CN113057955A

CN113057955A - Medicine for inhibiting stearoyl-CoA desaturase 1

Info

Publication number: CN113057955A
Application number: CN202110257342.1A
Authority: CN
Inventors: 吴国胜; 杨晨; 金忆媛; 唐春雷; 陈永泉; 崔国祯
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-07-02
Anticipated expiration: 2041-03-09
Also published as: CN113057955B

Abstract

The invention discloses a medicine for inhibiting stearoyl-CoA desaturase 1, belonging to the technical field of medical biotechnology. The invention firstThe compound with the structure shown in the formula I is discovered to have the effect of inhibiting stearoyl-CoA desaturase 1, can inhibit the activity of saturated fatty acid desaturase, plays a role in regulating fatty acid composition, and provides an effective new means and a new way for treating diseases targeted by SCD1 drugs.

Description

Medicine for inhibiting stearoyl-CoA desaturase 1

Technical Field

The invention relates to a medicine for inhibiting stearoyl-CoA desaturase 1, belonging to the technical field of medical biotechnology.

Background

Stearoyl-coa desaturase 1(SCD1) is highly expressed in metabolism-related cells, some tumor cells, and SCD1 has become a new target for drugs for studying these diseases. The research of the SCD1 inhibitor has important significance for inhibiting the synthesis of monounsaturated fatty acid and further effectively controlling the occurrence and development of tumors, obesity, various related metabolic syndromes and the like.

The SCD1 small molecule inhibitors mainly comprise CAY-10566, MF438, A939572, CVT-11127, CVT-12012, T-3764518, PluriSIn 1, SW203668, BZ36, SSI-4 and the like (structural formula is shown in 1-11), and do not have a mother ring structure similar to that of the compound A.

Disclosure of Invention

The invention discovers that the compound with the structure shown in the formula I has the function of inhibiting stearoyl-CoA desaturase 1, can inhibit the activity of saturated fatty acid desaturase and plays a role in regulating fatty acid composition. Based on the fact, the invention is applied to the preparation of the drug for inhibiting stearoyl-CoA desaturase 1, and provides an effective new means and a new way for treating the diseases of SCD1 drug targets.

The invention provides the following scheme:

the use of a compound of formula I for the manufacture of a medicament for inhibiting stearoyl-CoA desaturase 1,

in one embodiment of the present invention, the dosage form of the drug can be selected from traditional dosage forms, including 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.

In one embodiment of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, wherein the pharmaceutically acceptable excipients comprise a binder, a filler, a disintegrant, a lubricant, an antioxidant, a flavoring agent, an aromatic agent, a cosolvent, an emulsifier, a solubilizer, an osmotic pressure regulator, a colorant, and the like.

In one embodiment of the present invention, the drug further comprises a carrier, and the carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

The second purpose of the invention is to apply the compound with the structure shown in the formula I in the preparation or development of the medicine for treating the fatty acid metabolism diseases.

In one embodiment of the present invention, the fatty acid metabolism disorder is selected from obesity, fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis and diabetes.

The third purpose of the invention is to use the compound with the structure shown in the formula I in preparing or developing the medicine for treating cancer.

In one embodiment of the invention, the cancer is selected from breast cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, lung cancer, ovarian cancer, thyroid cancer.

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

(1) the compound A can be used for preparing an inhibitor of stearoyl-CoA desaturase 1, can inhibit the activity of SCD1, and is a novel SCD1 inhibitor. SCD1 is a key enzyme in cell lipid metabolism and is involved in the development of tumors. SCD1 has important effects in controlling lipid composition of tumor cells, regulating energy metabolism, endoplasmic reticulum stress, apoptosis, etc., and is expected to become a target for tumor therapy. The SCD1 inhibitor is effective in controlling tumor growth by inhibiting desaturation of saturated fatty acids, altering intracellular energy metabolism, etc. Therefore, the inhibitor of stearoyl-CoA desaturase 1 has obvious inhibition effect on the enzyme activity of SCD1, can inhibit the growth of tumor cells, influence the energy metabolism of the cells, promote autophagy and apoptosis, play a role in treating tumors, and has important clinical application prospect.

(2) The inhibitor of stearoyl-CoA desaturase 1 can regulate the proportion of fatty acids in cells and change the saturation proportion, and can be used for treating diseases related to fatty acid metabolism and the like.

Therefore, the compound A is an important medicament with a prospect of treating tumors, and provides a new treatment way for cancers and fatty acid metabolic diseases.

