A sulfathiazole selenide compound and its application in the preparation of hypoglycemic drugs

Abstract

The present invention discloses a sulfathiazole selenide compound and its application in the preparation of hypoglycemic drugs. The structural formula of the compound is shown in formula (I). The sulfathiazole selenide compound proposed by the present invention can not only retain insulin sensitivity but also reduce the side effects caused by PPARγ full agonists.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a sulfathiazole selenazole compound and its application in the preparation of hypoglycemic drugs. Background Art

[0002] Type II diabetes (T2DM) is one of the major chronic diseases that seriously endanger the health of humans in China and even the world. The incidence of type 2 diabetes in China shows an increasing trend year by year, and currently, the number of diabetes patients in China ranks first in the world. Existing drugs can only control blood sugar within a certain range to reduce and delay the occurrence of complications, and the prevention and treatment research of diabetes still has a long way to go. Insulin resistance is one of the important mechanisms of type 2 diabetes, and improving insulin resistance is the main strategy for clinical treatment of diabetes. Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in regulating the body's glucose and lipid metabolism. As one of the most effective targets for the development of diabetes treatment drugs, several highly effective hypoglycemic drugs have been successfully developed. For example, PPARγ full agonists - thiazolidinediones (TZDs), rosiglitazone (Rosi) and pioglitazone, lower blood sugar by increasing the sensitivity of peripheral tissues to insulin and improving insulin resistance, and have always shown powerful efficacy in the treatment of type 2 diabetes. The listing of TZDs drugs has created a new era of highly effective hypoglycemic treatment. Unfortunately, during clinical use, it will cause many side effects such as obesity, weight gain, edema, myocardial hypertrophy, congestive heart failure, liver toxicity, and an increased risk of fractures. The occurrence of these side effects is mainly due to the strong hydrogen bond interaction between such small molecules and key amino acids such as His323, His449, and Tyr473 on the helix 12 of PPARγLBD, which causes the activation function-2 (AF-2) to be in a closed state, resulting in the fully transcriptional activation state of PPARγ. These side effects have seriously hindered the clinical application of TZDs drugs. Therefore, the development of oral hypoglycemic drugs with good efficacy and low toxicity and side effects has become an urgent need in the medical field, and it is also a research hotspot in the current academic and pharmaceutical industries. Summary of the Invention

[0003] The present invention provides a sulfathiazole selenazole compound and its application in the preparation of hypoglycemic drugs. The sulfathiazole selenazole compound proposed by the present invention can not only retain insulin sensitivity but also act as a PPARγ selective regulator to reduce the side effects caused by PPARγ full agonists.

[0004] The first object of the present invention is to provide a sulfathiazole selenazole compound represented by formula (I), its tautomer, its optical isomer, its hydrate, its solvate, its pharmaceutically acceptable salt or its prodrug:

[0005]

[0006] The second object of the present invention is to provide a hypoglycemic pharmaceutical composition comprising an effective amount of the sulfathiazole selenide compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt.

[0007] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from one or more of binders, excipients, stabilizers and lubricants.

[0008] Preferably, the dosage form of the pharmaceutical composition is tablets, injections, suspensions or solutions.

[0009] The third object of the present invention is to provide a pharmaceutical preparation comprising an effective amount of the sulfathiazole selenide compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt or the pharmaceutical composition.

[0010] Preferably, the dosage form of the preparation is an oral dosage form, an injection dosage form or a transdermal dosage form.

[0011] The fourth object of the present invention is to provide the use of the sulfathiazole selenide compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt or the hypoglycemic pharmaceutical composition or the pharmaceutical preparation in the preparation of PPARγ regulators (including but not limited to covalent regulators), and / or in the preparation of drugs for treating and / or preventing diseases regulated by PPARγ regulators (including but not limited to agonists, antagonists).

[0012] Preferably, the diseases are selected from diabetes, elevated blood pressure, elevated lipids, metabolic syndrome with elevated cholesterol levels, or combinations thereof.

[0013] More preferably, the diabetes includes type II diabetes and non-insulin-dependent diabetes.

[0014] More preferably, the diabetes is type II diabetes targeted at PPARγ. The present invention protects the application of sulfaselenazole derivatives in type II diabetes (T2DM).

[0015] The present invention provides the use of the sulfathiazole selenide compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt or the hypoglycemic pharmaceutical composition or the pharmaceutical preparation in the preparation of hypoglycemic drugs.

