Quinazolinone carboline compound as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a quinazolinone carboline compound as well as a pharmaceutical composition and application thereof. It is found for the first time that the quinazolinone carboline compound with the structure shown in the formula I has the SCD1 inhibiting effect, the activity of saturated fatty acid desaturase can be inhibited, the quinazolinone carboline compound plays a role in adjusting fatty acid composition, and an effective new means and a new approach are provided for treating SCD1 drug target diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a quinazolinone carboline compound, a pharmaceutical composition thereof and an application thereof. Background Art

[0002] Stearoyl-CoA desaturase 1 (SCD1) is highly expressed in metabolism-related cells and some tumor cells, and SCD1 has become a new drug target for studying such diseases. SCD1 is a lipase that converts saturated fatty acids into monounsaturated fatty acids and is a key regulator of the fatty acid metabolic pathway. In several types of cancer, SCD1 expression is associated with poor prognosis. The study of SCD1 inhibitors is of great significance for inhibiting the synthesis of monounsaturated fatty acids and thus effectively controlling the occurrence and development of tumors, obesity, and various related metabolic syndromes.

[0003] The SCD1 small molecule inhibitors reported in the prior art mainly include CAY-10566, MF438, A939572, CVT-11127, CVT-12012, T-3764518, PluriSIn 1, SW203668, SW203668, BZ36, SSI-4, etc., none of which has a parent ring structure similar to the quinazolinone carboline compounds described in the present invention. Summary of the invention

[0004] The present invention finds that the quinazolinone carboline compound of the structure shown in the following formula I has the effect of inhibiting SCD1, can inhibit the activity of saturated fatty acid desaturase, and plays a role in regulating fatty acid composition. Based on this, the present invention and its application in the preparation of drugs for inhibiting SCD1 provide an effective new means and new approach for treating diseases that are targets of SCD1 drugs.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] A quinazolinone carboline compound, the structural formula of the quinazolinone carboline compound is as shown in Formula I:

[0007]

[0008] Furthermore, the preparation method of the quinazolinone carboline compound comprises the following steps:

[0009] S1. condensing D-tryptophan methyl ester hydrochloride and isatoic anhydride in a solvent to obtain methyl (2-aminobenzoyl)-D-tryptophan ester;

[0010] S2. reacting methyl (2-aminobenzoyl)-D-tryptophanate with a cyclization reagent in the presence of a catalyst to obtain methyl (7R,13bR)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole [2',3':3,4] pyrido [2,1-b] quinazoline-7-carboxylate;

[0011] S3. Methyl (7R,13bR)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazoline-7-carboxylate is reacted with chloroacetyl chloride to obtain a quinazolinone carboline compound (methyl (7R,13bS)-14-(2-chloroacetyl)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazoline-7-carboxylate).

[0012] Specifically, the solvent in step S1 includes one or more of methanol, ethanol, dichloromethane or toluene.

[0013] Specifically, triethylamine is added as an acid binding agent in step S1.

[0014] Specifically, the temperature of the condensation reaction in step S1 is 85-95°C.

[0015] Specifically, the molar ratio of D-tryptophan methyl ester hydrochloride to isatoic anhydride in step S1 is 1:(1-1.2).

[0016] Specifically, the cyclization reagent in step S2 is triethyl orthoformate.

[0017] Specifically, the catalyst in step S2 is one or more of sodium bisulfate monohydrate, ferric chloride, zinc chloride, zinc bromide, polyphosphoric acid, sodium bisulfate, potassium bisulfate or magnesium bisulfate.

[0018] Specifically, the reaction temperature in step S2 is 110-130°C.

[0019] Specifically, the molar ratio of methyl (2-aminobenzoyl)-D-tryptophan ester to the cyclization reagent in step S2 is 1:(3.0-3.1).

