CN117229231A - Sulfathiazole compound and application thereof in preparation of hypoglycemic drugs - Google Patents

Abstract

The invention discloses a sulfathiaselenazole compound and application thereof in preparation of hypoglycemic drugs. The structural formula of the compound is shown as formula (I). The sulfathiaselenazole compound provided by the invention can not only keep insulin sensitization, but also reduce side effects caused by PPAR gamma complete agonists.

Description

Sulfathiazole compound and application thereof in preparation of hypoglycemic drugs

Technical Field

The invention relates to the technical field of medicines, in particular to a sulfathiaselenazole compound and application thereof in preparation of hypoglycemic drugs.

Background

Type II diabetes mellitus (T2 DM) is one of serious chronic diseases seriously harming human health in China or even worldwide, the incidence rate of type 2 diabetes mellitus in China shows a trend of rising year by year, and the number of diabetes mellitus patients in China is the first place worldwide at present. The existing medicine can only control blood sugar in a certain range so as to reduce and delay the occurrence of complications, and the prevention and treatment of diabetes mellitus are far away. Insulin resistance is one of the important mechanisms of onset of type 2 diabetes, and improvement of insulin resistance is a major strategy for clinical treatment of diabetes. Peroxisome proliferator-activated receptor gamma (pparγ) plays an important role in regulating the body's glycolipid metabolic process, and has been successfully developed as one of the most effective targets for development of therapeutic drugs for diabetes. Such as pparγ full agonists, thiazolidinediones (TZDs), rosiglitazones (Rosi) and pioglitazone, have been shown to have powerful therapeutic effects on the treatment of type 2 diabetes by increasing insulin sensitivity in peripheral tissues, improving insulin resistance and lowering blood glucose, and the market of TZDs has opened a new era of high-efficiency hypoglycemic. Unfortunately, many side effects such as obesity, weight gain, edema, myocardial hypertrophy, congestive heart failure, hepatotoxicity, and increased risk of fracture can result during clinical use. These side effects are mainly caused by the fact that the activating function-2 (AF-2) is in a closed state due to the strong hydrogen bond interaction between the small molecules and key amino acids such as His323, his449 and Tyr473 on the PPARgamma LBD helix 12, so that the PPARgamma is in a complete transcriptional activation state, and the side effects have seriously hindered the clinical application of TZDs drugs, so that the development of oral hypoglycemic drugs with good curative effects and low toxic and side effects has become urgent demands of the medical community, and are also a research hot spot in the current academia and pharmaceutical industry.

Disclosure of Invention

The invention provides a sulfathiaselenazole compound and application thereof in preparation of hypoglycemic drugs, and the sulfathiaselenazole compound provided by the invention can not only keep insulin sensitization, but also be used as a PPAR gamma selective modulator for reducing side effects caused by a PPAR gamma complete agonist.

The first object of the present invention is to propose a sulfathiaselenazole compound represented by the formula (I):

the second object of the present invention is to provide a hypoglycemic pharmaceutical composition, which contains an effective amount of the sulfathiaselenazole compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from more than one of a binder, an excipient, a stabilizer and a lubricant.

Preferably, the pharmaceutical composition is in the form of a tablet, injection, suspension or solution.

A third object of the present invention is to provide a pharmaceutical preparation comprising an effective amount of the sulfathiaselenazole compound, its tautomer, its optical isomer, its hydrate, its solvate or a pharmaceutically acceptable salt thereof or the pharmaceutical composition.

Preferably, the preparation is in the form of oral preparation, injection or transdermal preparation.

A fourth object of the present invention is to propose the use of said sulfathiaselenazole compound, its tautomer, its optical isomer, its hydrate, its solvate or its pharmaceutically acceptable salt or said hypoglycemic pharmaceutical composition or said pharmaceutical formulation for the preparation of pparγ modulators (including but not limited to covalent modulators), and/or for the treatment and/or prevention of diseases modulated by pparγ modulators (including but not limited to agonists, antagonists).

Preferably, the disease is selected from diabetes, elevated blood pressure, elevated lipids, metabolic syndrome of cholesterol levels, or a combination thereof.

Further preferably, the diabetes includes type II diabetes and non-insulin dependent diabetes.

Further preferably, the diabetes is type II diabetes targeted to pparγ. The invention relates to application of a sulfamethylthiazole derivative in the aspect of type II diabetes mellitus (T2 DM).

The invention provides application of a sulfathiaselenazole compound, a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof or pharmaceutically acceptable salt thereof or a hypoglycemic pharmaceutical composition or pharmaceutical preparation in preparation of hypoglycemic drugs.

