CN116270622A

CN116270622A - Medicine for inhibiting stearoyl-CoA desaturase 1

Info

Publication number: CN116270622A
Application number: CN202310139259.3A
Authority: CN
Inventors: 朱升龙; 王威; 陈永泉
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-02-20
Filing date: 2023-02-20
Publication date: 2023-06-23
Anticipated expiration: 2043-02-20
Also published as: CN116270622B

Abstract

The invention discloses a medicine for inhibiting stearoyl-CoA desaturase 1, and belongs to the technical field of medical biology. The invention discloses a method for preparing the compound of formula IThe compound with the structure is used for preparing an inhibitor of stearoyl-CoA desaturase 1, can target liver-fat axis and inhibit the activity of SCD1, and is a novel SCD1 inhibitor. SCD1 is a key enzyme for lipid metabolism, and is involved in the development of metabolic syndromes such as non-alcoholic fatty liver disease.

Description

Medicine for inhibiting stearoyl-CoA desaturase 1

Technical Field

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

Background

stearoyl-CoA desaturase 1 (SCD 1) has been the focus of research in lipid metabolism since its discovery. SCD1 maintains the balance of MUFA and SFA composition in lipids by catalyzing the conversion of Saturated Fatty Acids (SFA) to monounsaturated fatty acids (MUFA). SCD is abundantly expressed in adipose tissue and liver, a key factor in fat metabolism and weight control. The gene deletion of SCD1 has been shown to lead to defective synthesis of cholesterol esters and triglycerides in the liver, to combat obesity, and to reduce liver steatosis in rodents. SCD1 deficiency may also improve glucose tolerance, possibly due to increased sensitivity of liver, adipose tissue and skeletal muscle to insulin. Furthermore, plasma very low density lipoprotein 18:1/18: the ratio of 0 is closely related to the expression of human liver SCD 1. Based on these findings, SCD1 is considered as a new potential target for the treatment of non-alcoholic fatty liver and metabolic diseases.

The liver-fat axis contributes to the development of non-alcoholic fatty liver. Adipose tissue acts as an energy buffering organ, storing and releasing triglycerides and fatty acids. Disorders of fat storage function may lead to excessive fatty acid influx into the liver, leading to liver steatosis. Clinical studies have found that the degree of fatty differentiation in non-alcoholic fatty liver patients is directly related to the degree of liver steatosis. Given that the liver-fat axis contributes to the development of non-alcoholic fatty liver, targeting this axis may contribute to improving and slowing the progression of non-alcoholic fatty liver conditions. However, to date, there is no SCD 1-specific inhibitor against the liver-fat axis in non-alcoholic fatty liver. Therefore, development of the liver-fat axis targeted SCD1 inhibitor has important significance for inhibiting biosynthesis of monounsaturated fatty acid and further improving occurrence and development of non-alcoholic fatty liver.

Disclosure of Invention

In order to solve the above problems, the present invention has found that a compound having a structure represented by the following formula I has an effect of inhibiting stearoyl-coa desaturase 1, can inhibit the activity of saturated fatty acid desaturase, and has a role in regulating fatty acid composition. Based on the above, the invention provides an effective new means and a new way for treating diseases of SCD1 drug targets by applying the invention to the preparation of drugs for inhibiting stearoyl-CoA desaturase 1.

The invention provides the following scheme:

the use of a compound of the structure shown in formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting stearoyl-coa desaturase 1.

Wherein R is mono-to trisubstituted selected from: H. halogen (F, cl, br), C1-4 alkyl, C1-4 alkoxy.

In one embodiment of the present invention, the compound of the structure shown in formula I is specifically selected from:

in one embodiment of the invention, the pharmaceutically acceptable salt comprises an inorganic salt or an organic salt; wherein the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.

In one embodiment of the present invention, the dosage form of the medicament may be selected from conventional dosage forms including decoction, pill, powder, paste, pellet, medicated wine, syrup, extract, lozenge, stick, suppository, qu Ji, moxibustion agent, etc.; also comprises modern dosage forms such as tablet, granule, bagged steeping preparation, oral liquid, capsule, dripping pill, mixture, tincture, aerosol, pellicle, injection, etc.