[0020] Specifically, the specific steps of step S3 are: dissolving methyl (7R, 13bR)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole [2',3':3,4] pyrido [2,1-b] quinazoline-7-carboxylate in dichloromethane, adding chloroacetyl chloride and sodium bicarbonate at 0° C., and reacting at room temperature to obtain quinazolinone carboline compounds.

[0021] Specifically, in step S3, the molar ratio of methyl (7R, 13bR)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole [2',3':3,4] pyrido [2,1-b] quinazoline-7-carboxylate to chloroacetyl chloride is 1: (1.0 to 1.5).

[0022] More specifically, the quinazolinone carboline compound is prepared according to the following reaction:

[0023]

[0024] The present invention also provides a pharmaceutical composition, wherein the medicine comprises the quinazolinone carboline compound and pharmaceutical excipients.

[0025] Another object of the present invention is to provide the use of the quinazolinone carboline compound or the pharmaceutical composition in the preparation of a drug for inhibiting stearoyl-CoA desaturase 1.

[0026] Another object of the present invention is to provide the use of the quinazolinone carboline compound or the pharmaceutical composition in the preparation or development of drugs for treating fatty acid metabolism diseases.

[0027] Specifically, the fatty acid metabolism disease is selected from obesity, fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis and diabetes.

[0028] Another object of the present invention is to provide the use of the quinazolinone carboline compound or the pharmaceutical composition in the preparation or development of drugs for treating cancer.

[0029] Specifically, the cancer is selected from breast cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, lung cancer, ovarian cancer, and thyroid cancer.

[0030] In some specific embodiments, the dosage form of the drug includes a traditional dosage form or a modern dosage form.

[0031] Specifically, the traditional dosage forms include decoctions, pills, powders, ointments, pills, wines, syrups, extracts, lozenges, sticks, suppositories, koji or roasted preparations; the modern dosage forms include tablets, granules, bags, oral liquids, capsules, pills, mixtures, tinctures, aerosols, films, injections or injections.

[0032] In some specific embodiments, the pharmaceutical excipients include binders, fillers, disintegrants, lubricants, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0033] In some specific embodiments, the drug further contains a carrier, and the carrier includes microcapsule, microsphere, nanoparticle and liposome.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The quinazolinone carboline compounds of the present invention have the activity of inhibiting stearoyl-CoA desaturase 1, and can be used to prepare inhibitors of stearoyl-CoA desaturase 1. The quinazolinone carboline compounds have obvious inhibitory effects on the enzyme activity of SCD1, can inhibit the growth of tumor cells, affect the energy metabolism of cells, promote apoptosis, and play a therapeutic effect on tumors, and have important clinical application prospects; the quinazolinone carboline compounds can also regulate the proportion of fatty acids in cells and change the saturation ratio, and are used to prepare drugs for treating diseases related to fatty acid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of compound I on the SCD1 enzyme activity of breast cancer MDA-MB-231 cells;

[0037] Figure 2 The effect of compound I on the expression of SCD1 protein in breast cancer MDA-MB-231 cells;

[0038] Figure 3 The effect of compound I on the proliferation of breast cancer cells;

[0039] Figure 4 The effect of compound I on the apoptosis of breast cancer MDA-MB-231 cells;

[0040] Figure 5 The effect of compound I on the expression of apoptosis-related proteins in MDA-MB-231 cells. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] Example 1

[0043] The synthetic reaction formula of compound I in Example 1 is as follows:

[0044]

[0045] S1. Preparation of methyl (2-aminobenzoyl)-D-tryptophan ester (Compound I-1):

[0046]

[0047] At room temperature, D-tryptophan methyl ester hydrochloride (2.54 g, 10.0 mmol) and isatoic anhydride (1.63 g, 10.0 mmol) and triethylamine (1.67 mL, 12.0 mmol) were added to ethanol and heated at 90°C for reaction. The reaction was monitored by TLC. The solvent was dried under vacuum, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and recrystallized from petroleum ether / ethyl acetate to obtain 2.1 g of a yellow solid with a yield of 60%.

