26-O-β-D-glucopyranosyl-(25R)-furostane-Δ5,20(22)-dien-3β,26-dihydroxy-3-O-β-D-glucopyranoside and its use

Abstract

The present application relates to 26-O-beta-D-glucopyranosyl-(25R)-furostane-5,20(22)-dien-3beta,26-dihydroxy-3-O-beta-D-glucopyranoside and its use, in particular to a selective SGLT-2 inhibitor and its use, the compound structural formula is shown in the following formula, the compound can selectively inhibit SGLT-2 (sodium-dependent glucose transporters 2, sodium-glucose cotransporter 2); the drug prepared by using the compound can be used for the treatment of diseases such as diabetes, heart failure.

Description

[0001] This application claims priority to Chinese application No. 202310689042.X, filed on June 12, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the pharmaceutical field, specifically to a compound with selective SGLT-2 (sodium-dependent glucose transporters 2) inhibitory activity and its uses. Background Technology

[0003] SGLT (sodium-dependent glucose transporters) are a class of sodium-glucose cotransporters, with two main subtypes: SGLT-1 and SGLT-2. SGLT-1 is primarily distributed in the brush border of the small intestine and the S3 segment of the proximal convoluted tubule of the kidney, with smaller amounts expressed in the heart and trachea. It is a high-affinity, low-capacity transporter. SGLT-2 is mainly distributed in the S1 segment of the proximal convoluted tubule of the kidney. It is a low-affinity, high-capacity transporter, whose main physiological function is to reabsorb 90% of glucose from glomerular filtrate in the proximal convoluted tubule of the kidney. Studies have found that loss of SGLT-1 activity can lead to severe diarrhea. Therefore, SGLT inhibitors with poor selectivity for both SGLT-1 and SGLT-2, which simultaneously inhibit both, have significant adverse reactions. SGLT-2 selective inhibitors, on the other hand, avoid inhibiting SGLT-1, specifically inhibiting renal glucose reabsorption, causing excess glucose to be excreted in the urine, thus lowering blood glucose levels and becoming a novel type of antidiabetic drug.

[0004] Diabetes is a common independent risk factor for cardiovascular and cerebrovascular diseases. Heart failure (HF) refers to a syndrome caused by impaired systolic and / or diastolic function of the heart, which fails to adequately pump venous blood back to the heart, leading to venous congestion and insufficient arterial perfusion. The vast majority of HF cases begin with left ventricular failure. Based on left ventricular ejection fraction (LVEF), HF is classified as: heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF), and heart failure with preserved ejection fraction (HFpEF).

[0005] Large-scale clinical data show that SGLT-2 inhibitors can reduce the risk of hospitalization for heart failure by 30-35% and improve clinical outcomes in patients with different types of heart failure, HFrEF and HFpEF. [1-5] SGLT-2 inhibitors have been included in European guidelines for the treatment of heart failure. [6] Furthermore, studies have shown that the significant effect of SGLT-2 inhibitors on heart failure is unrelated to the presence or absence of diabetes. [7-10] .

[0006] Natural products have always been an important source of drugs for disease prevention and treatment, with a long history and solid foundation. Although synthetic drugs account for a significant proportion of drug development, many drugs used clinically are directly or indirectly derived from natural products. Natural products remain one of the main sources of drugs or lead compounds for treating major diseases. Therefore, continuously searching for and obtaining novel selective SGLT-2 inhibitors based on natural products and other sources is of great significance.

[0007] References:

[0008] [1] Zinman, B. et al. Empagliflozin, cardiovascular outcomes, and mortality in type 2diabetes. N. Engl. J. Med. 373, 2117–2128 (2015).

[0009] [2]Neal,B.et al.Canagliflozin and cardiovascular and renal events intype 2diabetes.N.Engl.J.Med.377,644–657(2017).

[0010] [3]Wiviott,S.D.et al.Dapagliflozin and cardiovascular outcomes intype 2diabetes.N.Engl.J.Med.380,347–357(2019).

[0011] [4]Perkovic,V.et al.Canagliflozin and renal outcomes in type2diabetes and nephropathy.N.Engl.J.Med.380,2295–2306(2019).

[0012] [5]Das,S.R.et al.2020expert consensus decision pathway on noveltherapies for cardiovascular riskreduction in patients with type2diabetes.J.Am.Coll.Cardiol.76,1117–1145(2020).