Drawings

FIG. 1 is a graph of the effect of Compound A, Compound B, Compound C and Icaritin on SCD1 enzyme activity in PC-3 cells from prostate cancer;

FIG. 2 is a graph of the effect of Compound A on SCD1 expression in breast cancer MCF-7 cells and prostate cancer PC-3 cells;

FIG. 3 shows the effect of Compound A, Compound B, Compound C and Icaritin on the proliferation of various tumor cells;

FIG. 4 is a graph of the apoptotic effect of Compound A on breast cancer MCF-7 cells;

FIG. 5 is a flow cytometry result of the effect of Compound A on mitochondrial membrane potential of breast cancer MCF-7 cells.

Detailed Description

The present invention will be described in detail with reference to examples.

The icariin aglycone (ICT) raw material of the present invention can be prepared by the method described in the conventional method (CN 102167690A).

Example 1: preparation of the Compounds

Synthesis of compound a (structure of formula I):

in a 250mL round bottom flask, 800mg of ICT was added, 150mL of acetone, 324mg of 1-bromo-3-methyl-2-butene, 300mg of anhydrous potassium carbonate was added, reflux was carried out at 56 ℃ for 4 hours, cooling was carried out, a part of acetone was distilled off, the remaining reaction solution was adjusted to PH 4 to 5 with 1N HCl, and then extracted three times with 100mL of dichloromethane, and after the organic layer was dried over anhydrous sodium sulfate, separation was carried out by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain 130mg of compound a (yield ═ 13.71%).

¹H NMR(400MHz,Chloroform-d)：δ12.75(s,1H),8.12(d,J＝9.0Hz,2H),7.03(d,J＝9.1Hz,2H),6.31(s,1H),6.12(s,1H),5.44(t,J＝7.3Hz,1H),5.32(s,1H),4.58(d,J＝7.3Hz,2H),3.92(s,3H),3.59(d,J＝7.3Hz,2H),1.86(s,3H),1.79(s,3H),1.71(s,3H),1.65(s,3H).

Synthesis of Compound B:

in a 250mL round bottom flask, 800mg of ICT was added, 150mL of acetone, 324mg of 1-bromo-3-methyl-2-butene, 300mg of anhydrous potassium carbonate was added, reflux was carried out at 56 ℃ for 4 hours, cooling was carried out, a part of acetone was evaporated, the remaining reaction solution was adjusted to PH 4 to 5 with 1N HCl, and then extracted three times with 100mL of dichloromethane, and after the organic layer was dried over anhydrous sodium sulfate, separation was carried out by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain 200mg of compound B (yield ═ 18.25%).

¹H NMR(400MHz,Chloroform-d)：δ12.82(s,1H),8.15(d,J＝9.0Hz,2H),7.02(d,J＝9.0Hz,2H),6.40(s,1H),5.50(d,J＝6.5Hz,1H),5.45(t,J＝7.4Hz,1H),5.24–5.20(m,1H),4.61(d,J＝6.6Hz,2H),4.57(d,J＝7.3Hz,2H),3.91(s,3H),3.52(d,J＝7.0Hz,2H),1.82(s,3H),1.79(s,3H),1.77(s,3H),1.72(s,3H),1.70(s,3H),1.65(s,3H).

Synthesis of Compound C:

to a 250mL round bottom flask, 800mg of ICT was added, 150mL of acetone, 324mg of 1-bromo-3-methyl-2-butene, 300mg of anhydrous potassium carbonate was added, reflux was performed at 56 ℃ for 4 hours, cooling was performed, a part of acetone was evaporated, the remaining reaction solution was adjusted to PH 4 to 5 with 1N HCl, and then extracted three times with 100mL of dichloromethane, and after the organic layer was dried over anhydrous sodium sulfate, separation was performed by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain 90mg of compound C (yield ═ 9.49%).

¹H NMR(400MHz,Chloroform-d)：δ11.81(s,1H),8.21(d,J＝9.0Hz,2H),7.05(d,J＝9.0Hz,2H),6.61(s,1H),6.44(s,1H),5.52–5.48(m,1H),5.26–5.22(m,1H),4.63(d,J＝6.6Hz,2H),3.91(s,3H),3.56(d,J＝7.0Hz,2H),1.82(s,6H),1.78(s,3H),1.70(s,3H).

Example 2:

effect of compound a, compound B, compound C, Icaritin (ICT) on SCD1 enzyme activity in prostate cancer cells:

detecting the enzyme activity of SCD 1:

(1) logarithmic phase cells were collected, cell suspension concentration was adjusted, and the cells were plated at 4X10 on 10cm diameter format dishes⁶Preferably one cell/dish.