[0048] Characterization data: 1 H NMR (600MHz, CDCl3) δ8.27(s,1H),7.65(d,J=7.9Hz,1H),7.43(d,J=8.1Hz,1H),7.29-7.27(m,1H),7.25(s,1H),7.21-7.16(m,1H),7.08(d,J=2.4 Hz,1H),6.73(d,J=8.1Hz,1H),6.68(d,J=7.5Hz,1H),6.67-6.61(m,1H), 5.66-5.46(m,2H),5.18-5.14(m,1H),3.80(s,3H),3.51(t,J=5.0Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.5,168.7,148.7,136.1,132.5,127.5,127.5,122.8,122 .3,119.7,118.6,117.3,116.7,115.3,111.3,110.1,77.0,53.1,52.4,27.6.LC-MS M / Z(ESI):338.15[M+H] + ;

[0049] S2. Preparation of methyl (7R, 13bR)-5-carbonyl-5,7,8,13,13b,14-hexahydroindole [2',3':3,4] pyrido [2,1-b] quinazoline-7-carboxylate (Compound I-2):

[0050]

[0051] At room temperature, compound I-1 (702.0 mg, 2.0 mmol), triethyl orthoformate (1.0 mL, 6.0 mmol), and sodium bisulfate monohydrate (99.0 mg, 0.72 mmol) were added to N,N-dimethylformamide and heated to 120°C for reaction. The reaction was monitored by TLC, extracted with ethyl acetate, washed with water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed using petroleum ether and ethyl acetate in a volume ratio of 2:1 as eluent to obtain 584.0 mg of a white solid with a yield of 84%;

[0052] Characterization data: 1 H NMR(400MHz, CDCl3)δ8.29(dd,J=8.0,1.5Hz,1H),8.09(s,1H),7.74(ddd,J=8.5,7.1,1.6Hz,1H), 7.61(d,J=13.9Hz,2H),7.55(d,J=7.9Hz,1H),7.49(ddd,J=8.2,7.1,1.2Hz,1H),7.31(dt,J=8.1, 0.9Hz,1H),7.17(ddd,J=8.1,7.0,1.2Hz,1H),7.08(ddd,J=8.0,7.0,1.1Hz,1H),6.83(d,J=2.4Hz ,1H),5.35(dd,J=10.0,4.9Hz,1H),3.81(s,3H),3.79-3.73(m,1H),3.64(dd,J=15.3,10.0Hz,1H). 13 C NMR (101MHz, CDCl3) δ169.5,160.7,147.5,145.6,136.2,134.5,127.4,127.3,126. 8,126.6,123.1,122.5,121.7,119.9,118.0,111.5,109.7,59.2,53.0,25.9.LC-MS M / Z(ESI):348.13[M+H] + ;

[0053] S3. Preparation of methyl (7R, 13bS) -14- (2-chloroacetyl) -5-carbonyl -5,7,8,13,13b,14- hexahydroindole [2',3':3,4] pyrido [2,1-b] quinazoline-7-carboxylate) (Compound I):

[0054]

[0055] At room temperature, compound I-2 (69.4 mg, 0.2 mmol) was dissolved in dichloromethane, cooled to 0°C, sodium bicarbonate (20.2 mg, 0.24 mmol) and chloroacetyl chloride (19.1 μL, 0.24 mmol) were added, and the mixture was transferred to room temperature for reaction. After the reaction was completed by TLC monitoring, the mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography, using petroleum ether and ethyl acetate in a volume ratio of 4:1 as the eluent, to obtain 64.3 mg of a white solid with a yield of 75%;

[0056] Characterization data: 1 H NMR (400MHz, CDCl3) δ8.21(s,1H),8.11-8.06(m,1H),7.60-7.52(m,2H),7.48(d,J=7.8Hz,1H),7.36(t,J=7.4Hz,1H),7.30(d,J=8.1Hz,2H ),7.21-7.16(m,1H),7.11(t,J=7.5Hz,1H),5.97(t,J=4.2Hz,1H),4.40(s,1H),4.28(d,J=12.7Hz,1H),3.76(s,3H),3.39(d,J=2.2Hz,2H). 13 C NMR (101MHz, CDCl3) δ171.3,167.9,164.3,136.2,133.4,129.5,128.1,127.4,126 .5,123.6,123.2,120.1,118.7,111.5,110.6,64.8,54.3,52.9,41.9,22.2.LC-MS M / Z(ESI):424.10[M+H] + .