[0013] [6]McDonagh,T.A.et al.2021ESC Guidelines for the diagnosis andtreatment of acute and chronic heart failure:developed by the Task Force forthe diagnosis and treatment of acute and chronic heart failure of theEuropean Society of Cardiology(ESC)with the special contribution of the HeartFailure Association(HFA)of the ESC.Eur.Heart J.42,3599–3726(2021).

[0014] [7]Joshi, S.S., Singh, T., Newby, D.E. & Singh, J. Sodium-glucose co-transporter 2 inhibitor therapy: mechanisms of action in heart failure. Heart 107, 1032–1038 (2021).

[0015] [8]de Leeuw, A.E. & de Boer, R.A. Sodium-glucose cotransporter 2 inhibition: cardioprotection by treating diabetes—a translational viewpoint explaining its potential salutary effects. Eur. Heart J. Cardiovasc. Pharmacother. 2, 244–255 (2016).

[0016] [9]Arnold, J.M.O. et al. Prevention of heart failure in patients in the heart outcomes prevention evaluation (HOPE) study. Circulation 107, 1284–1290 (2003).

[0017]

[10] NAVIGATOR Study Group, McMurray, J.J. et al. Effect of valsartan on the incidence of diabetes and cardiovascular events. N. Engl. J. Med. 362, 1477–1490 (2010). Summary of the Invention

[0018] The technical problem this invention aims to solve includes the issue that when SGLT inhibitor drugs are used to treat diabetes and / or heart failure, the lack of selectivity for SGLT-2 and the high inhibitory activity of SGLT-1 by these drugs lead to severe side effects such as diarrhea, resulting in ineffective treatment of diabetes and / or heart failure symptoms. To address this problem, there is a need to provide more drugs selective for SGLT-2, particularly those based on natural medicines. This invention provides a compound with the structure shown in Formula I and a pharmaceutically acceptable salt thereof; uses of a natural product with selective SGLT-2 inhibitory activity; and uses of a natural product for preparing drugs and / or pharmaceutical compositions for treating diabetes and / or heart failure.

[0019] Technical solution (1): The compound represented by formula I or a pharmaceutically acceptable salt thereof,

[0020]

[0021] Technical solution (2): A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0022] Technical solution (3): The pharmaceutical composition according to claim (2) is either an injection or an oral preparation;

[0023] Preferably, the oral preparation is any one of the following: mixture, tincture, pill, drop pill, powder, granule, tablet, micro-pill, microcapsule, capsule, soft capsule, suspension, and syrup; wherein the capsule includes, for example, hard capsule dosage form.

[0024] Technical solution (4): According to the pharmaceutical composition of technical solution (2) or (3), the carrier is selected from any one or more substances in the group consisting of adhesives, fillers, disintegrants, lubricants, flow aids, surfactants, wetting agents, antioxidants, plasticizers, sweeteners, colorants and coating agents.

[0025] Specifically, examples of alternative carriers include: the adhesive may be selected from polyvinylpyrrolidone, hydroxypropyl cellulose, or hydroxypropyl methylcellulose; the filler may be selected from microcrystalline cellulose, silicified microcrystalline cellulose, lactose, calcium dihydrogen phosphate, mannitol, corn starch, or pregelatinized starch; the disintegrant may be selected from croscarmellose sodium, croscarmellose, or glycolic acid starch sodium; the lubricant may be selected from magnesium stearate or sodium stearoyl fumarate; and the flow aid may be selected from colloidal silica or talc.

[0026] Technical solution (5): Use of the compound or a pharmaceutically acceptable salt thereof according to technical solution (1), or the pharmaceutical composition according to any one of technical solutions (2)-(4) in the preparation of a selective inhibitory drug for SGLT-2.

[0027] Any of the aforementioned carriers that are known or commercially available and routinely used by those skilled in the art can be used in the drugs or drug compositions of the present invention to prepare drugs in various dosage forms.

[0028] Technical solution (6): According to the use described in technical solution (5), the drug is used to treat diabetes, diabetic nephropathy and / or cardiovascular and cerebrovascular diseases;

[0029] Preferably, the diabetes is type II diabetes, and the cardiovascular disease is heart failure.

[0030] More preferably, the heart failure is heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, or heart failure with preserved ejection fraction.

[0031] Terminology definition:

[0032] The following terms, etc., are used to describe the present invention. It should be understood that, as those skilled in the art will recognize, terms without a specific definition are given the same meaning as used in the context of the present invention.

