Selective SGLT-2 inhibition drug and application thereof

Abstract

The present invention relates to a selective SGLT-2 inhibition drug and uses thereof, the structural formula of the compound is as shown in the following formula, and the compound can selectively inhibit SGLT-2 (Sodium-Glucose Transporters 2, Sodium-Glucose Transporters 2), and can selectively inhibit SGLT-2 (Sodium-Glucose Transporters 2). The medicine prepared from the compound can be used for treating diseases such as diabetes mellitus and heart failure. # imgabs0 #

Description

[0001] This application claims priority to a Chinese application filed on June 12, 2023, with application number 202310689042.X, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to a compound with selective SGLT-2 (sodium-dependent glucose transporters 2, sodium-glucose co-transporter 2) inhibitory activity and use thereof. Background Art

[0003] SGLT (sodium-dependent glucose transporters) is a class of sodium-glucose co-transporters, the most important of which are SGLT-1 and SGLT-2. SGLT-1 is mainly distributed in the brush border of the small intestine and the S3 segment far away from the proximal tubule of the kidney, and is expressed in small amounts in the heart and trachea. It is a high-affinity, low-transport capacity transporter. SGLT-2 is mainly distributed in the S1 segment of the proximal tubule of the kidney. It is a low-affinity, high-transport capacity transporter. Its main physiological function is to complete the reabsorption of 90% of glucose in the glomerular filtrate in the proximal tubule of the kidney. Studies have found that the loss of SGLT-1 activity can lead to severe diarrhea. Therefore, SGLT inhibitors with poor selectivity for SGLT-1 and SGLT-2 have greater adverse reactions due to the simultaneous inhibition of SGLT-1 and SGLT-2. SGLT-2 selective inhibitors can avoid the inhibition of SGLT-1, specifically inhibit the reabsorption of glucose by the kidney, excrete excess glucose from the urine, lower blood sugar, and become a new type of anti-diabetic drug.

[0004] Diabetes is a common independent risk factor for cardiovascular and cerebrovascular diseases. Heart failure, also known as HF, refers to the inability to fully discharge venous blood from the heart due to the impairment of the heart's contractile and (or) diastolic functions, resulting in blood congestion in the venous system and insufficient blood perfusion in the arterial system, which in turn causes a syndrome of cardiac circulatory disorders. The vast majority of heart failures begin with left heart failure. Heart failure is divided into heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF), and heart failure with preserved ejection fraction (HFpEF) based on the left ventricular ejection fraction (LVEF).

[0005] Large-scale clinical data show that SGLT-2 inhibitors can reduce the risk of heart failure hospitalization by 30-35% and improve clinical outcomes in patients with different types of heart failure, including HFrEF and HFpEF. [1-5] SGLT-2 inhibitors have been included in European guidelines for the treatment of heart failure [6] ; And the study showed that the significant effect of SGLT-2 inhibitors on heart failure is not related to 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 considerable proportion in drug research and development, many drugs used in clinical practice are directly or indirectly derived from natural products. Natural products are still one of the main sources of drugs or lead compounds for the treatment of major diseases. Based on sources such as natural products, it is of great significance to continuously search for and obtain new structural types of selective SGLT-2 inhibitory ingredients.

[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 problems to be solved by the present invention include that when SGLT inhibitor drugs are used to treat diabetes and / or heart failure, due to the lack of selectivity for SGLT-2, SGLT inhibitor drugs also have high inhibition on SGLT-1, resulting in severe diarrhea and other side effects, and the problem that the symptoms of diabetes and / or heart failure cannot be effectively treated. In order to solve this technical problem, it is necessary to provide more drugs that are selective for SGLT-2, especially to provide drugs that are selective for SGLT-2 based on natural drugs. The present invention provides a compound with a structure as shown in Formula I and a pharmaceutically acceptable salt thereof; provides a use of a natural product with selective SGLT-2 inhibitory activity; and provides a use of a natural product for preparing a drug and / or pharmaceutical composition for treating diabetes and / or heart failure.

[0019] Technical solution (1): a 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 technical solution (2), which is any one of an injection or an oral preparation;

[0023] Preferably, the oral preparation is any one of a mixture, tincture, pill, pellet, powder, granule, tablet, micropill, microcapsule, capsule, soft capsule, suspension and syrup; wherein the capsule includes, for example, a hard capsule dosage form and the like.

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

[0025] Specifically, examples of optional carriers include: the binder can be selected from polyvinyl pyrrolidone, hydroxypropyl cellulose or hydroxypropyl methylcellulose; the filler can be selected from microcrystalline cellulose, silicified microcrystalline cellulose, lactose, calcium dihydrogen phosphate, mannitol, corn starch or pregelatinized starch; the disintegrant can be selected from cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone or sodium starch glycolate; the lubricant can be selected from magnesium stearate or sodium stearyl fumarate; the glidant can be selected from colloidal silicon dioxide or talc.

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

[0027] As for the above-mentioned various carriers, any known or commercially available carriers routinely used by those skilled in the art can be used in the medicine or pharmaceutical composition of the present invention to prepare medicines in various dosage forms.

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

[0029] Preferably, the diabetes is type 2 diabetes, and the cardiovascular and cerebrovascular disease is heart failure;

[0030] Further 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] Definition of terms:

[0032] The following terms and the like are used to describe the present invention. It should be understood that terms that are not specifically defined are given a meaning consistent with the use of the terms in the context of the present invention as understood by those of ordinary skill in the art.

[0033] As used herein, the term "pharmaceutically acceptable carrier" refers to any formulation carrier or excipient that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient.