[0057] Example 2 Effect of Compound I on SCD1 enzyme activity in breast cancer cells:

[0058] SCD1 enzyme activity detection:

[0059] (1) Collect cells in the logarithmic phase and adjust the concentration of the cell suspension to 4 x 10 cells per 10 cm diameter culture dish. 6 cells / dish is appropriate.

[0060] (2) Incubate at 5% CO2 and 37°C. After the cells have completely attached to the wall, discard the original culture medium and add 8 mL of 0.2 mM [d31]PA (isotope d31-labeled palmitic acid, linear molecular formula: CD3(CD2)) to each dish of cells. 14 CO2H) and culture medium with different concentrations of compound Ⅰ, and SCD1 inhibitor A939572 was used as a positive control.

[0061] (3) After incubation for 24 h, discard the supernatant, wash twice with PBS, add 1 mL of CH3OH, scrape the cells with a scraper, and disrupt them by ultrasonication into a lipid extraction bottle.

[0062] (4) Add 3 mL CH3OH, 2 mL CHCl3, and 50 μL 6 mol / L HCl to each sample, check for leaks, and shake for 1 hour. Then add 2 mL CHCl3 and 2 mL H2O, shake, and centrifuge at 2500 rpm for 10 minutes. Transfer the lower layer to a new fat extraction bottle and blow it dry with nitrogen.

[0063] (5) Add 1 mL of 0.5 mol / L NaOH methanol solution and incubate at 100 °C for 5 min to saponify the lipids.

[0064] (6) Add 1 mL of 14% BF3-CH3OH and incubate at 100°C for 5 min to prepare fatty acid methyl ester (FAME).

[0065] (7) Add 1 mL C6H 14 , 1mL saturated NaCl was shaken, centrifuged at 2000rpm for 2min, the upper layer of liquid nitrogen was blown to dryness, 300μL C6H 14 After re-dissolution, the samples were analyzed by GC-MS to determine the cellular fatty acid profile.

[0066] Definition of SCD1 enzyme activity: The conversion rate of [d31]PA is detected by converting isotope-labeled [d31]PA into isotope-labeled fatty acid methyl esters to measure the cellular fatty acid profile, and the ratio of [d31]C16:1 / ([d31]C16:1+[d31]C16:0) (where [d31]C16:0 and [d31]C16:1 are palmitic acid and palmitoleic acid labeled with isotope d31, respectively) in the test result is used as an indicator of SCD1 enzyme activity.

[0067] Detection of SCD1 and apoptosis-related protein expression:

[0068] Western-blot was used to detect the changes in the expression of SCD1 protein in breast cancer cells treated with compound I. The specific steps of Western-blot were as follows:

[0069] A. Preparation of solution:

[0070] (1) 10% (w / v) sodium dodecyl sulfate (SDS) solution: 0.1 g SDS, 1 mL H2O, deionized water, and stored at room temperature.

[0071] (2) Separation gel buffer: Weigh 18.15 g of Tris, dissolve it in 80 mL of water, adjust the pH to 8.8 with HCl, and dilute it to a final volume of 100 mL with water to obtain a 1.5 mmol / L Tris-HCl (pH 8.8) solution.

[0072] (3) Stacking gel buffer: 6.05 g Tris was dissolved in 80 mL water, adjusted to pH 6.8 with HCl, and diluted to a final volume of 100 mL with water to obtain a 0.5 mmol / L Tris-HCl (pH 6.8) solution.