(2) At 5% CO₂Incubating at 37 deg.C, removing culture medium after cells adhere completely, and adding 8mL of culture medium containing 0.2mM [ d31] into each dish of cells]PA (isotope d31 labeled palmitic acid, linear formula: CD)₃(CD₂)₁₄CO₂H) And culture media of compound a at different concentrations, with SCD1 inhibitor MF-438 as a positive control.

(3) After 24h incubation the supernatant was discarded, washed twice with PBS and 1ml CH was added₃OH scraper offCells were sonicated and broken into liposuction vials.

(4) 3ml of CH was added to each sample₃OH、2ml CHCl₃6mol/L HCl50 mu L, and performing leak detection and oscillation for 1 h. 2ml of CHCl were added₃、2mlH₂O, shaking, centrifuging at 2500rpm for 10min, transferring the lower layer into a new grease extraction bottle, and blowing nitrogen to dry.

(5) The lipid was saponified by adding 1ml of a 0.5mol/L NaOH methanol solution and solid-bathing at 100 ℃ for 5 min.

(6) 1ml of 14% BF was added₃-CH₃OH, solid bath at 100 ℃ for 5min to prepare Fatty Acid Methyl Ester (FAME).

(7) Adding 1ml of C₆H₁₄Shaking 1ml saturated NaCl, centrifuging at 2000rpm for 2min, collecting the upper layer liquid, blowing nitrogen to dry, 300 μ l C₆H₁₄Redissolving, and analyzing the sample by GC-MS to determine the cell fatty acid spectrum.

Definition of SCD1 enzyme activity: the conversion rate of [ d31] PA was examined by converting isotope-labeled [ d31] PA into isotope-labeled fatty acid methyl ester, thereby determining a cellular fatty acid profile, and the ratio of [ d31] C16: the ratio of 1/([ d31] C16: 1+ [ d31] C16: 0) (where [ d31] C16:0, [ d31] C16:1 are isotopic d 31-labeled palmitic acid and palmitoleic acid, respectively) was used as an indicator of SCD1 enzyme activity.

Detecting the expression level of SCD 1:

western-blot analysis of the changes in the expression level of SCD1 protein in breast cancer cells and prostate cancer cells treated with Compound A. The Western-blot comprises the following specific steps:

A. preparing a solution:

(1) 10% (w/v) sodium dodecyl sulfate SDS solution: 0.1g SDS, 1mL H₂Preparing deionized water O, and storing at room temperature.

(2) Separating gel buffer solution: 18.15g of Tris was weighed, dissolved in 80mL of water, adjusted to pH8.8 with HCl and diluted to a final volume of 100mL with water to give a solution of 1.5mmol/L Tris-HCl (pH 8.8).

(3) Concentrating the gel buffer: 6.05g Tris was dissolved in 80mL water, adjusted to pH6.8 with about HCI, and diluted to 100mL final volume with water to give a 0.5mmol/L Tris-HCl (pH6.8) solution.

(4) SDS-PAGE loading buffer: 8mL of 0.5mol/L Tris buffer solution with pH of 6.8, 6.4mL of glycerol, 12.8mL of 10 wt% SDS, 3.2mL of mercaptoethanol, 1.6mL of 0.05 wt% bromophenol blue, and H₂O32 mL, mix well for use.

(5) Tris-glycine electrophoresis buffer: 30.3g Tris, 188g glycine and 10g SDS were weighed, dissolved in distilled water to 1000ml and diluted 10-fold before use.

(6) And (3) membrane transfer buffer solution: 14.4g of glycine and 6.04g of Tris were weighed, 200ml of methanol was added thereto, and finally water was added to make a total volume of 1L.

(7) Tris Buffered Saline (TBS): containing 20mM Tris-HCl (pH7.5) and 500mM NaCl.

(8) Phosphate Buffer (PBS) 80g NaCl, 36.3g NaH were weighed₂PO₄·12H₂O, 2gKCl and 2.7gKH₂PO₄Dissolving in 900ml distilled water, adjusting pH to 7.4, adding water to total volume of 1L, and diluting 10 times before use.

B. Washing cancer cells with PBS for 3 times, adding lysis solution, boiling for 5min, cooling on ice, centrifuging at 12000rpm for 2min, collecting supernatant, and storing at-20 deg.C.