[0033] As used herein, the term "pharmaceutically acceptable carrier" means any formulation carrier or excipient capable of delivering an effective amount of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient.

[0034] Beneficial technical effects achieved:

[0035] This invention, through the study of preparing a new compound with selective SGLT-2 inhibitory activity, found that the compound can be used to prepare a drug with a high selective inhibition rate of SGLT-2, which can be used to treat type II diabetes and heart failure. Detailed Implementation

[0036] The following examples illustrate the preparation of the compounds of the present invention and demonstrate the use of the drug for treating type II diabetes and heart failure through experimental data on the selective inhibition rate of SGLT-2.

[0037] Example 1: Preparation and structural confirmation of compound I

[0038] Compound preparation:

[0039] Commercially available slices of *Dioscorea nipponica* (Chinese Pharmacopoeia 2020 Edition, Part I, pp. 279-280) were extracted three times with water. For the first extraction, 8 times the volume of drinking water was added, and the mixture was boiled for 4 hours. For the second and third extractions, 6 times the volume of drinking water was added, and the mixture was boiled for 3 hours each time. The extracts were combined, concentrated, and dried to obtain 1278g of *Dioscorea nipponica* extract. This 1278g extract was then dispersed in 11100ml of water and extracted sequentially with ethyl acetate (7500ml × 4 times) and n-butanol (7500ml × 5 times). The extracts were concentrated and dried to obtain 102g of ethyl acetate extract and 503g of n-butanol extract.

[0040] 200 g of the above n-butanol extract was subjected to silica gel column chromatography (Φ10.5 cm × 52 cm, 2000 g), eluted with chloroform-methanol-water (9:1:0.1), collecting 500 ml of eluent from each bottle, for a total of 160 fractions (bottles). Fractions determined by thin-layer chromatography were combined, and bottles 138–143 were combined, concentrated, and dried to obtain 2.6 g. Further silica gel column chromatography (Φ3.1 cm × 19 cm, 90 g), eluted with a chloroform-methanol (4:1) water-saturated solution, yielded 19 mg of the target product.

[0041] Structural confirmation:

[0042] ESI-MS m / z: 761 [M+Na] + 739[M+H] + The molecular weight was confirmed to be 738; high-resolution mass spectrometry result: 761.40919 [M+Na]. + Its molecular formula is confirmed to be: C 39 H 62 O 13 .

[0043] 1 H NMR (400MHz, C5D5N): δ0.75(3H,s,CH3-18), δ0.94(3H,s,CH3-19), δ1.05(3H,d,J=4,CH3-27), δ1.67(3H,s,CH3-21), δ4.87and 5.08(each 1H,d,J=8Hz,H-1'andH-1″), δ5.34(1H,br,s,H-6).

[0044] 13C NMR, carbon signals of sapogenins C-1 to C-27: δ=37.29,30.06,78.33,39.16,140.71,121.53,32.18,31.27,50.06,36.84,21.08,39.47,43.23,54.75,34.29,84.28,64.30,13.96,19.23,103.39,11.63,152.20,33.20,23.51,31.31,74.77,17.17.

[0045] 13 C NMR, carbon signals of sugar C-1' to C-6': δ=102.42,75.03,77.88,71.52,78.45,62.67; C-1” to C-6”: δ=104.73,75.17,78.36,71.52,78.45,62.67.

[0046] 13 The C10 NMR spectrum showed a total of 39 carbon signals, of which 12 were sugar carbon signals (2 hexoses) and 27 were diosgenin carbon signals. Acid hydrolysis revealed only glucose, and both hexoses were glucose. 1 HNMR (400MHz, C5D5N): δ0.75 (3H,s,CH3-18), δ0.94 (3H,s,CH3-19), δ1.67 (3H,s,CH3-21), δ1.05 (3H,d,J=4,CH3-27), showing four methyl proton signals of diosgenin. 13 C NMR spectra δ140.71 (C-5), δ121.53 (C-6) and 1 The HNMR spectrum at δ5.34 (1H, br, s, H-6) shows the signal of the 5-6 double bond of the steroidal saponin, and at δ103.39 (C-20) and δ152.20 (C-22) it shows the signal of the 20-22 double bond of the steroidal saponin. 13 The 1C NMR spectra at δ78.33 (C-3) and δ74.77 (C-26) show that the steroidal saponin is attached to sugars at the C-3 and C-26 positions. 13 The C NMR spectra at δ75.03 (C-2') and δ71.52 (C-4') show that there is no glucose at the C-2' and C-4' positions of the glucose at the C-3 position. 1The coupling constants of the terminal protons of the two sugars in the HNMR spectrum (δ 4.87 and 5.08, each 1H, d, J = 8 Hz, H-1' and H-1") indicate that both glucose molecules are β-configured. The compound was identified as 26-O-β-D-glucopyranosyl-(25R)-furoster-Δ5,20(22)-diene-3β,26-dihydroxy-3-O-β-D-glucopyranoside, with the following structural formula:

