CN113041243A

CN113041243A - Medicine for inhibiting fatty acid synthetase and preparation method thereof

Info

Publication number: CN113041243A
Application number: CN202010114795.4A
Authority: CN
Inventors: 陈永泉; 渠宏雁; 单锴; 崔国祯; 王小英; 朱升龙; 韦冷云
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2021-06-29
Anticipated expiration: 2040-02-25
Also published as: CN113041243B

Abstract

The invention discloses a medicine for inhibiting fatty acid synthetase and a preparation method thereof, belonging to the technical field of medical biology. The compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof is applied to preparation of the medicine for inhibiting the fatty acid synthetase, can obviously inhibit the activity of the fatty acid synthetase, can inhibit the synthesis of fatty acid in cells, has good tumor proliferation inhibition effect, can induce tumor cell apoptosis, exerts the treatment effect on tumors, can be used for treating the tumors and the diseases related to metabolism, and has important clinical application prospect.

Description

Medicine for inhibiting fatty acid synthetase and preparation method thereof

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a medicament for inhibiting fatty acid synthetase and a preparation method thereof.

Background

It is well known that fatty acid biosynthesis is an important metabolic pathway of the body, and plays an important role in many aspects, such as energy transport and storage, cell membrane structure, provision of intermediates for hormone synthesis, and participation in signal transduction and protein acylation. The fatty acid source in the human body mainly consists of two parts, wherein one part is exogenous fatty acid which is directly taken in by diet, and the other part is endogenous fatty acid which is synthesized by the human body.

Fatty Acid Synthase (FASN) is the only key enzyme for de novo synthesis of fatty acids in organisms, exists in the form of a multienzyme complex, and catalyzes all reaction steps of acetyl-CoA and malonyl-CoA (i.e., malonyl-CoA) to generate long-chain fatty acids with palmitic acid as the main component by the action of seven active sites. FASN is widely expressed in various tissue cells, and is abundantly expressed in mammalian liver, kidney, brain, lung, breast and adipose tissues. Its expression level and activity are easily affected by body's nutrition, hormone and development. When animals are stimulated by different nutrients or hormones, the expression or activity of FASN in liver and adipose tissue can vary greatly.

A plurality of reports show that FASN is closely related to a plurality of metabolic syndromes such as obesity, diabetes, atherosclerosis and the like, and the development of FASN inhibitors is expected to open up a new way for treating the metabolic syndromes such as obesity and the like. Small molecule inhibitors specific for FASN can reduce fatty acid synthesis by inhibiting FASN; and increasing activity of carnitine soft acyl transferase-1 in peripheral tissues such as liver and fat to enhance fatty acid oxidation and energy consumption, thereby reducing weight. In addition, the FASN inhibitor can also improve non-insulin dependent diabetes mellitus, reduce the symptoms of hypertension, coronary artery embolism and other obesity complications, and reduce the incidence rate.

Increased fatty acid production is one of the metabolic features of many cancer cells. The increased production of fatty acids may satisfy the cancer cell's excessive need for cellular components, lipid signaling molecules, and post-translational modifications of proteins. Thus, over-expression of Fatty Acid Synthase (FASN) has been reported in a variety of malignancies, including prostate, ovarian, breast, colorectal, and lung cancers, among others. Furthermore, high levels of FASN expression are associated with more tumor aggressiveness and poor prognostic outcome. Inhibition of FASN activity by drug molecules or silencing of it by small interfering rna (sirna) can disrupt lipid raft formation, thereby limiting oncogenic signaling pathways, depleting lipids required to maintain cell membranes, inducing toxic accumulation of malonyl-coa and ceramide, and thus inducing cancer cell apoptosis.

In conclusion, FASN has high expression in liver, fat cells and various tumor cells, and has become a new drug target for researching the diseases. The existing fatty acid synthetase inhibitors have the problems of undefined target, high effective concentration, poor solubility, serious side effect and the like. Therefore, the research of the novel FASN inhibitor has important significance for inhibiting the excessive biosynthesis of endogenous fatty acid and further effectively controlling the occurrence and development of tumors, obesity, various related metabolic syndromes and the like.