C. Protein concentration was determined using BCA method.

Adding a standard protein gradient of 0.5mg/ml into a pore plate, and adding PBS to make up to 20 mu l; add appropriate volume (3. mu.l) of protein sample to the well plate, make up to 20. mu.l with PBS; adding 200 μ l BCA working solution (prepared before use and used as prepared) into each well, and incubating at 37 ℃ for 30 min; the absorbance at 562nm was measured and the protein concentration was calculated from the standard curve and the sample volume.

SDS-PAGE gel electrophoresis

(1) The two cleaned glass plates are aligned and then clamped in a clamp, and then vertically clamped on a frame for glue pouring.

(2) Pouring 10 wt% of separation gel: adding TEMED (tetramethylethylenediamine) into the separating gel buffer solution, shaking, pouring into the gap between the two glass plates, sealing with ethanol solution, completely solidifying the gel, removing the ethanol on the gel upper layer, and drying with absorbent paper.

(3) 5 wt% of concentrated gum was poured: adding TEMED (tetramethylethylenediamine) into the concentrated gel buffer solution, shaking, pouring into the upper layer of the separation gel, filling the rest space with concentrated gel, and inserting a comb into the concentrated gel. After the concentrated gel is solidified, the concentrated gel is pulled out vertically and slightly upwards.

(4) And (4) putting the glass plate after the glue pouring into an electrophoresis tank, and adding sufficient electrophoresis liquid to prepare for sample loading. After the protein content of the protein sample is measured, 5 xSDS loading buffer is added, the mixture is boiled in boiling water for 3min and mixed evenly, and then the loading is carried out, wherein the total protein amount of the loading is 35 mu g.

(5) And (3) performing constant-pressure 80V electrophoresis gel running, performing constant-pressure 120V electrophoresis after the sample enters the lower layer gel until bromophenol blue reaches the bottom of the gel, and performing membrane rotation.

E. Rotary film

(1) Cutting the glue: prying off the glass plate, removing the small glass plate, scraping off the concentrated glue, and cutting the glue according to the molecular weight of the protein and the experimental requirement by taking a Marker as a contrast.

(2) Preparing a film: the PVDF membrane and filter paper were cut and the cut PVDF was activated for 30s in 80% methanol solution.

(3) Film loading: the film transfer clip was opened to keep the black side (negative electrode) horizontal. And (3) filling a sponge cushion on the upper surface, adding the membrane transferring liquid for soaking, soaking the filter paper in the upper cushion of the cushion, and then sequentially stacking the gel, the nitrocellulose membrane, the filter paper and the sponge cushion. And finally, covering the white plate (anode) and putting the white plate into a film transferring groove.

(4) Film transfer: the clip is placed in the transfer tank so that the black side of the clip faces the black side of the tank and the white side of the clip faces the red side of the tank. The film is rotated for 120min in ice bath and the constant current is 180 mA.

(5) After the rotation is finished, the film is taken down, and a corner is marked.

F. Immune response

(1) And (3) sealing: the membranes were rinsed 3 times for 5min each in TBST. After rinsing, the PVDF membrane is put into 5 percent of skimmed milk powder and shaken for 1h at room temperature.

(2) Plus primary antibody (SCD 1): the PVDF membrane after blocking was placed in a TBST-containing wash tank and rinsed 3 times for 5min each time on a shaker. The cells were incubated overnight at 4 ℃ in a dish to which primary antibody was added.

(3) Secondary antibody (mouse or rabbit antibody): recovering primary antibody, rinsing PVDF membrane in TBST washing jar for 5min 3 times at room temperature by shaking table, placing the rinsed PVDF membrane in a dish containing secondary antibody, and incubating for 45min at room temperature in dark. After incubation, the PVDF membrane was washed 3 times in TBST wash tank, 5min each time.

G. Chemiluminescence

The chromogenic reagent was mixed in a small centrifuge tube according to the protocol of the luminescence kit, added to the PVDF membrane and developed with a chemiluminescent imager.

FIG. 1 shows the variation of SCD1 enzyme activity of PC-3 cells from prostate cancer under different conditions. Wherein, the compound A has obvious inhibition effect on SCD1 activity in PC-3 cells, and the proportion of monounsaturated fatty acid palmitoleic acid containing isotope labeling is obviously reduced under the condition of exogenous addition of [ d31] PA. Specific inhibition results are shown in table 1.

TABLE 1 SCD1 enzymatic Activity of PC-3 cells from prostate cancer under different conditions

FIG. 2 shows the effect of compound A on the expression level of SCD1 in MCF-7 and PC-3 cells at different concentrations, and compared with the control group, compound A can effectively inhibit the expression of SCD1 and is dose-dependent.