In one embodiment of the present invention, the medicament further comprises pharmaceutically acceptable excipients including binders, fillers, disintegrants, lubricants, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc.

In one embodiment of the invention, the medicament further comprises a carrier, including microcapsules, microspheres, nanoparticles, and liposomes.

The second purpose of the invention is to provide the application of the compound with the structure shown in the formula I or the pharmaceutically acceptable salt thereof in preparing or developing the medicine for treating fatty acid metabolic diseases.

In one embodiment of the invention, the fatty acid metabolic disorder is selected from the group consisting of obesity, non-alcoholic fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis, and diabetes.

In one embodiment of the invention, the compound of the structure of formula I or a pharmaceutically acceptable salt thereof is used as a stearoyl-coa desaturase 1 inhibitor in such applications.

The invention has the beneficial effects that:

the compound with the structure shown in the formula I is used for preparing an inhibitor of stearoyl-CoA desaturase 1, can target liver-fat axis and inhibit the activity of SCD1, and is a novel SCD1 inhibitor. SCD1 is a key enzyme for lipid metabolism, and is involved in the development of metabolic syndromes such as non-alcoholic fatty liver disease. SCD1 plays an important role in catalyzing saturated fatty acid to form monounsaturated fatty acid, regulating triglyceride synthesis and the like, and is expected to become a target point of non-alcoholic fatty liver. Therefore, the inhibitors of stearoyl-CoA desaturase 1 have obvious effect of inhibiting the activity of SCD1 enzymes of liver-fat axes, can effectively inhibit the generation of monounsaturated fatty acids, reduce triglyceride synthesis, improve liver steatosis, play a role in treating non-alcoholic fatty liver, and have important clinical application prospects.

The inhibitors of stearoyl-CoA desaturase 1 of the present invention are useful in the treatment of fatty acid metabolism related disorders by modulating the composition and ratio of intracellular saturated and unsaturated fatty acids.

Therefore, the compound with the structure shown in the formula I is an important non-alcoholic fatty liver treatment prospect medicine, and provides a new treatment approach for non-alcoholic fatty liver and fatty acid metabolic diseases.

Drawings

FIG. 1 is the effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzymatic activity in mouse bone marrow stromal cells OP 9;

FIG. 2 is the effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzymatic activity in normal hepatocytes AML12 in mice;

FIG. 3 is the effect of compound 1, compound 2, compound 3 and compound 4 on the OP9 fat differentiation of mouse bone marrow stromal cells;

FIG. 4 is the effect of compound 1, compound 2, compound 3 and compound 4 on lipid production by normal hepatocytes AML12 in mice;

FIG. 5 effect of Compound 4 and SCD1 inhibitor A939572 on OP9 fat differentiation of mouse bone marrow stromal cells;

FIG. 6 effect of Compound 4 and SCD1 inhibitor A939572 on lipid production by mouse normal hepatocytes AML 12;

figure 7 effect of compound 4 on non-alcoholic fatty liver model mice.

Detailed Description

Example 1 preparation of Compounds

Synthesis of Compound 1:

to a 100mL single vial was added 2- (4-aminophenyl) -7- (2-morpholin-4-yl-ethoxy) imidazo [2,1-b ] - [1,3] benzothiazole (2.67 g,6.78 mmol), phenyl 5-t-butylisoxazol-3-ylcarbamate (1.94 g,7.4.5 mmol), DMAP (0.05 g,0.44 mmol), and DCM (26 g). Stirring was started and triethylamine (0.1 g,1.02 mmol) was added, DCM (0.5 g) was added. Complete dissolution was observed at 40 ℃ under reflux with stirring for at least 20 hours, after which the product crystallized from solution after-30 minutes. The single-necked flask was cooled to 0 ℃ and stirred for at least 2 hours. The contents of the single-necked flask were separated by a centrifuge. The mixture is rinsed with 0.2 to 03g of DCM at 0 ℃ and dried with nitrogen. The desired product was obtained in a yield of 3.505g, 92%.