Disclosure of Invention

In order to solve the problems, the invention provides application of a compound shown as a general formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting fatty acid synthetase,

wherein, X₁、X₂、X₃Each independently selected from N, O, CH; is a double bond or a single bond; r₁Selected from hydrogen, alkyl; r₂Any one of hydrogen, halogen, hydroxyl, alkyl and alkoxy;

ring A is

Is a double bond or a single bond; u, V, Z are each independently selected from CH, N, NH; r₃、R₄Each independently selected from any one of hydrogen, halogen, aryloxy and alkoxy;

q is at least one heteroatom or no heteroatomC of (A)_1～5A straight or branched chain hydrocarbyl group, the heteroatoms each independently selected from nitrogen, oxygen, and sulfur;

n is 0 to 4; y is selected from nitrogen, oxygen or sulfur.

In one embodiment of the invention, ring a is a substituted or unsubstituted phenyl, naphthyl, [1,8] naphthyridinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzotriazolyl, indolyl, benzo-1, 3-dioxolyl, benzodioxanyl, benzothiadiazolyl, indazolyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolo [5,4-b ] pyridyl or oxazolo [5,4-c ] pyridyl; wherein the substituent is any one of halogen, aryloxy and alkoxy.

In one embodiment of the invention, Q is a terminal group of-O-, -N (R) -, -S-, -SO-, -SO₂-、-NRC(O)-、 -C(O)NR-、-N(R)SO₂-、-SO₂C of N (R) -, -OC (O) -or-C (O) O-_1～5A straight or branched chain hydrocarbon group.

In one embodiment of the present invention, the pharmaceutically acceptable salts of the compounds represented by the general formula (I) include: lactate, hydrochloride, phosphate, acetate, malate, citrate, or aspartate.

In one embodiment of the present invention, the compound of formula (I) comprises a compound represented by the following structure:

the compound shown in the general formula (I) or pharmaceutically acceptable salt thereof can be used as a fatty acid synthetase ZZ inhibitor, is used for inhibiting the activity of the fatty acid synthetase and plays a role in regulating the fatty acid composition.

The second purpose of the invention is to provide a pharmaceutical composition, which comprises any one or more of the compounds shown in the general formula (I) or pharmaceutically acceptable salts, solvates, esters, prodrugs, tautomers and stereoisomers thereof.

The composition also contains other medically acceptable auxiliary materials, including adhesive, filler, disintegrating agent, lubricant, antioxidant, flavoring agent, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc.

The third purpose of the invention is to provide a medicament for treating fatty acid metabolic diseases, which comprises any one or more of the compounds shown in the general formula (I) or pharmaceutically acceptable salts, solvates, esters, prodrugs, tautomers and stereoisomers thereof.

The fatty acid metabolism disorder is selected from obesity, cardiovascular and cerebrovascular diseases, hyperlipidemia, primary obesity, pulmonary hypertension, Hodgkin's disease, irritable bowel syndrome, cerebrovascular accident, atherosclerosis and diabetes, glomerulonephritis, and viral infection.

The fourth purpose of the invention is to provide a medicament for treating cancer, which comprises any one or more of the compounds shown in the general formula (I) or pharmaceutically acceptable salts, solvates, esters, prodrugs, tautomers and stereoisomers thereof.

The cancer is selected from ovarian cancer, breast cancer, uterine cancer, colon cancer, cervical cancer, lung cancer, prostate cancer, testicular cancer, thymus cancer, skin cancer, bladder cancer, pancreatic cancer, leukemia, lymphoma, non-small cell lung cancer, multiple tumor cancer, squamous cell cancer, kidney cancer, urinary tract cancer, bronchial cancer, esophageal cancer, bone cancer, throat cancer, bladder cancer, thyroid cancer, liver cancer, head and neck cancer, eye cancer, skin cancer, oral cancer, stomach cancer, colon cancer, rectal cancer, brain cancer, and central nervous system cancer.