[0016] The present invention also provides an anti-diabetic drug, which uses sulfathiazole selenide compounds (especially compound 2n), their tautomers, their optical isomers, their hydrates, their solvates or their pharmaceutically acceptable salts as active ingredients. The anti-diabetic drug is specifically a type II diabetes drug. At the same time, the compounds synthesized in the present invention can effectively reduce fasting blood glucose and random blood glucose in diabetic model mice (including but not limited to HFD, db / db, ob / ob mice) without causing weight gain. This indicates that the compounds proposed in the present invention have good hypoglycemic effects and relatively few side effects.

[0017] The above drug composition or pharmaceutical preparation also has one or more properties selected from the following groups:

[0018] (a) Weak PPARγ activation ability;

[0019] (b) Strong PPARγ binding ability;

[0020] (c) Weak ability to transform preadipocyte 3T3-L1 into adipocytes.

[0021] (d) The compounds synthesized in the present invention weakly activate / inhibit the expression of PPARγ downstream adipogenic genes and insulin resistance genes (including but not limited to PTP1B and SOCS3).

[0022] (e) The compounds synthesized in the present invention up-regulate the expression of PPARγ downstream insulin-sensitive genes (including but not limited to Glut4 and Adiponectin).

[0023] The present invention targets PPARγ and hopes to find a class of potential small molecule ligands of PPARγ through high-throughput screening. They bind to the ligand binding pocket (LBP) of PPARγ in a unique way, enabling them to stabilize the AF-2 fragment in specific states of transcriptional activation and transcriptional inhibition, resulting in different cofactor recruitment or replacement, and reducing the transcriptional activation activity of PPARγ, so that it can both retain insulin sensitivity and reduce the side effects caused by PPARγ full agonists, this "specific selectivity".

[0024] Compared with the prior art, the beneficial effects of the present invention are: the sulfathiazole selenide compounds proposed in the present invention, especially compound 2n, can both retain insulin sensitivity and reduce the side effects caused by PPARγ full agonists. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 . Screening sulfathiazole selenide derivatives as PPARγ ligands by TR-FRET experiment.

[0026] Figure 2. Detection of the binding ability of compound 2n to PPARγ by TR-FRET assay, ** P < 0.01 compared with the DMSO group.

[0027] Figure 3 . Detection of the activation ability of compound 2n to PPARγ by dual-luciferase reporter gene assay.

[0028] Figure 4 . Detection of the ability of compound 2n to induce adipogenic differentiation of 3T3-L1 cells by Oil Red O staining. Compared with the positive drug Rosi, compound 2n only weakly induced adipocyte differentiation; *** P < 0.001 compared with the DMSO group; ## P < 0.01 compared with the Vehicle group.

[0029] Figure 5 . Detection of the expression of adipogenic genes and insulin-sensitive genes by real-time fluorescence quantitative PCR. * P < 0.05, ** P < 0.01 compared with the DMSO group; # P < 0.05, ## P < 0.01 compared with Rosi.

[0030] Figure 6 . Preliminary evaluation of the hypoglycemic activity and safety of compound 2n in vivo. ### P < 0.001 compared with the db / m group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the Vehicle group. Specific implementation manners

[0031] The present invention will be further described in detail below with reference to the embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, are regarded as raw materials and reagents that can be obtained through commercial channels such as the conventional market. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention fall within the scope of the present invention claimed.

[0032] Example 1

[0033] Sulfathiazole selenide compounds were prepared according to the method disclosed in CN 115677621 A.

[0034]

[0035] The following is a preparation method of the specific disclosed compound 2n (1-oxide-1-(4-(p-toluenesulfonyloxy)phenyl)-1λ 4 -benzo[d][1,3,2]thiaselenazol-5-yl 4-methylbenzenesulfonate derivative), which comprises the following steps:

[0036]

[0037] Step 1: Add a magnetic stir bar into a dry 100 mL eggplant-shaped flask. Weigh 4,4'-dihydroxydiphenyl sulfide (1.0 equiv.) into the reaction flask and add DCM (0.1 M) to dissolve it. Slowly add TsOCl (3.0 equiv.) and triethylamine (3.0 equiv.) portionwise at 0 °C, and then slowly warm up to room temperature and react overnight. Monitor the reaction by TLC until completion, terminate the reaction, extract three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, and evaporate and concentrate to obtain the corresponding diaryl sulfide, which is directly used in the next step of the reaction.

[0038] Step 2: Take a 100 mL round-bottom flask, dissolve the diaryl sulfide in methanol solvent, then weigh ammonium carbamate (2.0 equiv.) and iodobenzene diacetate (2.5 equiv.) and add them together to the reaction solution. React the reaction solution at room temperature overnight. Monitor the reaction by TLC until completion, terminate the reaction. Evaporate the methanol solution, add dichloromethane to dissolve, extract with water, dry the organic phase with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain a viscous substance. Then purify the crude product by column chromatography (PE / EA = 3 / 1) to obtain the required diaryl sulfonimide substrate.