[0073] (4) SDS-PAGE loading buffer: 8 mL of 0.5 mol / L Tris buffer (pH 6.8), 6.4 mL of glycerol, 12.8 mL of 10 wt % SDS, 3.2 mL of mercaptoethanol, 1.6 mL of 0.05 wt % bromophenol blue, and 32 mL of H 2 O were mixed and set aside.

[0074] (5) Tris-glycine electrophoresis buffer: Weigh 30.3 g Tris, 188 g glycine and 10 g SDS, dissolve to 1000 mL with distilled water, and dilute 10-fold before use.

[0075] (6) Transfer buffer: Weigh 14.4 g of glycine and 6.04 g of Tris, add 200 mL of methanol, and finally add water to a total volume of 1 L.

[0076] (7) Tris buffered saline (TBS): contains 20 mM Tris-HCl (pH 7.5) and 500 mM NaCl.

[0077] (8) Phosphate buffer saline (PBS): Weigh 80 g NaCl, 36.3 g NaH2PO4·12H2O, 2 g KCl, and 2.7 g KH2PO4, dissolve in 900 mL distilled water, adjust the pH to 7.4, add water to a total volume of 1 L, and dilute 10-fold before use.

[0078] B. Wash the cancer cells three times with PBS, add lysis buffer, boil directly for 5 minutes, cool on ice, centrifuge at 12000rpm for 2 minutes, take the supernatant, and store at -20℃ for later use.

[0079] C. Protein concentration was determined using the BCA method.

[0080] Add 0.5 mg / mL standard protein gradient to the well plate and add PBS to make up to 20 μL; add an appropriate volume (3 μL) of protein sample to the well plate and add PBS to make up to 20 μL; add 200 μL of BCA working solution (prepare before use, prepare and use immediately) to each well, incubate at 37°C for 30 min; measure the absorbance at a wavelength of 562 nm, and calculate the protein concentration using the standard curve and sample volume.

[0081] D. SDS-PAGE gel electrophoresis

[0082] (1) After cleaning, align the two glass plates and clamp them in the clamp, then clamp them vertically on the rack and prepare for glue filling.

[0083] (2) Pouring 10 wt% separation gel: Add TEMED (tetramethylethylenediamine) to the separation gel buffer and immediately shake to mix well. Pour the mixture into the gap between two glass plates and then seal with ethanol. When the gel is fully solidified, pour off the ethanol on the top of the gel and dry it with absorbent paper.

[0084] (3) Pour 5 wt% stacking gel: Add TEMED (tetramethylethylenediamine) to the stacking gel buffer and shake well immediately. Pour it to the upper layer of the separation gel, fill the remaining space with stacking gel, and then insert the comb into the stacking gel. After the stacking gel solidifies, gently pull it out vertically.

[0085] (4) Place the glass plate after gel filling into the electrophoresis tank, add enough electrophoresis solution and start loading. After the protein content of the protein sample is measured, add 5×SDS loading buffer, boil in boiling water for 3 minutes to mix well and then load the sample. The total protein amount loaded is 35μg.

[0086] (5) Run the gel at a constant voltage of 80 V. When the sample enters the lower gel, run the gel at a constant voltage of 120 V until bromophenol blue reaches the bottom of the gel, and then transfer to the membrane.

[0087] E. Transfer

[0088] (1) Gel cutting: Pry off the glass plate, remove the small glass plate, scrape off the concentrated gel, and cut the gel according to the molecular weight of the protein based on the experimental needs, using the marker as a control.

[0089] (2) Membrane preparation: Cut the PVDF membrane and filter paper, and activate the cut PVDF in 80% methanol solution for 30 seconds.