The invention also provides an antidiabetic drug, which takes the sulfathiaselenazole compound (especially compound 2 n), a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof as an active component. The antidiabetic agent is specifically a type II diabetes agent. Meanwhile, the compound synthesized by the invention can effectively reduce fasting blood glucose and random blood glucose in diabetic mice (including but not limited to HFD, db/db and ob/ob mice) without causing weight gain. The compound provided by the invention has good hypoglycemic effect and small side effect.

The above pharmaceutical composition or pharmaceutical formulation further has one or more characteristics selected from the group consisting of:

(a) Weak activation ability of pparγ;

(b) Pparγ has strong binding ability;

(c) Weak capacity of preadipocytes for 3T3-L1 transformation into adipocytes.

(d) The compounds synthesized in the present invention weakly activate/inhibit the expression of the downstream adipogenic genes and insulin resistance genes of pparγ (including but not limited to PTP1B and SOCS 3).

(e) The compounds synthesized in the present invention up-regulate the expression of insulin sensitive genes downstream of pparγ, including but not limited to Glut4 and adionectin.

The invention takes PPARgamma as a target, and a potential PPARgamma small molecule ligand is expected to be found through high-throughput screening and combined with a Ligand Binding Pocket (LBP) of PPARgamma in a unique mode, so that the ligand binding pocket can stabilize an AF-2 fragment under specific states of transcriptional activation and transcriptional inhibition, different cofactors are recruited or replaced, and the transcriptional activation activity of PPARgamma is reduced, thereby not only retaining insulin sensitization, but also reducing the side effect caused by a PPARgamma full agonist.

Compared with the prior art, the invention has the beneficial effects that: the sulfathiaselenazole compound, especially compound 2n, provided by the invention can not only keep insulin sensitization, but also reduce side effects caused by PPAR gamma complete agonists.

Drawings

FIG. 1 TR-FRET experiment screening of a sulfathiaselenazole derivative as PPARgamma ligand.

FIG. 2.TR-FRET assay to determine the binding capacity of Compound 2n to PPARgamma, ^** P<0.01 compared to DMSO group.

FIG. 3 shows the ability of compound 2n to activate PPARgamma by a dual luciferase reporter assay.

FIG. 4 oil red O staining test compound 2n for its ability to induce adipogenic differentiation of 3T3-L1 cells. Compound 2n only weakly induced adipocyte differentiation compared to the positive drug Rosi; ^*** P<0.001 compared to DMSO group; ^## P<0.01 compared to the veccle group.

FIG. 5 real-time fluorescent quantitative PCR assay of expression of adipogenic and insulin sensitive genes. ^* P<0.05， ^** P<0.01 compared to DMSO group; ^# P<0.05， ^## P<0.01 compared to Rosi.

FIG. 6 preliminary evaluation of hypoglycemic activity and safety in vivo of Compound 2n. ^### P<0.001 compared to db/m group; ^* P<0.05， ^** P<0.01， ^*** P<0.001 compared to the veccle group.

Detailed Description

The present invention will be described in further detail with reference to examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods without specific conditions noted in the examples below are generally in accordance with conventional conditions in the art or in accordance with manufacturer's recommendations; the raw materials, reagents and the like used, unless otherwise specified, are considered to be commercially available through conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art in light of the above teachings are intended to be within the scope of the invention as claimed.

Example 1

The sulfathiaselenazole compound is prepared by a method disclosed by CN 115677621A.

The following specifically discloses the compound 2n (1-oxide-1- (4- (p-toluenesulfonyloxy) phenyl) -1λ ⁴ Benzo [ d ]][1,3,2]A thiaselenazol-5-yl 4-methylbenzenesulfonate derivative) comprising the steps of:

step 1: into a dry 100mL eggplant-shaped bottle was added a rotor, 4' -dihydroxydiphenyl sulfide (1.0 eq) was weighed into a reaction bottle and dissolved by adding DCM (0.1M), tsOCl (3.0 eq) and triethylamine (3.0 eq) were added slowly in portions at 0℃and then allowed to react overnight at a slow temperature. The reaction is monitored to be complete by thin layer chromatography, the reaction is ended, ethyl acetate is extracted for three times, the organic phase is dried by anhydrous sodium sulfate, and the corresponding diaryl sulfide is obtained by evaporation and concentration and is directly put into the next reaction.