[0039] Step 3: Take a 5 mL reaction flask, weigh sulfonimide (1 mmol, 1.0 equiv.), elemental selenium (3 mmol, 3.0 equiv.), [Cp*Rh(MeCN)3(SbF6)2] (5 mol%), and silver fluoride (2.5 mmol, 2.5 equiv.) into the reaction flask, add 1,2-dichloromethane (5 mL) to dissolve, without the need to exclude air or moisture additionally, stir and react at 100 °C for 10 hours. Monitor the reaction by TLC until completion, filter out the metal residue through a silica gel column, rotary evaporate the filtrate, and then purify it through a silica gel plate to obtain the sulfonothiaselenazole compound 2n. Yield: 38% (248 mg, 1 mmol); yellow viscous substance; Rf = 0.3 (PE / EA = 3 / 1).

[0040] 11H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 8.7 Hz, 2H), 7.74 (t, J = 7.2 Hz, 4H), 7.38 - 7.33 (m, 4H), 7.31 (s, 1H), 7.26 (d, J = 1.9 Hz, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.6 Hz, 1H), 2.47 (s, 6H). 13 13C NMR (101 MHz, CDCl3): δ 153.9, 152.1, 146.3, 144.0, 138.6, 132.5, 131.9, 131.83, 131.76, 130.3, 128.6, 126.8, 123.4, 121.2, 118.0, 21.9.

[0041] HRMS (ESI) calculated for C 26 H 22 NO7S3Se ([M + H] + ): 653.9718; found: 653.9716.

[0042] The following experimental examples are used to illustrate the uses of the sulfathiazole selenide compounds provided by the present invention, especially the use of compound 2n.

[0043] Experimental Example 1

[0044] I. Detection of the binding ability of sulfathiazole selenide derivatives to PPARγ by TR-FRET experiment, and the specific steps are as follows:

[0045] 1) Prepare Complete TR-FRET PPAR Assay Buffer: Add 5 μL of 1 M DTT to 1 mL of TR-FRET PPAR Assay Buffer to make the final concentration of DTT 5 mM;

[0046] 2) Prepare 2× test compounds, negative control group, and positive control drug: Dilute the test compounds to 2× concentration with the Complete TR-FRET PPAR Assay Buffer prepared in step 1), and the DMSO with the same concentration is used as the negative control, and the Rosi with the same concentration is used as the positive control (the final 1× concentration is 10 μM);

[0047] 3) Prepare 4× Fluormone TM Pan-PPAR Green (20 nM): Add 10 μL of 2 μM Fluormone to 1 mL of 4× Fluormone TM Pan-PPAR Green TMPan-PPAR Green was gently inverted and mixed well in 990 μL of Complete TR-FRET PPAR Assay Buffer;

[0048] 4) Prepare 4×PPARγ-LBD / Tb-anti-GST Ab (20 nM): For 1 mL of 4×PPARγ-LBD / Tb-anti-GST Ab, 5.71 μL of Tb-anti-GST Ab (stock solution concentration is 3.5 μM) and 0.12 μL of PPARγ-LBD (stock solution concentration is 17300 nM) were added to 994.2 μL of Complete TR-FRET PPAR Assay Buffer, and gently inverted and mixed well.

[0049] 5) Add the reagents to the measurement wells in the order listed in Table 1 below, and gently mix on a shaker for 30 s.

[0050] Table 1

[0051]

[0052]

[0053] 6) Incubate in the dark at room temperature for 1 - 6 h. During this period, multiple measurements can be taken to ensure that the binding of the test compound reaches equilibrium.

[0054] 7) Use a multi-functional microplate reader to detect the fluorescence absorption signals at 495 nm and 520 nm respectively. The instrument parameters are set as shown in Table 2 below:

[0055] Table 2

[0056]

[0057] 8) Calculate the TR-FRET value using the ratio of the fluorescence signal at 520 nm to the fluorescence signal at 495 nm; Use the formula K i =IC 50 / (1 + [tracer] / K D ) to calculate the inhibition constant K i . Where IC 50 is the concentration at which the compound produces 50% competitive substitution of the tracer, [tracer] is the concentration of fluormonone TM Pan-PPAR Green (5 nM), and K D is the binding constant of fluormonone TM Pan-PPAR Green to PPARγ-LBD (2.8 ± 0.8 nM).