[0090] (3) Installing the membrane: Open the transfer clamp to keep the black side (negative electrode) horizontal. Place a sponge pad on top and soak it with transfer solution. Place the soaked filter paper on the pad and then stack them in the order of gel-nitrocellulose membrane-filter paper-sponge pad. Finally, cover the white plate (positive electrode) and install it in the transfer tank.

[0091] (4) Transfer: Place the clip into the transfer tank, with the black side of the clip facing the black side of the tank, and the white side of the clip facing the red side of the tank. Transfer the film in an ice bath for 120 minutes, with a constant current of 180 mA.

[0092] (5) After rotation, remove the membrane and mark one corner.

[0093] F. Immune response

[0094] (1) Blocking: Rinse the membrane in TBST for 3 times, 5 min each time. After rinsing, place the PVDF membrane in 5% skimmed milk powder and shake at room temperature for 1 h.

[0095] (2) Adding primary antibodies (SCD1, PARP, Bcl-2, Bax): Place the blocked PVDF membrane in a wash tank containing TBST and rinse on a shaker three times, 5 minutes each time. Place the membrane in a dish containing primary antibodies and incubate overnight at 4°C.

[0096] (3) Adding secondary antibody (mouse or rabbit antibody): Recover the primary antibody, rinse the PVDF membrane in a TBST washing tank for 3 times at room temperature on a shaker, 5 min each time, then place the rinsed PVDF membrane in a dish with secondary antibody, incubate at room temperature for 45 min in the dark. After incubation, wash the PVDF membrane in a TBST washing tank for 3 times, 5 min each time.

[0097] G. Chemiluminescence

[0098] Mix the color developing reagents in a small centrifuge tube according to the instructions of the luminescence kit, add them to the PVDF membrane, and develop the color using a chemiluminescence imager.

[0099] Figure 1 The changes in SCD1 enzyme activity of breast cancer MDA-MB-231 cells under different conditions. Among them, compound I has a significant inhibitory effect on SCD1 activity in breast cancer MDA-MB-231 cells. When exogenously added [d31]PA, the proportion of isotope-labeled monounsaturated fatty acid palmitoleic acid is significantly reduced. The specific inhibition results are shown in Table 1.

[0100] Table 1 SCD1 enzyme activity of breast cancer MDA-MB-231 cells under different conditions

[0101]

[0102] Figure 2 The figure shows the effect of compound Ⅰ at different concentrations on the expression of SCD1 in breast cancer MDA-MB-231 cells. Compared with the control group, compound Ⅰ can effectively inhibit the expression of SCD1 in a dose-dependent manner.

[0103] The above results indicate that compound I can be used as a new type of SCD1 inhibitor to effectively inhibit SCD1, suggesting that compound I can be used as a therapeutic drug for tumors, obesity and other related metabolic diseases to prevent and / or treat the occurrence and development of diseases.

[0104] Example 3 Effect of Compound I on the proliferation of tumor cells (MDA-MB-231, MDA-MB-468):

[0105] (1) Collect cells in the logarithmic phase and adjust the concentration of the cell suspension to 100 μL per well of a 96-well plate. Seed the plate so that the cell density to be tested is adjusted to 1,000 to 10,000 cells per well, and fill the edge wells with sterile PBS.

[0106] (2) Incubate the cells at 37°C in the presence of 5% CO2. After the cells have completely adhered to the wall, discard the original culture medium and add 100 μL of 10% FBS culture medium containing 1.25, 2.5, 5, 10, or 20 μM of Compound I to each well. The number of replicate wells is 4.

[0107] (3) After incubation for 24 h, take photos to record the cell status, discard the original culture medium, and add 20 μL of 5 mg / mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, trade name: Thiazole Blue) to each well.

[0108] (4) After incubation for 4 h, the original culture medium was discarded, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the cells were shaken at 300 rpm for 10 min to fully dissolve the purple crystals.