¹ H-NMR DMSO-d 6δ9.6(br,1H),8.9(br,1H),8.61(s,1H),7.86(d,J＝8.9Hz,1H),7.76(d,J＝8.0Hz,2H),7.69(d,J＝1.3Hz,1H),7.51(d,J＝8.0Hz,2H),7.18(dd,J＝1.3and 8.9Hz,1H),6.52(s,1H),4.16(t,J＝5.7Hz,2H),3.59(t,J＝4.2Hz,4H),3.36(overlapping,4H),2.72(t,J＝5.7Hz,2H),1.30(s,9H).

Synthesis of Compound 2:

to a 100mL single vial was added a mixture of 2- (3- { [7- (3-chloropropoxy) quinazolin-4-yl ] amino } -1H-pyrazol-5-yl) -N- (2, 3-difluorophenyl) acetamide (0.300 g,0.634 mmol), potassium iodide (0.210 g,1.27 mmol), DMA (2 mmol) and 2- (ethylamino) ethanol (0.226 g,2.54 mmol) and combined, heated to 50℃for 72 hours. The mixture was diluted with dichloromethane (20 ml) and then eluted directly with dichloromethane, then dichloromethane: methanol (9:1), finally with dichloromethane: methanol: ammonia (9:1:0.8) afforded compound 20.181g in 54% yield.

¹ H NMR(DMSO):δ12.31(m,1H),10.39(s,1H),10.15(m,1H),8.51(s,2H),7.62(d,1H),7.35(m,2H),7.16(m,2H),6.90(t,1H),6.78(m,1H),4.29(m,1H),4.20(t,2H),3.76(s,2H),3.45(m,2H),3.30(m,4H),2.61(t,2H),1.89(t,2H),0.95(t,3H).

Synthesis of Compound 3:

to a 250mL round bottom flask was added N- (6- (4- (5-amino-1, 3, 4-thiadiazol-2-yl) butyl) pyridazin-3-yl) -2- (3- (trifluoromethoxy) phenyl) acetamide (5.5 g,12.3 mmol), 2-pyridylacetic acid (2.56 g,14.8 mmol). Propylphosphonic anhydride (11.0 g of a 50% solution in ethyl acetate, 17.3 mmol) was added to a 25mL addition funnel and added dropwise to the reaction solution at a rate of 5 mL/min. During the addition, the internal temperature increased from 20.1 ℃ to 26.1 ℃. After 4 hours, the reaction is generally complete. The reaction solution was then cooled at 0deg.C and diluted with methyl ethyl ketone (50 mL). To the stirred reaction solution was added water (50 mL). The pH was adjusted to 6 with 2.5N sodium hydroxide (28 mL). The precipitate was collected and rinsed with isopropyl alcohol and water (1:1, 50 mL). The air-dried solid was then transferred to a 100mL round bottom flask and slurried in isopropanol and water (9:1, 50 mL). The slurry was heated to 65.1 ℃ for 8 hours and then cooled to room temperature for 16 hours. The precipitate was collected and rinsed with isopropanol (10 mL). Drying in vacuo afforded compound 45.27g in 76% yield.

¹ H NMR(DMSO)δ12.67(s,1H),11.32(s,1H),8.53-8.49(m,1H),8.22-8.19(d,J＝9.12H z,1H),7.78-7.76(t,1H),7.58-7.26(m,7H),4.01(s,2H),3.87(s,2H),3.01(b s,2H),2.90(b s,2H),1.73(b s,4H)。

Synthesis of Compound 4:

to a 100mL single vial was added 6- (3-chloropropoxy) -2- (4- (3-chloropropoxy) phenyl) benzo [ d ] oxazole (13.4 g, 0.0352M) and piperidine (14 mL, 0.14M), ethanol (100 mL, 2M) and heated at 100deg.C for 18 hours. TLC showed the reaction was complete. The mixture was concentrated and excess piperidine was removed by azeotropic concentration with toluene (three times in 50 ml). The residue was dissolved in water, meOH was added, then 1N NaOH was added until no further precipitate formed with adequate stirring. The mixture was concentrated to remove any residual MeOH, filtered and washed with water. The collected solid was dried to give 16.55g of a compound.