The fifth purpose of the invention is to provide a medicine for treating immune diseases, wherein the medicine comprises any one or more of the compounds shown in the general formula (I) or pharmaceutically acceptable salts, solvates, esters, prodrugs, tautomers and stereoisomers thereof.

The immune disease is selected from multiple sclerosis, central nervous system injury, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, graft-versus-host disease, asthma and chronic obstructive pulmonary disease.

The dosage forms of the medicine comprise traditional dosage forms, such as decoction, pill, powder, paste, pellet, medicated wine, syrup, extract, lozenge, stick, suppository, leaven, moxibustion agent, etc.; also comprises modern dosage forms, such as tablet, granule, bagged steeping drug, oral liquid, capsule, dripping pill, mixture, tincture, aerosol, pellicle, injection, etc.

The medicine also contains other medically acceptable auxiliary materials, including adhesive, filler, disintegrating agent, lubricant, antioxidant, flavoring agent, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc.

The technical scheme provided by the invention has the following advantages:

(1) the compound with the structure shown in the general formula I or the pharmaceutically acceptable salt thereof can be used for preparing a fatty acid synthetase inhibitor, can inhibit the enzymatic activity of FASN, and is a novel FASN inhibitor. FASN is a key enzyme in de novo fatty acid synthesis in mammals and is involved in tumor development and progression. The excessive synthesis of endogenous fatty acid of tumor cells is expected to be effectively reduced through the function of the FASN inhibitor, so that the energy metabolism, the cell cycle regulation and the epithelial-mesenchymal transition of the tumor cells are influenced. Therefore, the fatty acid synthetase inhibitor has obvious inhibition effect on the enzyme activity of FASN, inhibits the proliferation of tumor cells, promotes the apoptosis of the tumor cells, exerts the treatment effect on tumors, and has important clinical application prospect.

(2) The fatty acid synthetase inhibitor can regulate the fatty acid composition and proportion, and can be applied to treating fatty acid metabolism diseases or immune system diseases.

(3) The pharmaceutical composition prepared from the fatty acid synthetase inhibitor has obvious inhibition effect on the fatty acid synthetase, can cause the cell cycle arrest of tumor cells, obviously reduce the transplanted tumor in the endosome of a living mouse and inhibit the formation of lipid droplets. Therefore, the medicine composition is an important medicine with a prospect of treating tumors, and provides a new treatment way for cancers or fatty acid metabolic diseases.

Drawings

FIG. 1 is a graph showing the effect of compounds of formula I on the proliferation of various tumor cells;

FIG. 2 shows that the compound of formula I can induce apoptosis of PC3 of prostate cancer cells;

FIG. 3 is a graph showing the effect of compounds of formula I on the fatty acid composition of PC3 from prostate cancer cells;

FIG. 4 shows the effect of compounds of formula I on lipid droplet synthesis in preadipocytes op 9;

FIG. 5 is a graph showing the effect of compounds of formula I on transplanted tumors of mouse prostate cancer cells PC 3.

Detailed Description

Halogen as referred to herein is fluorine, chlorine, bromine, iodine; the alkyl is C1-8 straight chain or branched chain alkyl; alkoxy is C1-8 straight chain or branched chain alkoxy; aryloxy is a phenyl group in which the aryl group is unsubstituted or contains the following substituents: halogen, (C)₃-C₁₀) -cycloalkyl, (C)₁-C₁₀) Alkyl radicals, (C)₂-C₆) -alkenyl, (C)₂-C₆) -alkynyl, O- (C)₁-C₆) Alkyl, O-CO- (C)₁-C₆) -an alkyl group.

Sources of Compounds 1-3, comparative Compound C-1: commercially available from Chemdiv (http:// www.chemdiv.com /), wherein Compound I-1 is given product number Y200-4916; the product number of the compound I-2 is Y207-4406; the product number of the compound I-3 is Y200-5086.