[0047]

[0048] The instrument used for the ESI-MS testing mentioned above is the Waters Vion. IMS QTof ultra-high resolution liquid chromatography-mass spectrometry; 1 HNMR and 13 The instrument used for the CNMR nuclear magnetic resonance experiment was a Bruker Avance III 400M nuclear magnetic spectrometer.

[0049] Example 2: SGLT-1 and SGLT-2 activity inhibition experiments of compounds of formula I

[0050] 1.1 Instruments and Materials

[0051] Experimental equipment: PerkinElmer 1450-023

[0052] Experimental materials:

[0053] Compound of Formula I (prepared in Example 1), Dapagliflozin (commercially available standard);

[0054] Stable cell lines of NIH3T3-hSGLT1 and NIH3T3-hSGLT2 (both cell lines were provided by the National Center for Drug Screening);

[0055] Methyl-α-D-[U- 14 C] Gluranoside (methyl-α-D-[U-) 14 C]glucopyranoside)(PerkinElmer);

[0056] Methyl sulfoxide (Genebase, ProdNo: 0231);

[0057] DMEM (High Glucose and Fetal Bovine Serum) (gibico).

[0058] 1.2 Sample Preparation

[0059] The sample was dissolved in DMSO and stored at low temperature. The concentration of DMSO in the final system was controlled within a range that would not affect the detection activity.

[0060] 1.3 Experimental Procedure

[0061] Construction of experimental cell lines: Human SGLT-1 and SGLT-2 genes were cloned into eukaryotic expression vectors and transfected into NIH3T3 cells respectively. NIH3T3 cells (NIH3T3-hSGLT1 and NIH3T3-hSGLT2) that stably expressed SGLT1 and SGLT2 were obtained by antibiotic selection and identification by RT-PCR and Western blot.

[0062] Sample activity assay:

[0063] (1) Spread 0.2% gelatin on a 96-well plate and store at 37°C. Wash with PBS before use.

[0064] (2) NIH3T3-hSGLT1 and NIH3T3-hSGLT2 cells were inoculated into 96-well plates at 30,000 cells / well and 100 μl of culture medium per well.

[0065] (3) Change the medium once on the second day, and add sodium butyrate to the culture medium at the same time, with a final concentration of 2mM.

[0066] (4) Change the medium once on the third day, and add sodium butyrate to the culture medium at the same time, with a final concentration of 2mM.

[0067] (5) Wash the cells three times with 100 μl of KRH-Na+ and KRH-acetylcholine solution respectively, and then incubate the cells with 50 μl for 30 minutes.

[0068] (6) Compound solution preparation:

[0069] Single-concentration inhibition rate determination: Compound I and Dapagliflozin (positive control drug) were dissolved separately in DMSO to prepare stock solutions; then the stock solutions of compound I were subjected to KRH-Na... + Dilute to 100 μM and use KRH-Na to dilute the Dapagliflozin (positive control) stock solution. + Dilute to 100 nM, at which point KRH-Na + The concentration of the compound in the sample is the test concentration, and the DMSO content is 0.1%.

[0070] IC50 determination: Dissolve the compound of formula I and Dapagliflozin (positive control) in DMSO at appropriate concentrations, and then dilute the test compound and positive control with DMSO to create six concentration gradients; then add the above compound solutions to KRH-Na +In this case, KRH-Na + The concentration of the compound in the sample is the test concentration, and the DMSO content is 0.1%.

[0071] (7) Replace the cells with 50 μl of KRH-Na containing the target concentration of the compound or positive control drug. + Or KRH-choline chloride, plus uptake buffer (KRH-Na) + Methyl-α-D-[U-] 14 C] Gluranoside (methyl-α-D-[U-) 14 [C]glucopyranoside and 1mM methyl-α-D-glucopyranoside; for nonspecific absorption, KRH-acetylcholine solution was used instead of KRH-Na. + 10 μl per well.