[0109] (5) In the enzyme-linked immunosorbent assay (ELISA) 450nm The absorbance of each well was measured and the result was calculated by the following formula:

[0110] Cell inhibition rate = (OD 处理 -OD 空白 ) / (OD 对照 -OD 空白 )*100%

[0111] Among them, OD 处理 OD of each treatment group 450nm Value; OD 空白 :Blank group OD 450nm Value; OD 对照 : OD of negative control group 450 value.

[0112] IC 50 Value determination:

[0113] IC 50(half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It can indicate that a certain concentration of a drug or substance (inhibitor) induces 50% apoptosis of tumor cells. This concentration is called the 50% inhibitory concentration, that is, the concentration corresponding to the ratio of apoptotic cells to the total number of cells equals 50%. IC 50 The value can be used to measure the ability of a drug to induce apoptosis, that is, the stronger the induction ability, the lower the value. It can also reversely indicate the degree of tolerance of a certain cell to a drug.

[0114] (1) Collect cells in the logarithmic phase and adjust the concentration of the cell suspension to 100 μL per well of a 96-well plate. Seed the plate so that the cell density to be tested is adjusted to 1,000 to 10,000 cells per well, and fill the edge wells with sterile PBS.

[0115] (2) Incubate the cells at 37°C in the presence of 5% CO2. After the cells have completely adhered to the wall, discard the original culture medium and add 100 μL of 10% FBS culture medium containing 1.25, 2.5, 5, 10, or 20 μM of Compound I to each well. The number of replicate wells is 4.

[0116] (3) After incubation for 24 h, take photos to record the cell status, discard the original culture medium, and add 20 μL of 5 mg / mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, trade name: Thiazole Blue) to each well.

[0117] (4) After incubation for 4 h, the original culture medium was discarded, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the cells were shaken at 300 rpm for 10 min to fully dissolve the purple crystals.

[0118] (5) In the enzyme-linked immunosorbent assay (ELISA) 450nm The absorbance of each well was measured and the result was calculated by the following formula:

[0119] Cell inhibition rate = (OD 处理 -OD 空白 ) / (OD 对照 -OD 空白 )*100%

[0120] Among them, OD 处理 OD of each treatment group 450nm Value; OD 空白 :Blank group OD 450nm Value; OD 对照 : OD of negative control group 450 value.

[0121] (6) The inhibition rate results were processed using GraphPad 7.0 software according to the concentration gradient, and the IC 50 value.

[0122] Table 2 IC of compound Ⅰ on different tumor cells 50 value:

[0123] Cell Type MDA-MB-468 MDA-MB-231 <![CDATA[IC 50 / μM]]> 14.21 8.15

[0124] Figure 3 The effect of compound I on the proliferation of two different tumor cells, MDA-MB-231 and MDA-MB-468, is shown. With the increase of concentration, especially at 5 μM and above, the inhibitory effect of compound I on tumor cells increases significantly, indicating that compound I can effectively inhibit different types of tumor cells. And according to the IC of compound I on tumor cells in Table 2 50 The values ​​show that compound Ⅰ has a more obvious inhibitory effect on breast cancer MDA-MB-231 and MDA-MB-468 cells.

[0125] Example 4 Detection of Compound I inducing apoptosis in breast cancer cells MDA-MB-231:

[0126] (1) Take cancer cells in the logarithmic growth phase and seed them in a 6-well plate at a density of 10 6 cells, incubated at 37°C with 5% CO2.

[0127] (2) After the cells adhered to the wall, 2 mL of 10% FBS medium containing compound I at different concentration gradients (0 μM, 2.5 μM, 5 μM and 10 μM) was added to each dish.

[0128] (3) After incubation for 24 h, discard the culture medium, digest with EDTA-free trypsin for 2 min, terminate the digestion with 2 to 4 times the volume of trypsin and complete culture medium, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0129] (4) After washing twice with 1 mL of PBS, resuspend the cells in 1 mL of 1X binding buffer in the Annexin V / PI detection kit.