Wherein, the structures of the compounds 1-3 are shown as follows:

example 1

Detecting the effect of the compound shown in the following formula I-1 on the FASN activity in HCT116 of colon cancer cells:

detecting FASN enzyme activity:

(1) logarithmic phase cells were collected, cell suspension concentration was adjusted to 6cm diameter petri dish per 2x10⁶Preferably one cell/dish.

(2) At 5% CO₂Incubation is carried out at 37 ℃, the original culture medium is discarded after the cells are completely attached, 6ml of 1% FBS culture medium containing 20 mu M of compound I-1 is respectively added into each dish of cells, and 6ml of 1% FBS culture medium containing 20 mu M of cerulenin is added as a positive control.

(3) And after incubation for 4 hours, removing the supernatant, carrying out trypsinization for 2min, stopping digestion by using complete culture medium with 2-4 times of the volume of the pancreatin, centrifuging for 5min at 1000rpm, removing the supernatant, carrying out cell resuspension on 5ml of DPBS (platelet-rich plasma lysate), taking 500-1.5 ml of EP (Epstein-Barr) tube for protein quantification, and centrifuging the rest to remove the supernatant.

(4) 1ml of 10mM KH₂PO₄The cells were resuspended in KOH pH6.5 buffer (containing 4mM DTT, 0.3mg/ml BSA, 2.5mM EDTA), NADPH, acetyl-CoA and malonyl-CoA were added to give final concentrations of 0.14mM, 0.18mM and 0.09mM, respectively, and their absorbance values were measured at 340nm for 1 min.

Definition of FASN enzyme Activity: definition of activity units: 1 μmol NADPH per minute per mg protein at 37 ℃ is oxidized to 1U;

FASN (U/mg protein) [ (Delta A)_{Measuring tube}-ΔA_{Blank tube})]÷ε÷d×V_{General assembly}×10⁶]÷(Cpr×V_{Sample (A)})÷T＝1.16×(ΔA_{Measuring tube} -ΔA_{Blank tube})÷Cpr；

The effect of inhibitors on the activity of FASN enzyme (inhibition rate) is defined as: FASN (%) - (1- (FASN)_Inhibitors÷FASN _Control)]×100％；

Wherein epsilon: NADPH molar extinction coefficient, 6.22X 10³l/mol/cm; d: the optical path of the cuvette is 1 cm; v_{General assembly}: the total volume of the reaction system was 1000. mu.L-0.001L; cpr: supernatant protein concentration, mg/ml; and V sample: adding the volume of the supernatant in the reaction system, wherein 100 mu l of the supernatant is 0.1 ml; t: reaction time, 1 min; FASN_Inhibitors: (ii) cellular FASN enzyme activity treated with each inhibitor; FASN_Control: cellular FASN enzyme activity without inhibitor treatment.

IC50 value determination:

IC50(half maximum inhibition concentration) refers to the half inhibitory concentration of the antagonist measured. It indicates that a certain drug or substance (inhibitor) induces apoptosis of tumor cells by 50% at a concentration, referred to as the 50% inhibitory concentration, i.e., the concentration corresponding to a ratio of apoptotic cells to total cell number equal to 50%. The IC50 value can be used to measure the ability of a drug to induce apoptosis, i.e., the greater the ability to induce, the lower the value, and can be used to reverse the degree of tolerance of a cell to a drug.

(1) Collecting logarithmic phase cells, adjusting the concentration of cell suspension, preferably using a 96-pore plate with 100 mu l per hole, adjusting the density of the cells to be detected to 1000-10000 cells per hole by using the plate, and filling the edge holes with sterile PBS.

(2) 5% CO2, 37 ℃ incubation of cells, after complete cell adhesion and discard of original medium, 200. mu.l of 1 wt% FBS medium containing 0.01. mu.M, 0.1. mu.M, 1. mu.M, 5. mu.M and 10. mu.M Compound I-1 or Cerulenin (Cerulenin) was added to each well, and the number of duplicate wells was 6. An equal volume of DMSO was added as a negative control.