[0058] The experimental results are asFigure 1-2 as shown in Table 3:

[0059] Table 3

[0060]

[0061] Data are presented as mean ± standard deviation. **P < 0.01 compared with the DMSO group, n = 3.

[0062] Figure 1 and Figure 2 Among them, the smaller the value, the stronger the binding ability. It can be seen from Figure 1-2 and Table 3 that compound 2n has a relatively strong binding affinity with PPARγ.

[0063] II. Detection of the activation ability of compound 2n on PPARγ by dual-luciferase reporter gene assay. The specific steps are as follows:

[0064] Transfection of PPARγ plasmid into Cos-7 cells: Cos-7 cells were purchased from ATCC and cultured in DMEM without antibiotics containing 10% FBS in a 37°C, 5% CO2 incubator. When the cells entered the logarithmic growth phase, they were seeded in 24-well plates at a density of 2×10 5 cells, and the transfection experiment was carried out when the cells reached 70% confluence. The experimental operations are as follows:

[0065] 1) Prepare solution A: 640 μL of serum-free medium + 25.6 μL of Lipo2000 (for one 24-well plate);

[0066] 2) Prepare solution B: 750 μL of serum-free medium + 2.38 μL of 3 μg PPRE×3TK-luciferase plasmid + 0.83 μL of 1.5 μg hPPARγ expression plasmid + 0.25 μL of 0.3 μg Renilla luciferase expression plasmid;

[0067] 3) Mix at a ratio of A:B = 1:1 (640 μL + 640 μL) and incubate at room temperature for 5 min. Add the transfection solution (the mixture of A and B) to the cells containing 450 μL of serum-free medium, 50 μL / well.

[0068] 4) After 24 h of transfection, treat the cells with drugs for 24 h (1 μM, 10 μM of the test compound, 1 μM, 10 μM of Rosi, and the same concentration of DMSO).

[0069] III. Dual-luciferase reporter gene assay:

[0070] 1) Preparation of 1× cell lysis buffer: Mix 1 volume of 5× Passive Lysis Buffer (PLB) with 4 volumes of ddH2O evenly, add it to a 24-well plate (100 μL / well), and incubate at room temperature for 20 min;

[0071] 2) Preparation of Luciferase Assay Reagent II: Resuspend 1 vial of lyophilized Luciferase Assay Substrate with 10 mL of Luciferase Assay Buffer II and mix well (it can be stored at -20°C for one month and at -70°C for one year after dissolution);

[0072] 3) Preparation of Stop& Reagent: Add 1 volume of 50× Stop& Substrate to 50 volumes of Stop& Buffer and mix well. Prepare it freshly before use (it can be stored at -20°C for 15 days);

[0073] 4) Add 20 μL of PLB lysis buffer containing cell lysate to a white opaque 96-well plate, then add 100 μL of Luciferase Assay Reagent II, gently pipette and mix well, and use a multi-functional microplate reader to detect the activity of firefly luciferase;

[0074] 5) After the first detection, add 100 μL of Stop& Reagent, gently pipette and mix well, and detect the activity of Renilla luciferase.

[0075] Calculate the ratio of the activity of firefly luciferase detected in the first detection to the activity of Renilla luciferase detected in the second detection, which is the PPARγ transcriptional activation data.

[0076] The experimental results are as Figure 3 shown in and Table 4. Compared with the full agonist Rosi, compound 2n only weakly activates PPARγ.

[0077] Table 4

[0078]

[0079] IV. Detection of the ability of compound 2n to induce adipogenic differentiation of 3T3-L1 cells by Oil Red O staining. The specific steps are as follows:

[0080] 3T3-L1 preadipocytes were purchased from ATCC and cultured in DMEM medium containing 10% FBS. The cells were incubated at 37°C in a 5% CO2 incubator. They were seeded in 6-well plates, and after confluence, on the 2nd day, induction medium (10% FBS DMEM containing 0.5 mmol / L IBMX (3-isobutyl-1-methylxanthine), 1 μmol / L DEX (dexamethasone), and 850 nmol / L insulin) and compound 2n were added. After 72 h, the medium was changed to 10% FBS high-glucose DMEM medium containing 850 nmol / L insulin, and the medium was changed every 2 days for a total of 6 days. 10 μM rosiglitazone was used as the positive control, DMSO as the negative control, and the sample group of 2n was at 1 μM. On the 9th day after the start of induction, Oil Red O staining was performed, and photographs were taken with a microscope (OLYMPUS), and the adipocyte differentiation rate was calculated.

[0081] The experimental results are as Figure 4 shown that compound 2n only weakly induced adipocyte differentiation.