¹ H NMR(CD3OD)δ8.12(d,J＝8.9Hz,2H),7.57(d,J＝8.8Hz,1H),7.28(d,J＝2.2Hz,1H),7.12(d,J＝8.9Hz,2H),7.01(dd,J＝2.2,8.8Hz,1H),4.18(t,J＝5.86Hz,2H),4.15(t,J＝6.15Hz,2H),3.07-2.94(m,12H),2.15(m,4H),1.96(m,8H):MS(ES+)m/z 450.4.

Example 2

Effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzyme activity in mouse bone marrow stromal cells OP 9;

(1) Collecting logarithmic phase cells, adjusting cell suspension concentration, and culturing with 4cm diameter culture dish per 1×10 ⁶ Individual cells/dishes are preferred.

(2) At 5% CO ₂ After incubation at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 1. Mu.M rosiglitazone and 10. Mu.M of Compound 1, compound 2, compound 3 and Compound 4, respectively, was added to each dish of cells, and the culture medium without the addition of Compound was used as a control.

(3) The medium was changed every three days, incubated for 15d, the supernatant was discarded, washed twice with PBS, and 1ml of CH was added ₃ The cells were scraped off with an OH spatula and sonicated into lipid-extracting flasks.

(4) 3ml CH was added to each sample ₃ OH、2ml CHCl ₃ 50 μl of 6mol/L HCl is used for detecting leakage and oscillating for 1h. Then add

2ml CHCl ₃ 、2mlH ₂ O, shaking and centrifuging at 2500rpm for 10min, transferring the lower layer into a new lipid extraction bottle, and blowing nitrogen to dryness.

(5) 1ml of 0.5mol/L NaOH methanol solution was added, and the mixture was subjected to a solid bath at 100℃for 5 minutes to saponify the lipid.

(6) 1ml of 14% BF was added ₃ CH ₃ OH, solid bath at 100deg.C for 5min to prepare Fatty Acid Methyl Ester (FAME).

(7) 1ml of C was added ₆ H ₁₄ 1ml saturated NaCl is shaken, centrifuged at 2000rpm for 2min, the upper liquid nitrogen is taken and blown to dryness, 300 mu l C H14 is redissolved, and a sample is analyzed by GC MS to determine the fatty acid spectrum of the cells.

(8) Definition of SCD1 enzyme activity: the conversion of PA and OA was measured by measuring the fatty acid profile of the cells and by measuring the C16:1/C16:0 and C18:1/C18: the ratio of 0 is used as an index of SCD1 enzyme activity.

FIG. 1 is the effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzymatic activity in mouse bone marrow stromal cells OP 9. The corresponding results are shown in Table 1.

TABLE 1 influence of Compounds 1-4 on the activity of SCD1 enzyme in mouse bone marrow stromal cells OP9

	C16：1/C16：0	C18:1/C18:0 (0)
			Control group	0.692	1.838
Compound 1	0.340	1.330
			Compound 2	0.324	1.100
Compound 3	0.256	0.910
			Compound 4	0.187	0.768

Compound 4 was effective in inhibiting SCD1 activity compared to the control group.

Example 3

Effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzyme activity in normal hepatocytes AML12 in mice.

SCD1 enzyme Activity assay

(2) At 5% CO ₂ After incubation at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 0.125. Mu.M PA and 0.25. Mu.M OA and 10. Mu.M Compound 1, compound 2, compound 3 and Compound 4 were added to each dish of cells, respectively, and the culture medium without the addition of Compound was used as a control.

(3) Co-incubation was performed for 48h, the supernatant was discarded, washed twice with PBS, and 1ml CH was added ₃ The cells were scraped off with an OH spatula and sonicated into lipid-extracting flasks.

(8) Definition of SCD1 enzymatic activity: fatty liver AML12 cell model to detect C16 in the results: 1 as an index of SCD1 enzyme activity.

FIG. 2 is the effect of compound 1, compound 2, compound 3 and compound 4 on SCD1 enzymatic activity in normal hepatocytes AML12 in mice. The corresponding results are shown in Table 2.