(3) After 72h incubation, the cell status was recorded by photography and the stock culture was discarded, and 100. mu.l of serum-free medium containing 0.5mg/ml MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide, trade name: thiazole blue) was added to each well.

(4) After incubation for 2-4 h, the stock culture solution is discarded, 150. mu.l of dimethyl sulfoxide (DMSO) is added into each well, and the mixture is shaken at 300rpm for 10min to fully dissolve the purple crystals.

(5) In an enzyme linked immunosorbent assay (ELISA) detector at OD_570nmThe absorbance of each well was measured and expressed as OD_630nmAnd (3) taking the light absorption value as reference, carrying out dual-wavelength determination, and calculating the survival rate result by the following formula:

cell survival rate ═ OD₅₇₀-OD₆₃₀)/(OD_{570 negative control}-OD_{630 negative control})；

Wherein, OD₅₇₀: OD of each treatment group₅₇₀A value; OD₆₃₀: OD of each treatment group₆₃₀A value; OD₅₇₀Comparison: OD of negative control group₅₇₀A value; OD₆₃₀Comparison: OD of negative control group₆₃₀The value is obtained.

(6) Survival results were processed with the software graphpad6.0 at a concentration gradient in the analysis mode log (inhibitor) vs. Nonlinear regression (curve fit), and the calculated IC50 values were expressed as x ± SD.

Example 2

Detecting the effect of the compound shown in formula I-1 on the proliferation of tumor cells (PC3, HT-29, Hela, HepG 2):

(2) 5% CO2, 37 ℃ incubation of cells, after cells complete adherence, abandoning the original culture medium, adding 200 μ l of 1 wt% FBS culture medium containing 10 μ M compound I-1 into each well, the number of the multiple wells is 6. The same concentrations of fatty acid synthetase inhibitor C75, Cerulenin (Cerulenin) and GSK2194069 (hereinafter referred to as GSK) were added as positive controls, and the same volume of DMSO was added as a negative control, respectively.

(5) In an enzyme linked immunosorbent assay (ELISA) detector at OD_570nmThe absorbance of each well was measured and expressed as OD_630nmTaking the light absorption value as reference, carrying out dual-wavelength measurement, and calculating the result by the following formula:

Wherein, OD₅₇₀: OD of each treatment group₅₇₀A value; OD₆₃₀: OD of each treatment group₆₃₀A value; OD₅₇₀Comparison: OD of negative control group₅₇₀A value; OD₆₃₀Comparison: negative controlOD of group₆₃₀The value is obtained.

FIG. 1 shows the effect of fatty acid synthase inhibitor C75, cerulenin and GSK, and of compound I-1 on the proliferation of 7 different tumor cells in total 22RV1, PC3, HT-29, Hela, HepG2, CaCo-2 and HCT 116. As can be seen from fig. 1, C75, cerulenin and GSK, which are fatty acid synthase inhibitors, have the effect of inhibiting tumor cell proliferation, but have a large difference in effect among different types of tumor cells, have no significant effect on proliferation of some tumor cells, and have poor stability. Compared with the three inhibitors, the compound I-1 has obviously improved inhibition effect on tumor proliferation, and can effectively inhibit various tumor cells.

Example 3

Detecting the effect of the compound shown as the formula I-1 on the apoptosis of the prostate cancer cell PC 3:

(1) taking cancer cells in logarithmic growth phase, inoculating the cancer cells in 6cm culture dishes, wherein the inoculation amount of each dish is 2x10⁶Single cell, 5% CO₂Incubation at 37 ℃.

(2) After the cells were completely attached, 6ml of 2% FBS medium containing 10. mu.M of the structural compound of formula I was added.

(3) After 24h and 48h incubation respectively, the supernatant is discarded, pancreatin without EDTA is digested for 2min, digestion is stopped by complete culture medium with 2-4 times of pancreatin volume, centrifugation is carried out at 1000rpm for 5min, the supernatant is discarded, 1ml of precooled DPBS is washed twice, and the supernatant is discarded.