Step 2: a100 mL round-bottomed flask was taken, diaryl sulfide was dissolved in a methanol solvent, and ammonium carbamate (2.0 equivalents) and iodobenzene acetate (2.5 equivalents) were weighed into the reaction solution together, and the reaction solution was allowed to react overnight at room temperature. The reaction was monitored by thin layer chromatography and ended. The methanol solution was removed by evaporation, dissolved in methylene chloride, extracted with water, and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure to give a viscous product. The crude product was then purified by column chromatography (PE/ea=3/1) to give the desired diaryl sulfonimide substrate.

Step 3: a5 mL reaction flask was used to weigh the sulfonimide (1 mmol,1.0 eq.), elemental selenium (3 mmol,3.0 eq.), [ Cp. Rh (MeCN) ₃ (SbF ₆ ) ₂ ](5 mol%) and silver fluoride (2.5 mmol,2.5 eq.) were placed in a reaction flask and 1, 2-dichloromethane (5 mL) was added to dissolve, and the reaction was stirred at 100℃for 10 hours without additional removal of air or moisture. And monitoring the reaction completely by thin layer chromatography, filtering out metal residues by a silica gel column, spin-drying filtrate, and purifying by a silica gel plate to obtain the sulfathiaselenazole compound 2n. Yield: 38%. (248 mg,1 mmol); yellow sticky; rf=0.3 (PE/ea=3/1).

¹ H NMR(400MHz,CDCl ₃ ):δ7.90(d,J＝8.7Hz,2H),7.74(t,J＝7.2Hz,4H),7.38-7.33(m,4H),7.31(s,1H),7.26(d,J＝1.9Hz,1H),7.21(d,J＝8.7Hz,2H),6.93(d,J＝8.6Hz,1H),2.47(s,6H). ¹³ C NMR(101MHz,CDCl ₃ ):δ153.9,152.1,146.3,144.0,138.6,132.5,131.9,131.83,131.76,130.3,128.6,126.8,123.4,121.2,118.0,21.9.

HRMS(ESI)calculated for C ₂₆ H ₂₂ NO ₇ S ₃ Se([M+H] ⁺ ):653.9718；found:653.9716.

The following experimental examples are used for illustrating the application of the sulfathiaselenazole compound provided by the invention, in particular to the application of the compound 2n.

Experimental example 1

1. The TR-FRET experiment detects the binding capacity of the sulfathiaselenazole derivative and PPARgamma, and comprises the following specific steps:

1) Preparation of Complete TR-FRET PPAR Assay Buffer:1mL TR-FRET PPAR Assay Buffer was added with 5. Mu.L of 1M DTT to give a final concentration of 5mM DTT;

2) Preparing 2 x test compound, negative control group and positive control drug: diluting the compound to be tested to 2X concentration by using the Complete TR-FRET PPAR Assay Buffer prepared in the step 1), taking DMSO with equal concentration as a negative control, and taking Rosi with equal concentration as a positive control (final 1X concentration is 10 mu M);

3) Preparation of 4X fluorone ^TM Pan-PPAR Green(20nM)：1mL 4×Fluormone ^TM Pan-PPAR Green requires 10. Mu.L of 2. Mu.M fluorone ^TM Add Pan-PPAR Green to 990. Mu.L Complete TR-FRET PPAR Assay Buffer and mix gently upside down;

4) Preparation of 4 XPPARgamma-LBD/Tb-anti-GSTAb (20 nM): 1mL of 4 XPPARgamma-LBD/Tb-anti-GST Ab 5.71. Mu.L of Tb-anti-GST Ab (stock concentration 3.5. Mu.M) and 0.12. Mu.L of PPARgamma-LBD (stock concentration 17300 nM) were added to 994.2. Mu.L of Complete TR-FRET PPAR Assay Buffer, and mixed gently and inverted.

5) The reagents were added to the measurement wells in the order listed in Table 1, and gently mixed on a shaker for 30s.

TABLE 1

6) Incubation at room temperature for 1-6h in the absence of light can be measured multiple times to ensure that the binding of the test compound reaches equilibrium.

7) Fluorescence absorption signals at 495nm and 520nm were detected using a multifunctional microplate reader, respectively, with the following table 2 set as the instrument parameters:

TABLE 2

8) The TR-FRET value is calculated using the ratio of the fluorescence signal at 520nm to the fluorescence signal at 495 nm; using formula K _i ＝IC ₅₀ /(1+[tracer]/K _D ) To calculate the suppression constant K _i . Wherein IC ₅₀ Is the concentration of the compound that produces 50% competitive substitution of tracer, [ tracer ]]Is fluoromonomer ^TM Concentration of Pan-PPAR Green (5 nM), K _D Is fluoromonomer ^TM Binding of Pan-PPAR Green to PPARgamma-LBDNumber (2.8.+ -. 0.8 nM).