TABLE 2 influence of Compounds 1-4 on the enzymatic Activity of SCD1 in mouse Normal hepatocytes AML12

	And C16:1 content (. Times.10) ⁸ )
		Control group	2.647
Compound 1	2.160
		Compound 2	2.417
Compound 3	2.183
		Compound 4	1.347

Example 4

Effect of compound 1, compound 2, compound 3 and compound 4 on OP9 fat differentiation of mouse bone marrow stromal cells;

and (3) detecting the content of triglyceride:

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 0.125. Mu.M PA and 0.25. Mu.M OA and 10. Mu.M Compound 1, compound 2, compound 3 and Compound 4, respectively, was added to each dish of cells, and the culture medium without the addition of Compound was used as a control.

(3) Incubate for 48h, discard supernatant, wash twice with PBS, scrape cells with 200. Mu.L PBS spatula, and sonicate.

(4) The triglyceride content was determined by GPO-PAP method.

(5) Taking a proper volume (2.5) of protein sample into an orifice plate, adding 250 mu L of GPO-PAP working solution into each orifice, and incubating for 30min at 37 ℃; the absorbance at a wavelength of 510nm was measured, and the concentration of triglyceride was calculated from the absorbance of the standard and control wells.

(6) Protein concentration was determined using BCA method.

(7) A standard protein gradient of 0.5mg/ml was added to the well plate and PBS was added to make up to 20. Mu.l; add appropriate volume (3 μl) of protein sample to the well plate and add PBS to make up to 20 μl; 200 μl BCA working solution (prepared before use and as-prepared) was added to each well and incubated at 37deg.C for 30min; the absorbance at wavelength 562nm was measured, and the protein concentration was calculated from the standard curve and the sample volume.

(9) Sample triglyceride content: the ratio of the concentration of triglyceride to the concentration of sample protein is used as an indicator of the content of triglyceride in the sample.

Oil red staining:

(4) Hematoxylin counterstain for 1min, and tap water returns to blue.

(5) And photographing and observing under a microscope.

Lipid production gene detection:

(3) Incubating for 48h, discarding the supernatant, washing twice with PBS, adding 1mL of Trizol dye solution, blowing thoroughly to lyse the cells, and standing in ice bath for 5min.

(4) Collecting in a 1.5mL centrifuge tube, adding 200 μl chloroform, shaking vigorously for 15s, standing in ice bath for 10min, standing at 4deg.C, 12000g, and centrifuging for 15min;

(5) After centrifugation, the solution was divided into three layers, the lower layer was red Trizol reagent, the middle layer was aqueous phase, the upper layer was organic phase, and RNA was distributed on the upper layer. The RNA-containing upper aqueous phase was aspirated at about 400. Mu.l and transferred to a new centrifuge tube.

(6) Adding equal volume of isopropanol, gently reversing and mixing, standing in ice bath for 5min,4 ℃,12000g, and centrifuging for 10min to obtain RNA precipitate.

(7) Discarding the supernatant, adding 1ml of 75% ethanol, mixing uniformly, washing RNA at 4 ℃ and 7500g, and centrifuging for 5min;

(8) The supernatant was discarded and the RNA pellet was dried in air for 10-20min until the white pellet was completely removed, indicating the completion of drying after a colorless gummy solid.

(9) RNA was dissolved by adding 30. Mu.l to 50. Mu.l of DEPC water.

(10) After dissolution, the purity and concentration of RNA were measured by a spectrophotometer, and the RNA was diluted appropriately according to the concentration of RNA and used directly for reverse transcription to synthesize cDNA.

(11) qRT-PCR detects the expression level of the lipid production gene in the cDNA.

Figure 3 shows the effect of compound 1, compound 2, compound 3 and compound 4 on OP9 fat differentiation of mouse bone marrow stromal cells. The corresponding results are shown in Table 3.

TABLE 3 Effect of lipogenic Gene Compounds 1-4 on the differentiation of OP9 fat in mouse bone marrow stromal cells

The compound 4 can obviously inhibit the generation of triglyceride and inhibit the expression of lipid generation genes. As can be seen from fig. 3C, the number of OP9 cell lipid droplets treated with compound 4 was significantly reduced compared to the control group.

Example 5

Effect of compound 1, compound 2, compound 3 and compound 4 on lipid production of normal hepatocytes AML12 in mice.

Triglyceride content detection

(1) The log phase cells are collected and the cell suspension concentration is adjusted, preferably every 1X106 cells/dish in a 4cm diameter dish.