[0072] (8) Wash 3 times with 100 μl PBS and dry; add 50 μl lysis buffer to each well and lyse for about 15 minutes, then add 150 μl scintillation buffer to each well, cover with membrane, flatten, shake well on a shaker, and centrifuge at 1200 r for 5 min at 4 °C. Take out and measure the reading using the liquid scintillation counting method.

[0073] 1.4 Calculation and processing of inhibition rate and IC50 value

[0074] (1) Method for calculating inhibition rate:

[0075] Set the reading value of the DMSO reference orifice to L. Control The reading of the compound pore is L. Sample The background choline orifice reading was L. Background The inhibition rate (Inhibition%) of each sample under each concentration condition was calculated using the following formula:

[0076]

[0077] (2) IC50 calculation method:

[0078] The IC50 value of the sample was calculated using a nonlinear fitting method with GraphpadPrism4 software, employing the sigmoidaldose-response (variable slope) model, and setting the bottom and top of the fitted curve to 0 and 100, respectively.

[0079] 1.5 Experimental Results

[0080] The activity data of the compound of formula I in inhibiting SGLT-1 and SGLT-2 obtained from the above experiments are shown in Table 1 below.

[0081] Table 1. Inhibition of SGLT-1 and SGLT-2 activity by single-concentration Formula I compounds.

[0082] compound SGLT-2 inhibition rate SGLT-1 inhibition rate Compound of Formula I 61.3％ 11.6％ Dapagliflozin (positive control drug) 95.2％ 11.0％

[0083] 1.6 Experimental Conclusions

[0084] Dapagliflozin (positive control) has an IC50 value of 5.6 nM for SGLT-2; Compound I has almost no inhibitory activity against SGLT-1, and its IC50 value cannot be detected; Compound I has significant inhibitory activity against SGLT-2, with an IC50 value of 89.1 μM for SGLT-2, and Compound I has highly selective SGLT-2 inhibitory activity.

Claims

1. The compound of Formula I that is a selective inhibitor of SGLT-2, or a pharmaceutically acceptable salt thereof. Formula I; in, The SGLT-2 selective inhibitor refers to an inhibitor whose inhibitory activity against SGLT-2 is higher than that against SGLT-1; and the SGLT-2 selective inhibitor is used to treat type II diabetes and / or cardiovascular and cerebrovascular diseases, and to reduce diarrhea caused by the inhibition of SGLT-1 activity, wherein the cardiovascular and cerebrovascular diseases are heart failure.

2. A pharmaceutical composition as a selective inhibitor of SGLT-2, comprising an active ingredient and a pharmaceutically acceptable carrier, said active ingredient being the compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The SGLT-2 selective inhibitor refers to an inhibitor whose inhibitory activity against SGLT-2 is higher than that against SGLT-1; and the SGLT-2 selective inhibitor is used to treat type II diabetes and / or cardiovascular and cerebrovascular diseases, and to reduce diarrhea caused by the inhibition of SGLT-1 activity, wherein the cardiovascular and cerebrovascular diseases are heart failure.

3. The pharmaceutical composition according to claim 2, wherein it is either an injectable or oral formulation.

4. The pharmaceutical composition according to claim 3, wherein the oral preparation is any one of the following: mixture, tincture, pill, drop pill, powder, granule, tablet, micro-pill, microcapsule, capsule, soft capsule, suspension, and syrup.

5. The pharmaceutical composition according to any one of claims 2-4, wherein the carrier is selected from one or more substances selected from the group consisting of binders, fillers, disintegrants, lubricants, flow aids, surfactants, wetting agents, antioxidants, plasticizers, sweeteners, colorants, and coating agents.

6. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 2-5, in the preparation of a selective SGLT-2 inhibitor, wherein selective SGLT-2 inhibition means that the inhibitory activity against SGLT-2 is higher than that against SGLT-1; and wherein the selective SGLT-2 inhibitor is used to treat type II diabetes and / or cardiovascular and cerebrovascular diseases, and to reduce diarrhea caused by inhibition of SGLT-1 activity, wherein the cardiovascular and cerebrovascular diseases are heart failure.

7. The use according to claim 6, characterized in that, The heart failure referred to here includes heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, and heart failure with preserved ejection fraction.

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