(4) 300. mu.l of 1 XBinding Buffer resuspended cells.

(5) Add 5. mu.l Annexin V-FITC and mix well, incubate for 15min at room temperature in the dark.

(6) Adding 5 μ l PI dye solution, mixing, and incubating at room temperature in dark for 5 min;

(7) adding 200 mul of 1 × Binding Buffer and then performing upper-level detection by using a flow cytometer.

FIG. 2 is a graph showing the effect of compounds of formula I on apoptosis of PC3 in prostate cancer cells. Fig. 2 shows that the proportion of PC3 cells undergoing initial apoptosis and late apoptosis of prostate cancer cells treated with cerulenin at 10 μ M and compound I-1 for 24h and 72h, respectively, is significantly increased compared to PC3 cells of prostate cancer cells of a control group not treated with an inhibitor, and particularly that of the test group treated with compound I-1, the total proportion of apoptosis (initial apoptosis + late apoptosis) at 24h is 29.9%, and the total proportion of apoptosis at 72h is 38.5%, which is not only higher than that of the control group (0.498% and 8.20%), but also significantly higher than that of the cerulenin-treated group as a positive reference (23.87% and 34.5%). Therefore, the structural compound shown in the formula I can induce PC3 cell apoptosis, and the compound with the structure shown in the formula I can inhibit the occurrence and development of tumors and is applied to clinical treatment of tumors as a novel therapeutic drug.

Example 4

Detecting the influence of the compound with the structure shown in the formula I-1 on the content and the composition of fatty acid synthesized by tumor cells (prostate cancer cells PC 3):

(1) taking cancer cells in logarithmic growth phase, and plating on 6cm cell culture dish plate with density of 2x10⁶Single cell, 5% CO₂Incubation at 37 ℃.

(2) After the cells adhered to the wall, 6ml of 1% FBS medium containing 20. mu.M of the compound of formula I was added to each dish, and the medium was used as a negative control and cerulenin was used as a positive control.

(3) After incubation for 24h, the medium is discarded, the digestion is carried out for 2min by pancreatin, the digestion is stopped by completely culturing the medium with 2-4 times of the volume of the pancreatin, the centrifugation is carried out for 5min at 1000rpm, and the supernatant is discarded.

(4) Resuspending 10ml of DPBS, taking 1ml of cell suspension to a 1.5ml ep tube for protein quantification, centrifuging the rest 9ml again, discarding supernatant, adding an appropriate volume of internal standard, and freeze-drying.

(5) Adding 1ml of 0.5M NaOH-methanol solution, charging nitrogen, performing vortex oscillation for 30s, performing solid bath at 100 ℃ for 5min, and cooling to room temperature.

(6) Adding 1ml of 40% boron trifluoride-methanol solution, charging nitrogen, performing vortex oscillation for 30s, performing solid bath at 100 ℃ for 5min, and cooling to room temperature.

(7) Adding 4ml of n-hexane and 2ml of saturated sodium chloride solution, carrying out vortex oscillation, centrifuging at 2000rpm for 10min, taking the supernatant, blowing nitrogen to be complete, resuspending 500 mu l of n-hexane, transferring to a sample bottle, and detecting on a gas chromatography-mass spectrometer.

FIG. 3 shows the effect of compounds of formula I on the content and composition of fatty acids PC3 in prostate cancer cells, including myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1) and linoleic acid (18: 2). As can be seen from FIG. 3, the total fatty acid content and 16:0 content of PC3 cells treated by the compound of formula I are both significantly lower than those of the control group, which indicates that the compound of formula I can inhibit the fatty acid synthesis of PC3, change the fatty acid distribution in tumor cells, thereby reduce the structural lipid and energy required by cell proliferation, and is an important reason for inhibiting tumor proliferation and inducing tumor cell apoptosis, therefore, the compound of formula I has an important clinical application prospect in the aspect of tumor treatment.