[0130] (5) Take 100 μL of cell suspension, add 5 μL of Annexin V and 5 μL of PI, mix gently, and incubate at room temperature (25°C) in the dark for 15 min.

[0131] (6) Add 400 μL 1X binding buffer and perform flow cytometry within 1 hour

[0132] Note: A control group and a single positive tube must be set up at the same time to adjust the compensation.

[0133] Figure 4This is the apoptosis test of compound I on MDA-MB-231. The results show that compound I can induce apoptosis of tumor cells. When the drug concentration is 2.5μM, the apoptotic cell rate is about 16%, when it is 5μM, the apoptotic cell rate is about 20%, and when the drug concentration is 10μM, the apoptotic cell rate is about 44%. Especially at a concentration of 10μM, the proportion of live cells decreased significantly, and the total proportion of early apoptotic and late apoptotic cells increased significantly, indicating that compound I achieves anti-tumor effects by inducing apoptosis of tumor cells.

[0134] Therefore, compound I can cause obvious apoptosis in MDA-MB-231 cells and inhibit the proliferation of tumor cells, indicating that compound I can inhibit the occurrence and development of tumors and be used as a new therapeutic drug in the clinical treatment of tumors.

[0135] From the above results, we know that compound I can be used as a new type of SCD1 inhibitor to inhibit the enzyme activity of SCD1 and reduce the expression of SCD1, thereby effectively inhibiting SCD1. Figure 5 By counting the expression of related apoptotic proteins in MDA-MB-231, it can be found that compound I can induce the decrease of PARP total protein, promote the expression of Bax protein, and have little effect on Bcl-2 protein, resulting in a decrease in the ratio of Bcl-2 / Bax. These results all indicate that compound I can induce the occurrence of tumor cell apoptosis and has a significant inhibitory effect on the proliferation of tumor cells. SCD1, as a key enzyme regulating lipid metabolism, can affect the composition of intracellular membrane lipids, leading to damage and dysfunction of related structures, affecting the survival and rapid proliferation of tumor cells, and achieving the purpose of anti-tumor. At the same time, SCD1 inhibits the formation of monounsaturated fatty acids, can regulate fatty acid composition, and affect the occurrence and development of lipid metabolism-related diseases. Therefore, compound I, as a new type of SCD1 inhibitor, has important application prospects in the clinical treatment of tumors and the treatment of metabolic diseases such as obesity.

[0136] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A quinazolinone carboline compound, characterized in that The structural formula of the quinazolinone carboline compound is shown in Formula I:

2. A pharmaceutical composition, characterized in that The invention comprises the quinazolinone carboline compound as claimed in claim 1 and pharmaceutical excipients.

3. Use of the quinazolinone carboline compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for inhibiting stearoyl-CoA desaturase 1.

4. Use of the quinazolinone carboline compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation or development of drugs for treating fatty acid metabolism diseases.

5. Use of the quinazolinone carboline compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation or development of drugs for treating cancer.

6. The use according to claim 4, characterized in that: The fatty acid metabolism disease is selected from obesity, fatty liver, insulin resistance, hyperlipidemia, primary obesity, cardiovascular and cerebrovascular diseases, atherosclerosis or diabetes.

7. The use according to claim 5, characterized in that The cancer is selected from breast cancer, prostate cancer, liver cancer, lung cancer, colon cancer, lung cancer, ovarian cancer or thyroid cancer.

8. The pharmaceutical composition according to claim 2, characterized in that: The pharmaceutical excipients include one or more of a binder, a filler, a disintegrant, a lubricant, an antioxidant, a flavoring agent, a fragrance, a cosolvent, an emulsifier, a solubilizer, an osmotic pressure regulator or a colorant.

9. The use according to any one of claims 3 to 5, characterized in that: The dosage form of the drug includes traditional dosage form or modern dosage form.

10. The use according to any one of claims 3 to 5, characterized in that: The drug further contains a carrier, which includes microcapsules, microspheres, nanoparticles or liposomes.

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