from tetrahydrofuran or N,N-dimethylformamide.

4. The production method according to claim 2, characterized by, The catalyst is a combination of diethyl azodicarbonate and triphenylphosphine or K2CO3.

5. The preparation method according to claim 2, characterized in that, The reaction temperature is 10℃~100℃.

6. The preparation method according to claim 2, characterized in that, The reaction time is 4~24h.

7. The preparation method according to claim 2, characterized in that, The molar ratio of reactant 1 to reactant 2 is 0.6~1: 1~0.

6.

8. A pharmaceutical composition, characterized in that, This includes the compound as described in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

9. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting fatty acid synthesis, a drug for treating abnormal fatty acid synthesis, or a drug for treating breast tumors.

Images