Example 5

Examining the effect of a compound of the structure shown in formula I-1 on preadipocytes OP 9:

(2) After the cells are attached to the wall, 6ml of culture medium containing 0.5 mu M rosiglitazone is added into each dish to induce OP9 cells to differentiate, the group without rosiglitazone is taken as a complete control, and the culture medium added in the inhibitor group not only contains 0.5 mu M rosiglitazone, but also contains 20 mu M cerulenin or the compound shown in the formula I.

(3) After incubation for 5 days, removing the culture medium, washing 1ml of DPBS once in each hole, adding 1ml of 4% paraformaldehyde solution into each hole, and incubating for half an hour at room temperature in a dark place;

(4) discarding the supernatant, washing once with 1ml DPBS per hole, and adding 500 mul of oil red O working solution; water bath is carried out at 60 ℃ in dark for 1 h;

(5) discarding the supernatant, and washing with 1ml DPBS for three times in each hole; photographing and recording an experimental result;

(6) the supernatant was discarded, 500. mu.l of isopropanol was added to each well, incubated at 37 ℃ with shaking for 10min, and the OD was measured at 510 nm.

FIG. 4 shows the effect of compounds of formula I on lipid droplet synthesis by preadipocytes op 9. OP9 cell OD treated with a Compound of formula I₅₁₀The value is obviously reduced, the reduction amplitude is obviously greater than that of a cerulenin treatment group, and the structure shown in the formula I can inhibit the synthesis of OP9 cell lipid droplets。

Example 6

Detecting the effect of a compound having the structure shown in formula I on the growth of a mouse graft:

(1) culturing cancer cells to a density of 80% (logarithmic phase), performing cell passage experiment, washing with PBS, digesting, centrifuging to remove supernatant, and then suspending and washing the cells twice with a serum-free culture medium; adjusting the cell suspension concentration to 2X10 with serum-free medium⁷Per ml; adding matrigel which is melted overnight at four degrees in an equal volume, and uniformly mixing by shaking for later use (all the steps containing the matrigel are operated on ice);

(2) inoculating 100 μ l of mixed solution of cells and matrigel subcutaneously on the back of each nude mouse, observing the growth condition of tumor every day, determining whether the tumor model is transplanted successfully, and waiting for the tumor volume to be about 200mm³Starting drug treatment;

(3) experiment selection of tumor transplantation model successful nude mice (tumor formation rate 100%), nude mice adapted to be fed for 7 days, subcutaneous cancer cell transplantation experiment, after tumor bodies are visible by naked eyes, intraperitoneal injection is carried out every 2 days, the injection dose is 5mg/kg of compound I-1, the injection solvent group is used as a control, and 5 mice in each group are used. After 4 weeks of administration, tumor bodies were weighed.

FIG. 5 is a graph showing the effect of compounds of formula I on transplanted tumors of mouse prostate cancer cells PC 3. As shown in the figure, the tumor quality of mice treated by the compound shown in the formula I-1 for four weeks is obviously lower than that of a control group, which shows that the compound shown in the formula I can inhibit the proliferation of tumor cells in vivo, and has important application prospect as a novel antitumor drug.

Example 7

The inhibitory effect of the compounds I-1 to I-5 and the comparative compound C-1 on FASN activity in HCT116, as well as on the proliferation of tumor cells PC3, were examined.

The influence of different substances on the activity of FASN enzyme was examined by the method for measuring the activity of FASN enzyme shown in Experimental example 1, the influence of different substances on the proliferation of tumor cells was examined by the method shown in Experimental example 2, and the inhibition (%) of FASN enzyme activity and the inhibition of tumor proliferation by different substances were calculated using cerulenin as a controlInhibition rate (IC) of₅₀nM). The results are shown in table 1 below:

TABLE 1 inhibitory Effect of different substances on FASN enzymatic Activity and tumor proliferation

	Inhibition ratio of FASN enzyme Activity (%)	PC3(IC₅₀nM)
			Cerulenin	20.52	19±2.5
Compound I-1	55.26	10±1.2
			Compound I-2	34.37	16±1.9
Compound I-3	41.77	13±1.1