The experimental results are shown in FIGS. 1-2 and Table 3:

TABLE 3 Table 3

Data are expressed as mean ± standard deviation. * P <0.01 compared to DMSO group, n=3.

In FIGS. 1 and 2, smaller values represent stronger binding forces, as can be seen from FIGS. 1-2 and Table 3, compound 2n has stronger binding affinity to PPARy.

2. The dual luciferase reporter gene assay detects the ability of compound 2n to activate pparγ, and comprises the following specific steps:

PPARgamma plasmid transfection Cos-7 cells: cos-7 cells were purchased from ATCC, in 10% FBS, antibiotic free DMEM,37 ℃,5% CO ₂ Culturing in an incubator. To enter the logarithmic phase of cell growth by 2X 10 ⁵ The density of individual cells was seeded in 24 well plates and transfection experiments were performed when the cells were up to 70% confluence, as follows:

1) Preparing solution A: 640. Mu.L of serum-free medium+25.6. Mu.L of Lipo2000 (a 24-well plate);

2) Preparing a solution B: 750. Mu.L of serum-free medium+2.38. Mu.L of 3. Mu.g of PPRE×3TK-luciferase plasmid+0.83. Mu.L of 1.5. Mu.g of hPPARgamma expression plasmid+0.25. Mu.L of 0.3. Mu. g Renilla luciferase expression plasmid;

3) Mix at a: b=1:1 ratio (640 μl+640 μl), incubate at room temperature for 5min. The transfection solution (mixture of A and B) was added to cells containing 450. Mu.L of serum-free culture, 50. Mu.L/well.

4) After 24h transfection, cells were treated with drug for 24h (1. Mu.M, 10. Mu.M test compound, 1. Mu.M, 10. Mu.M Rosi and equal concentration of DMSO).

3. Dual luciferase reporter gene assay:

1) Preparation of 1 x cell lysate: 1 volume 5X Passive Lysis Buffer (PLB) +4 volume ddH ₂ O is mixed evenly, added into a 24-well plate (100 mu L/well) and incubated for 20min at room temperature;

2) Preparation Luciferase Assay Reagent II: 1via lyophilized Luciferase Assay Substrate was resuspended in 10mL Luciferase Assay Buffer II (one month at-20 ℃ C. And one year at-70 ℃ C.) after dissolution;

3) Preparing Stop&Reagent: 1 volume of 50 x Stop&/>Substrate is added to 50 volumes of Stop&/>Mixing the above materials, and storing at (-20deg.C for 15 days);

4) Adding 20 mu L of PLB lysate containing cell lysate into a white opaque 96-well plate, then adding 100 mu L Luciferase Assay Reagent II, lightly blowing and mixing by a pipetting gun, and detecting firefly luciferase activity by using a multifunctional enzyme-labeling instrument;

5) After the first detection is completed, 100 mu L Stop is added&Reagent, lightly blowing and mixing by a pipetting gun, and detecting the activity of the Renilla luciferase.

Calculating the ratio of the firefly luciferase activity detected for the first time to the Renilla luciferase activity detected for the second time to obtain PPARgamma transcriptional activation data.

The experimental results are shown in fig. 3 and table 4, where compound 2n only weakly activated pparγ compared to the full agonist Rosi.

TABLE 4 Table 4

4. The oil red O staining test compound 2n induces the fat differentiation ability of 3T3-L1 cells, and comprises the following specific steps:

3T3-L1 preadipocytes were purchased from ATCC and cultured with 10% FBSDMEMCulture medium, placing cells at 37deg.C, 5% CO ₂ Culturing in an incubator. Inoculated in 6-well plate, induction solution (containing 0.5mmol/L IBMX (3-isobutyl-1-methylxanthine), 1. Mu. Mol/LDEX (dexamethasone), 850nmol/L insulin 10% FBSDMEM) and compound 2n were added 2d after confluence. After 72h, the medium was changed to 10% FBS high glucose DMEM medium containing 850nmol/L insulin, once every 2d for 6 days. 10. Mu.M rosiglitazone was used as positive control, DMSO was used as negative control, and sample set 2n was 1. Mu.M. Induction began 9d and oil red O staining was performed, and a photograph was taken with a microscope (OLYMPUS) to calculate the adipocyte differentiation rate.

The experimental results are shown in fig. 4, in which compound 2n only weakly induced adipocyte differentiation.