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 1. Mu.M rosiglitazone and 10. Mu.M of Compound 1, compound 2, compound 3 and Compound 4, respectively, was added to each dish of cells, and the culture medium without the addition of Compound was used as a control.

(3) The medium was changed every three days, incubated for 15d, the supernatant was discarded, washed twice with PBS, and cells were scraped with 200. Mu.L PBS and sonicated.

(4) The triglyceride content was determined by GPO-PAP method.

(6) Protein concentration was determined using BCA method.

Oil red staining:

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4mL of medium containing 1. Mu.M rosiglitazone and 10. Mu.M of compound 1, compound 2, compound 3 and compound 4, respectively, was added to each dish of cells, and the culture medium without the addition of the compound was used as a control.

(3) The medium was changed every three days, incubated for 15d, the supernatant was discarded, washed twice with PBS, 1mL of oil red dye solution was added, the mixture was stained in the dark for 30min, and washed with 65% isopropyl alcohol for 10s.

(4) Hematoxylin counterstain for 1min, and tap water returns to blue.

(5) And photographing and observing under a microscope.

Lipid production gene detection:

(3) The medium was changed every three days, incubated for 15d, the supernatant was discarded, washed twice with PBS, 1mL of Trizol dye was added, and the cells were lysed by blowing thoroughly, and allowed to stand in an ice bath for 5min.

(8) Removing supernatant, drying RNA precipitate in air for 10-20min until white precipitate completely disappears to colorless

After the gummy solid of (2) indicated that drying was complete.

(9) RNA was dissolved by adding 30. Mu.l to 50. Mu.l of DEPC water.

Fig. 4 shows the effect of compounds 1 to 4 on lipid production of normal hepatocytes AML12 in mice. The corresponding results are shown in Table 4.

TABLE 4 Effect of Compounds 1-4 on the lipid production of mouse Normal liver cells AML12

The compound 4 can obviously inhibit the generation of triglyceride and inhibit the expression of lipid generation genes. As can be seen from fig. 3C, the number of AML12 cell lipid droplets treated with compound 4 was significantly reduced compared to the control group.

The above results demonstrate that compound 4 is capable of acting as a novel inhibitors of stearoyl-coa desaturase 1, resulting in effective inhibition of SCD 1. The compound 4 is suggested to be used as a therapeutic agent for obesity and other related metabolic diseases, and can prevent and/or treat occurrence and development of the diseases.

Example 6

Effect of compound 4 and SCD1 inhibitor a939572 on mouse bone marrow stromal cell OP9 fat differentiation.

And (3) detecting the content of triglyceride:

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 0.125. Mu.M PA and 0.25. Mu.M OA and 10. Mu.M Compound 4 and SCD1 inhibitor A939572 were added to each dish of cells, respectively, with no compound added as a control.

(4) The triglyceride content was determined by GPO-PAP method.

(6) Protein concentration was determined using BCA method.

Oil red staining:

(4) Hematoxylin counterstain for 1min, and tap water returns to blue.

(5) And photographing and observing under a microscope.

Lipid production gene detection:

(9) RNA was dissolved by adding 30. Mu.l to 50. Mu.l of DEPC water.

(11) And detecting the expression quantity of the lipid generating genes in the sample by qRT-PCR.

Figure 5 shows the effect of compound 4 and on OP9 fat differentiation of SCD1 inhibitor a939572 mouse bone marrow stromal cells. Compared with the SCD1 inhibitor A939572, the compound 4 can obviously inhibit the generation of triglyceride and the expression of lipid generating genes, and reduce the number of lipid droplets of OP9 cells.

Example 7

Effect of compound 4 and SCD1 inhibitor a939572 on lipid production by normal hepatocytes AML12 in mice.

Triglyceride content detection

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 1. Mu.M rosiglitazone and 10. Mu.M of Compound 4 and SCD1 inhibitor A939572 were added to each dish of cells, respectively, with no compound added as a control.

(4) The triglyceride content was determined by GPO-PAP method.

(6) Protein concentration was determined using BCA method.