As can be seen from the above Table 1, the compounds I-1 to I-5 provided by the present invention all have the effect of effectively inhibiting the activity of fatty acid synthetase, wherein the highest inhibition rate of the compound I-1 on Fatty Acid Synthetase (FASN) is 55.26%, which is significantly higher than the inhibition rate of cerulenin, which is a positive control, by 20.52%; according to the result of IC50, the effective concentration of compound I-1 is obviously lower than cerulenin, and the death rate of prostate cancer cell PC3 can reach 50% at 10 +/-1.2 nM. In addition, the applicant also found that if the hydroxyl group on the A ring is substituted, the inhibition rate is only 18.22%, the IC50 result is higher, and the blue pigment is not obviously improved or even is not enough.

The Fatty Acid Synthetase (FASN) participates in the metabolism and cell cycle process of tumor fatty acid, plays an important role in the growth, invasion and migration of tumor, and the compound with the structure shown in the general formula I provided by the application can be used as a FASN inhibitor, influences the synthesis and distribution of fatty acid in tumor cells, leads the cell cycle to be stopped at the interphase, organizes the mitosis of the tumor cells, and realizes the inhibition of tumor proliferation. Therefore, the compound with the structure shown in the general formula I as a novel fatty acid synthetase inhibitor has important application prospect in clinical treatment of tumors and treatment of metabolic diseases such as obesity and the like.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof in preparing the medicament for inhibiting the fatty acid synthetase,

wherein, X₁、X₂、X₃Each independently selected from N, O, CH;

is a double bond or a single bond; r₁Selected from hydrogen, alkyl; r₂Selected from hydrogen, halogen, hydroxy, alkyl, alkoxyAny one of the above groups;

ring A is

q is at least one heteroatom or C containing no heteroatom_1～5A straight or branched chain hydrocarbyl group, the heteroatoms each independently selected from nitrogen, oxygen, and sulfur;

n is 0 to 4; y is selected from nitrogen, oxygen or sulfur.

2. Use according to claim 1, wherein the aromatic ring of ring a in formula (I) is phenyl, naphthyl, [1,8] naphthyridinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzotriazolyl, indolyl, benzo-1, 3-dioxolyl, benzodioxinyl, benzothiadiazolyl, indazolyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolo [5,4-b ] pyridyl or oxazolo [5,4-c ] pyridyl, substituted or unsubstituted as substituent; wherein the substituent is any one of halogen, aryloxy and alkoxy.

3. Use according to claim 1, wherein Q in formula (I) is selected from the group consisting of terminal groups-N (R) -, -S-, -O-, -SO₂-、-NRC(O)-、-C(O)NR-、-N(R)SO₂-、-SO₂C of N (R) -, -OC (O) -or-C (O) O-_1～5A straight or branched chain hydrocarbon group.

4. The use according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt of the compound of formula (I) comprises: lactate, hydrochloride, phosphate, acetate, malate, citrate, or aspartate.

5. A pharmaceutical composition for inhibiting fatty acid synthase, comprising a compound represented by the general formula (I) of claim 1 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further comprises other pharmaceutically acceptable excipients, including binders, fillers, disintegrants, lubricants, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, tonicity adjusting agents, and colorants.

7. Use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disorder of fatty acid metabolism.

8. Use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer or immune disorders.

9. The use according to claim 1 or 7 or 8, wherein the medicament is in the form of decoction, pill, powder, paste, pellet, medicated wine, syrup, extract, lozenge, stick, suppository, yeast, moxibustion, etc.; also comprises modern dosage forms, such as tablet, granule, bagged steeping drug, oral liquid, capsule, dripping pill, mixture, tincture, aerosol, pellicle, injection, and injection.

10. The use according to claim 1 or 7 or 8, wherein the medicament further comprises other medically acceptable auxiliary materials, including binding agents, fillers, disintegrating agents, lubricating agents, antioxidants, flavoring agents, aromatizing agents, solubilizing agents, emulsifying agents, solubilizing agents, tonicity adjusting agents, coloring agents.