5. Real time PCR (polymerase chain reaction) determination of expression of lipid-forming genes comprises the following specific steps:

inducing and differentiating 3T3-L1 preadipocytes according to the scheme, extracting total RNA of the cells, performing Real time PCR according to the scheme of a kit (takara), and calculating the relative expression of mRNA by using beta-actin as an internal reference by using a method of delta Ct.

The results of the experiment are shown in FIG. 5, and the results show that the compound 2n can inhibit the expression of adipocyte differentiation related genes and insulin resistance genes (PTP 1B and SOCS 3) and up-regulate the expression of insulin sensitive genes (Glut 4 and adionectin).

TABLE 5 primer sequences

6. The hypoglycemic effect of compound 2n was tested in db/db model mice as follows:

six week old db/db diabetic model mice were purchased and randomized. Solvent group (Vehicle, n=6), compound 2n administration group (2.5 mg/kg/day, n=6, 5mg/kg/day, n=6, 10mg/kg/day, n=6), GW9662 control group (10 mg/kg/day, n=6) for intraperitoneal injection; the positive control group Rosi (10 mg/kg/day, n=6) was administered orally once daily and three weeks later mice of each experimental group were tested for fasting blood glucose, random blood glucose, fasting body weight, and glucose tolerance level.

The experimental results are shown in fig. 6, and the results show that the compound 2n can obviously reduce the fasting blood glucose and the random blood glucose of db/db mice and does not cause weight gain; in addition, compound 2n was able to significantly improve db/db mouse glucose tolerance.

The above results show that:

1. the compound 2n of the present invention has weak activation ability to pparγ, and the compound 2n of the present invention activates pparγ only 25% and 18% at concentrations of 1 μm and 10 μm with the pparγ full agonist rosiglitazone as a positive control (prescribed as 100%), suggesting that the compound of the present invention has less side effects.

2. The compound 2n of the invention has stronger binding force with PPARgamma, and the binding capacity with PPARgamma is equivalent to that of the positive medicine rosiglitazone at the concentration of 10 mu M.

3. The compound 2n of the invention extremely weakly induces adipocyte differentiation, and takes the oral hypoglycemic agent rosiglitazone as a positive control (test value is 44.8%), and the adipocyte differentiation rate of the compound 2n of the invention is only 9.4% under the same treatment concentration (solvent control group is 8.9%).

4. The compound 2n of the present invention can inhibit the expression of adipocyte differentiation-related genes and insulin resistance genes (PTP 1B and SOCS 3), and up-regulate the expression of insulin sensitive genes (Glut 4 and adionectin).

5. The compound 2n can effectively reduce the fasting blood sugar and the random blood sugar of the mice in db/db diabetes model mice without causing weight increase, and can obviously improve the glucose tolerance of the mice. The compound 2n of the invention has good hypoglycemic effect and small side effect.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (10)

1. A sulfathiaselenazole compound represented by formula (I), a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, a pharmaceutically acceptable salt thereof or a prodrug thereof:

2. a hypoglycemic pharmaceutical composition comprising an effective amount of the sulfathiaselenazole compound of claim 1, a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

3. The hypoglycemic pharmaceutical composition according to claim 2, further comprising a pharmaceutically acceptable carrier selected from one or more of a binder, an excipient, a stabilizer and a lubricant.

4. A pharmaceutical formulation comprising an effective amount of a sulfathiaselenazole compound of claim 1, a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, or a hypoglycemic pharmaceutical composition of claim 2.

5. The pharmaceutical formulation of claim 4, wherein the formulation is an oral dosage form, an injectable dosage form, or a transdermal dosage form.

6. Use of a sulfathiaselenazole compound of claim 1, a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof or a hypoglycemic pharmaceutical composition of claim 2 or a pharmaceutical formulation of claim 5 for the preparation of a pparγ modulator and/or a medicament for the treatment and/or prevention of a disease modulated by a pparγ modulator.

7. The use according to claim 6, wherein the disease is selected from diabetes, elevated blood pressure, elevated lipids, metabolic syndrome of cholesterol levels, or a combination thereof.

8. The use according to claim 7, wherein said diabetes comprises type II diabetes and non-insulin dependent diabetes.

9. The use according to claim 8, wherein the diabetes is type II diabetes targeted at pparγ.

10. Use of a sulfathiaselenazole compound of claim 1, a tautomer thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, or a hypoglycemic pharmaceutical composition of claim 2, or a pharmaceutical preparation of claim 5, for the preparation of a hypoglycemic drug.