Oil red staining:

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4mL of medium containing 1. Mu.M rosiglitazone and 10. Mu.M of Compound 4 and SCD1 inhibitor A939572 were added to each dish of cells, respectively, with no compound added medium as a control.

(4) Hematoxylin counterstain for 1min, and tap water returns to blue.

(5) And photographing and observing under a microscope.

Lipid production gene detection:

After the gummy solid of (2) indicated that drying was complete.

(9) RNA was dissolved by adding 30. Mu.l to 50. Mu.l of DEPC water.

IC50 value determination:

IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It indicates that a certain drug or substance (inhibitor) induces a 50% decrease in SCD1 activity at a concentration called 50% inhibitory concentration. The IC50 value can be used to measure the ability of a drug to induce a decrease in SCD1 activity, i.e., the greater the induction, the lower the value.

SCD1 enzyme Activity assay

(2) After incubation at 5% CO2 at 37℃and complete cell attachment, the original medium was discarded, and 4ml of medium containing 0.125. Mu.M PA and 0.25. Mu.M OA and 0.08,0.4,2, 10. Mu.M Compound 4 and SCD1 inhibitor A939572, respectively, was added to each dish of cells, and the culture medium without the addition of the compound was used as a control.

(3) Incubate for 48h, discard supernatant, wash twice with PBS, scrape cells with 1ml CH3OH spatula, sonicate into lipid-extracting flask.

(4) Each sample was then added with 3ml CH3OH, 2ml CH Cl3, 6mol/L HCl 50. Mu.l, followed by leak detection and shaking for 1h. Then add

2ml of CHCl3, 2ml of H2O, shaking and centrifuging at 2500rpm for 10min, the lower layer was transferred to a fresh lipid extraction flask and nitrogen was blown dry.

(6) Adding 1ml14%BF3 CH3OH,100 deg.C for 5min to obtain Fatty Acid Methyl Ester (FAME).

(7) 1ml of C6H14 and 1ml of saturated NaCl are added, the mixture is centrifuged for 2min at 2000rpm, the upper liquid nitrogen is taken and blown to dryness, 300 mu l C H14 is redissolved, and a sample is analyzed by GC MS to determine the fatty acid spectrum of cells.

(9) The inhibition results were processed with graphpad7.0 software according to concentration gradient and the IC50 values calculated.

The corresponding results are shown in Table 5.

Table 5 IC50 values of compound 4 and SCD1 inhibitor a939572 for AML12

Medicament	Compound 4	SCD1 inhibitor A939572
			IC50/μM	0.98μM	2.8μM

Figure 6 shows the effect of compound 4 and SCD1 inhibitor a939572 on lipid production by normal hepatocytes AML12 in mice. Compared with the SCD1 inhibitor A939572, the compound 4 can obviously inhibit the generation of triglyceride and the expression of lipid production genes, and reduce the number of lipid droplets of AML12 cells. And from the IC50 results, the effective concentration of compound 4 was also significantly lower than SCD1 inhibitor a939572.

The above results demonstrate that compound 4 has a more potent SCD1 inhibitory capacity. The compound 4 can inhibit occurrence and development of related metabolic diseases such as obesity and the like, and is used as a novel therapeutic drug applied to clinical treatment of the related metabolic diseases such as non-alcoholic fatty liver and the like.

Example 8

Effect of compound 4 on non-alcoholic fatty liver model mice.

(1) Male C57BL/6 mice of 6-8 weeks old are divided into a control group, a non-alcoholic fatty liver model mouse and a non-alcoholic fatty liver model mouse compound 4 treatment group.

(2) Non-alcoholic fatty liver model mice were fed a 60% high fat diet for 12 weeks, and then were given 20mg/kg of compound 4 and gavaged for six weeks.

(3) Six weeks later, mice were sacrificed under anesthesia after weight and blood glucose measurement, blood was taken for biochemical detection, and liver was stained with oil red and HE.

Oil red staining:

(1) The tissue stored at-80℃was cut into 8 μm thick sections using a frozen microtome and fixed with 4% formaldehyde for 30 minutes. (2) Oil red dye liquor, shading dyeing for 30min, washing and decoloring with 60% isopropanol solution for 10s.

(3) The slices are stained with hematoxylin dye solution for 3-5min, washed with tap water to turn blue, and washed with running water.