[0082] V. Real-time PCR was used to measure the expression of adipogenic genes, and the specific steps were as follows:

[0083] 3T3-L1 preadipocytes were induced to differentiate according to the above protocol, and total cellular RNA was extracted. Real-time PCR was performed according to the protocol of the kit (takara). Using the -ΔΔCt method and with β-actin as the internal reference, the relative expression of mRNA was calculated.

[0084] The experimental results are shown in Figure 5 , and the results showed that compound 2n could inhibit the expression of adipocyte differentiation-related genes and insulin resistance genes (PTP1B and SOCS3), and up-regulate the expression of insulin-sensitive genes (Glut4 and Adiponectin).

[0085] Table 5. Primer sequences

[0086]

[0087] VI. To detect the hypoglycemic effect of compound 2n in db / db model mice, the specific steps were as follows:

[0088] Six-week-old db / db diabetic model mice were purchased and randomly grouped. The vehicle group (Vehicle, n = 6), the compound 2n administration groups (2.5 mg / kg / day, n = 6, 5 mg / kg / day, n = 6, 10 mg / kg / day, n = 6), and the GW9662 control group (10 mg / kg / day, n = 6) were administered by intraperitoneal injection; the positive control group Rosi (10 mg / kg / day, n = 6) was administered orally, once a day. After three weeks, the fasting blood glucose, random blood glucose, fasting body weight, and glucose tolerance levels of the mice in each experimental group were measured.

[0089] The experimental results are shown in Figure 6 , and the results show that compound 2n can significantly reduce the fasting blood glucose and random blood glucose of db / db mice without causing weight gain; in addition, compound 2n can significantly improve the glucose tolerance of db / db mice.

[0090] The above results show that:

[0091] 1. The compound 2n of the present invention has a weak ability to activate PPARγ. Using rosiglitazone, a full agonist of PPARγ, as a positive control (specified as 100%), the activation values of the compound 2n of the present invention at concentrations of 1 μM and 10 μM are only 25% and 18% respectively, which indicates that the side effects of the compound of the present invention are relatively small.

[0092] 2. The compound 2n of the present invention has a strong binding ability to PPARγ, and its binding ability to PPARγ at a concentration of 10 μM is comparable to that of the positive drug rosiglitazone.

[0093] 3. The compound 2n of the present invention very weakly induces adipocyte differentiation. Using the oral hypoglycemic drug rosiglitazone as a positive control (test value of 44.8%), the adipocyte differentiation rate of the compound 2n of the present invention at the same treatment concentration is only 9.4% (the solvent control group is 8.9%).

[0094] 4. The compound 2n of the present invention can inhibit the expression of adipocyte differentiation-related genes and insulin resistance genes (PTP1B and SOCS3), and up-regulate the expression of insulin-sensitive genes (Glut4 and Adiponectin).

[0095] 5. The compound 2n of the present invention can effectively reduce the fasting blood glucose and random blood glucose of mice in the db / db diabetic model mouse body without causing weight gain, and at the same time can significantly improve the glucose tolerance of mice. It shows that the compound 2n of the present invention has good hypoglycemic effects and relatively small side effects.

[0096] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and retouches can also be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A hypoglycemic pharmaceutical composition, characterized in that, Containing an effective amount of sulfathiazole selenide compounds, the sulfathiazole selenide compounds are shown as formula (I):

2. The hypoglycemic pharmaceutical composition according to claim 1, wherein The hypoglycemic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is selected from one or more of binders, excipients, stabilizers and lubricants.

3. A pharmaceutical preparation, characterized in that, Containing an effective amount of the hypoglycemic pharmaceutical composition according to claim 1.

4. The pharmaceutical preparation according to claim 3, wherein, The dosage form of the preparation is an oral dosage form, an injection dosage form or a transdermal dosage form.

5. Use of the hypoglycemic pharmaceutical composition according to claim 1 or the pharmaceutical preparation according to claim 3 in the preparation of a PPARγ regulator, and / or a drug for treating and / or preventing diseases regulated by a PPARγ regulator.

6. The application according to claim 5, wherein The diseases are selected from diabetes, elevated blood pressure, elevated lipids, metabolic syndrome with elevated cholesterol levels, or a combination thereof.

7. The application according to claim 6, wherein The diabetes includes type II diabetes and non-insulin-dependent diabetes.

8. The application according to claim 7, wherein The diabetes is type II diabetes targeting PPARγ.

9. Use of the hypoglycemic pharmaceutical composition according to claim 1 or the pharmaceutical preparation according to claim 3 in the preparation of a hypoglycemic drug.

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