The above results indicate that compound a is capable of acting as a novel stearoyl-coa desaturase 1 inhibitor, resulting in an effective inhibition of SCD 1. The suggestion shows that the compound A can be used as a therapeutic drug for relevant metabolic diseases such as tumor, obesity and the like, and can prevent and/or treat the occurrence and development of the diseases.

Example 3:

detecting the effect of compound A of formula I on tumor cell proliferation (MCF-7, MDA-MB-231, PC-3, and LNCaP):

(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% CO₂Incubating the cells at 37 ℃, discarding the original culture medium after the cells are completely attached, and adding 100 mu l of 10% FBS culture medium containing 2.5, 5, 10, 20, 40 and 80 mu M of compound A into each well, wherein the number of the multiple wells is 4.

(3) After 24 hours of incubation, the cell status was recorded by photographing, and the stock culture was discarded, and 20. mu.l of MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide salt, trade name: thiazole blue) was added to each well at a concentration of 5 mg/ml.

(4) After 4h incubation, the stock culture was discarded and 150. mu.l of dimethyl sulfoxide (DMSO) was added to each well and shaken at 300rpm for 10min to dissolve the purple crystals sufficiently.

(5) In an enzyme linked immunosorbent assay (ELISA) detector at OD_450nmMeasuring the light absorption value of each hole, and calculating the result according to the following formula:

cell inhibition rate (OD)_{Treatment of}-OD_{Blank space})/(OD_Control-OD_{Blank space})*100％

Wherein, OD_{Treatment of}: OD of each treatment group_450nmA value; OD_{Blank space}: blank group OD_450nmA value; OD_Control: OD of negative control group₄₅₀The value is obtained.

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.

(2) 5% CO₂Incubating the cells at 37 deg.C, removing the original culture medium after the cells are completely adhered to the wall, adding 100 μ l of culture medium containing 2.5, 5, 10, 20, 40, 80 μ MCompound a in 10% FBS medium with a well number of 4.

(6) The inhibition results were processed by graphpad7.0 software in concentration gradient and the resulting IC50 values were calculated.

Table 2 IC50 values for compound a on different tumor cells:

cell species	MCF-7	MDA-MB-231	PC-3	LNCaP
					IC50/μM	46.17	44.83	14.99	20.46

FIG. 3 shows the effect of Compound A, Compound B, Compound C and Icaritin (ICT) on the proliferation of 4 different tumor cells, MCF-7, MDA-MB-231, PC-3, and LNCaP. With the increase of the concentration, especially at 20 μ M or more, the inhibitory effect of compound a on various tumor cells was significantly increased, and it was found that compound a can effectively inhibit various tumor cells. And according to the IC50 value of the compound A on each tumor cell in the table 2, the compound A has obvious inhibition effect on the prostate cancer cells PC-3 and LNCaP.

Example 4:

detecting the apoptosis of breast cancer cells MCF-7 induced by the compound A shown in the formula I:

(1) taking cancer cells in logarithmic growth phase, and plating on 6-well plate with density of 10⁶Single cell, 5% CO₂Incubation at 37 ℃.

(2) After the cells were attached, 2ml of 10% FBS medium containing Compound A at different concentration gradients (0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M) was added to each dish.

(3) After incubation for 24h, the medium is discarded, the trypsin is digested for 2min without EDTA, the digestion is stopped by completely culturing the medium with 2-4 times of the volume of the trypsin, the mixture is centrifuged for 5min at 1000rpm, and the supernatant is discarded.

(4) After washing twice with 1ml PBS, the cells were resuspended in 1X binding buffer in 1ml Annexin V/PI detection kit.

(5) Mu.l of the cell suspension was added with 5. mu.l of Annexin V and 5. mu.l of PI, mixed gently, and incubated at room temperature (25 ℃) for 15min in the absence of light.

(6) Adding 400 μ l of 1X binding buffer, and detecting on a flow cytometer in 1h

Note: a control group and a single male tube are required to be arranged at the same time for adjusting compensation.

FIG. 4 shows the result of apoptosis of MCF-7 by compound A. The results show that the compound A can induce apoptosis of tumor cells, particularly, when the concentration is 20 mu M, the proportion of living cells is obviously reduced, and the total proportion of early apoptosis cells and late apoptosis cells is obviously improved, which indicates that the compound A achieves the anti-tumor effect by inducing apoptosis of the tumor cells.