(4) The glycerogelatin tablet sealing tablet is sealed.

(5) After the sections were completely air-dried, histopathological photographic observations were made using an optical microscope. .

HE staining:

(1) Fresh mouse liver tissue is taken and fixed for 48 hours in 4% paraformaldehyde, samples are dehydrated by 70%, 80% and 90% ethanol solutions for 30 minutes each, and then 95% and 100% ethanol solutions are put into the samples for 2 times each for 15 minutes each time, and gradient dehydration is carried out. After the tissue is transparent in xylene solution (1/2 pure alcohol, 1/2 xylene equivalent mixed solution for 15min, xylene I for 15min and xylene II for 15 min), paraffin I and paraffin II are immersed for 60 min respectively, and finally the immersed tissue is embedded and stored (the largest surface is arranged at the bottom layer, so that the surface of the cut surface tissue occupies the largest surface).

(2) Thin sections (5 μm) were prepared for hematoxylin and eosin staining,

(3) Eosin staining was used for 30min, hematoxylin staining for 1min.

(4) Differentiating with 1% ethanol solution of hydrochloric acid until the slice turns red and the color is lighter. The slices are rinsed, counterstained, dehydrated, and then sealed by neutral resin after being transparent.

(5) After the sections were completely air-dried, histopathological photographic observations were made using an optical microscope.

Figure 7 shows that compound 4 can significantly reduce insulin resistance, liver weight and liver triglyceride, total cholesterol, low density lipoprotein, aspartate aminotransferase and alanine aminotransferase levels in non-alcoholic fatty liver model mice. HE and oil red staining showed that compound 4 improved liver steatosis and reduced liver lipid droplet accumulation in non-alcoholic fatty liver model mice.

The specific results are shown in Table 6.

Table 6 effect of compound 4 on non-alcoholic fatty liver mice

From the above results, it is known that compound 4 can act as a novel stearoyl-coa desaturase 1 inhibitor, targeting the liver-fat axis, inhibiting the enzymatic activity of SCD1, and thus forming an effective inhibition of SCD 1. Meanwhile, the compound 4 can inhibit fat differentiation and lipid generation, and can remarkably improve liver steatosis and liver lipid drop accumulation of a non-alcoholic fatty liver model mouse. Therefore, the compound 4 serving as a novel stearoyl-CoA desaturase 1 inhibitor targeting liver-fat axis has important application prospect in the treatment of metabolic diseases such as non-alcoholic fatty liver and the like.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 a compound with a structure shown in a formula I or pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting stearoyl-CoA desaturase 1,

wherein R is mono-to trisubstituted selected from: H. halogen, C1-4 alkyl, C1-4 alkoxy.

2. The use according to claim 1, wherein the compound of the structure of formula I is:

3. the use according to claim 1 or 2, wherein the pharmaceutically acceptable salt comprises an inorganic or organic salt; wherein the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.

4. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is a conventional dosage form, including decoction, pill, powder, paste, pellet, medicated wine, syrup, extract, lozenge, stick, suppository, qu Ji, and broil.

5. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is a modern dosage form, such as a tablet, granule, bubble, oral liquid, capsule, drop pill, mixture, tincture, aerosol, film, injection.

6. The use according to claim 1 or 2, wherein the medicament further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable auxiliary materials comprise adhesive, filler, disintegrating agent, lubricant, antioxidant, flavoring agent, aromatic, cosolvent, emulsifying agent, solubilizer, osmotic pressure regulator, colorant, etc.

7. The use according to claim 1 or 2, wherein the medicament further comprises a carrier, said carrier comprising microcapsules, microspheres, nanoparticles and liposomes.

8. The use of a compound of the structure shown in formula I in claim 1 or a pharmaceutically acceptable salt thereof for the preparation or development of a medicament for the treatment of fatty acid metabolic diseases.

9. The use according to claim 8, wherein the fatty acid metabolic disorder is selected from the group consisting of obesity, non-alcoholic fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis and diabetes.

10. The use according to claim 8, wherein the compound of formula I as set forth in claim 1 or a pharmaceutically acceptable salt thereof is used as a stearoyl-coa desaturase 1 inhibitor.