Therefore, the compound A can cause MCF-7 to have obvious apoptosis phenomenon, inhibit the proliferation of tumor cells, and prompt that the compound A can inhibit the generation and development of tumors, and can be used as a novel therapeutic drug for clinical treatment of tumors.

Example 5:

effect of compound a on the Mitochondrial Membrane Potential (MMP) of breast cancer cells MCF-7:

(1) taking cancer cells in logarithmic growth phase, inoculating the cancer cells in a 6-well plate, wherein the inoculation amount of each dish is 10⁶Single cell, 5% CO₂Incubation at 37 ℃.

(2) After the cells were fully adherent, 2ml of 10% FBS medium containing compound A at different concentration gradients (0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M) was added.

(3) After 24h incubation, the supernatant was discarded, trypsinized for 2min, digestion was stopped with complete medium 2 times the volume of pancreatin, centrifuged at 1000rpm for 5min, discarded, washed twice with 1ml serum-free medium and discarded.

(4)1ml of JC-1 working solution (5. mu.g/ml, diluted in serum-free medium) was added and incubated at 37 ℃ in the dark for 30 min.

(5)1ml PBS washed twice, 0.3ml PBS heavy suspension flow cytometry machine detection.

FIG. 5 is a graph showing the effect of Compound A on the Mitochondrial Membrane Potential (MMP) of MCF-7 cells. Flow cytometry results show that after MCF-7 cells are not treated by the compound A at a concentration, JC-1 aggregate content is remarkably reduced, monomer content is increased, green fluorescence/red fluorescence values are increased, mitochondrial membrane potential is remarkably reduced, and the results show that the compound A can cause mitochondrial damage of tumor cells, mitochondrial dysfunction and apoptosis induction.

From the above results, it is known that compound a can be used as a novel stearoyl-coa desaturase 1 inhibitor, inhibit the enzymatic activity of SCD1, and reduce the expression of SCD1, thereby forming an effective inhibition on SCD 1. Meanwhile, the compound A can cause damage to mitochondrial structure function, so that the membrane potential is reduced, the occurrence of tumor cell apoptosis is induced, and the compound A has a remarkable inhibiting effect on the proliferation of each tumor cell. SCD1 as a key enzyme for regulating lipid metabolism can affect the composition of intracellular membrane lipid, cause damage and dysfunction of related structures, affect the survival and rapid proliferation of tumor cells, and achieve the purpose of resisting tumors. Meanwhile, SCD1 inhibits the formation of monounsaturated fatty acid, can regulate fatty acid composition, and influences the occurrence and development of diseases related to lipid metabolism. Therefore, the compound A as a novel stearoyl-CoA desaturase 1 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 use of a compound of formula I for the manufacture of a medicament for inhibiting stearoyl-CoA desaturase 1,

2. a medicament for inhibiting stearoyl-coa desaturase 1, comprising a compound having the structure of formula I of claim 1 and a pharmaceutically acceptable excipient.

3. The medicine for treating the fatty acid metabolism diseases is characterized by comprising a compound with a structure shown in a formula I in claim 1 and a pharmaceutical excipient.

4. The medicine according to claim 2 or 3, wherein the dosage form of the medicine comprises decoction, pill, powder, ointment, pellet, wine, syrup, extract, lozenge, stick, suppository, leaven, moxibustion agent and the like; also comprises modern dosage forms, such as tablet, granule, bagged steeping drug, oral liquid, capsule, dripping pill, mixture, tincture, aerosol, pellicle, injection, and injection.

5. The medicament according to claim 2 or 3, wherein the pharmaceutic adjuvant comprises a binder, a filler, a disintegrant, a lubricant, an antioxidant, a flavoring agent, an aromatic agent, a cosolvent, an emulsifier, a solubilizer, an osmotic pressure regulator and a coloring agent.

6. The drug according to claim 2 or 3, further comprising a carrier selected from the group consisting of microcapsules, microspheres, nanoparticles, and liposomes.

7. The use of a compound having the structure shown in formula I in claim 1 for the preparation or development of a medicament for the treatment of disorders of fatty acid metabolism.

8. The use according to claim 7, wherein the disorders of fatty acid metabolism are selected from the group consisting of obesity, fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis and diabetes.

9. Use of a compound having the structure of formula I as defined in claim 1 for the preparation or development of a medicament for the treatment of cancer.

10. The use according to claim 9, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, lung cancer, ovarian cancer, thyroid cancer.