[0034] Beneficial technical effects achieved:

[0035] The present invention prepares a new compound with selective SGLT-2 inhibitory activity and finds that the compound can be used to prepare a drug with a high SGLT-2 selective inhibition rate, and the drug can be used to treat type II diabetes and heart failure. DETAILED DESCRIPTION

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

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

[0038] Compound preparation:

[0039] 6kg of commercially available Rhizoma Dioscoreae (Chinese Pharmacopoeia 2020, Part I, pages 279-280) slices were extracted three times with water. The first time, 8 times of drinking water was added and boiled for 4 hours. The second and third times, 6 times of drinking water were added and boiled for 3 hours. The extracts were combined and concentrated and dried to obtain 1278g of Rhizoma Dioscoreae extract. 1278g of the Rhizoma Dioscoreae extract was taken and dispersed in 11100ml of water. It was extracted with ethyl acetate (7500ml×4 times) and n-butanol (7500ml×5 times) in turn. The ethyl acetate extract was concentrated and dried to obtain 102g and 503g of n-butanol extract.

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

[0041] Structure confirmation:

[0042] ESI-MS m / z:761[M+Na] + , 739[M+H] + , confirmed molecular weight is 738; high resolution mass spectrum 761.40919 [M+Na] + Confirm its molecular formula: 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 sapogenin 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 C NMR spectrum showed a total of 39 carbon signals, of which 12 were carbon signals of sugars (2 hexoses) and 27 were carbon signals of diosgenin. Only glucose was detected by acid hydrolysis, and both hexoses were glucose. 1 HNMR (400 MHz, 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 the four methyl proton signals of dioscorea steroidal sapogenin. 13 C NMR spectrum δ140.71 (C-5), δ121.53 (C-6) and 1 The HNMR spectrum δ5.34 (1H, br, s, H-6) shows the signal of the steroidal sapogenin 5-6 double bond, δ103.39 (C-20), δ152.20 (C-22) show the signal of the steroidal sapogenin 20-22 double bond. 13 C NMR spectra δ78.33 (C-3) and δ74.77 (C-26) showed that the steroidal sapogenin C-3 and C-26 positions were connected with sugars. 13 C NMR spectra of δ75.03 (C-2') and δ71.52 (C-4') showed that there was no sugar attached to the C-2' and C-4' positions of the C-3 glucose. 1The coupling constants of the two sugar terminal protons δ4.87 and 5.08 (each 1H, d, J = 8 Hz, H-1' and H-1") in the HNMR spectrum show that the configurations of the two glucoses are both β-type. The compound was identified as: 26-O-β-D-pyranoglucopyranosyl-(25R)-furosta-Δ5,20(22)-diene-3β,26-dihydroxy-3-O-β-D-pyranoglucopyranoside, and its structural formula is as follows:

[0047]

[0048] The ESI-MS instrument model is Waters Vion IMS QTof ultra-high resolution liquid-mass spectrometer; 1 HNMR and 13 The instrument model used in the CNMR nuclear magnetic resonance experiment is: BrukerAvance III 400M nuclear magnetic spectrometer.

[0049] Example 2: SGLT-1 and SGLT-2 Activity Inhibition Experiment of Formula I Compound

[0050] 1.1 Instruments and Materials

[0051] Experimental instrument: PerkinElmer1450-023

[0052] Experimental Materials:

[0053] Compound of formula I (prepared in Example 1), Dapagliflozin (commercial standard);

[0054] NIH3T3-hSGLT1 stable strain, NIH3T3-hSGLT2 stable strain (cell lines were provided by the National Center for Drug Screening);

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

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

[0057] DMEM-(High glucose and FBS) (gibico).

[0058] 1.2 Sample processing

[0059] The sample was dissolved with 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 process

[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 stably expressing SGLT1 and SGLT2 (NIH3T3-hSGLT1, NIH3T3-hSGLT2) were obtained through antibiotic screening, RT-PCR and Western-blot identification.

[0062] Sample activity determination:

[0063] (1) 96-well plates were coated with 0.2% gelatin and kept at 37°C. They were then washed once with PBS before use.

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

[0065] (3) The medium was changed on the second day and sodium butyrate was added to the culture medium at a final concentration of 2 mM.

[0066] (4) On the third day, the medium was changed and sodium butyrate was added to the culture medium at a final concentration of 2 mM.

[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 preparation:

[0069] Single concentration inhibition rate determination: The compound of formula I and Dapagliflozin (positive drug) were dissolved in DMSO to prepare a stock solution; then the stock solution of the compound of formula I was stained with KRH-Na + Dapagliflozin (positive drug) stock solution was diluted to 100 μM with KRH-Na + Dilute to 100 nM, then KRH-Na + The compound concentration in the assay was the test concentration, and the DMSO content was 0.1%.

[0070] IC50 determination: Take the compound of formula I and Dapagliflozin (positive drug), dissolve them in DMSO at appropriate concentrations, and dilute the compound to be tested and the positive drug with DMSO to 6 concentration gradients; then add the above compound solutions to KRH-Na +At this time, KRH-Na + The compound concentration in the assay was the test concentration, and the DMSO content was 0.1%.

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

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

[0073] 1.4 Calculation of inhibition rate and IC50 value

[0074] (1) Calculation method of inhibition rate:

[0075] Set the reading of the DMSO control well to L Control The reading of the compound well is L Sample The choline well reading as background is L Background The inhibition rate (Inhibition%) of each sample at each concentration was calculated according to the following formula:

[0076]

[0077] (2) IC50 calculation method:

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

[0079] 1.5 Experimental Results

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

[0081] Table 1 Inhibition of SGLT-1 and SGLT-2 activity by single concentration of compound of formula I

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

[0083] 1.6 Experimental Conclusion

[0084] The IC50 value of Dapagliflozin (positive drug) for SGLT-2 is 5.6nM; the compound of formula I has almost no inhibitory activity on SGLT-1, and the IC50 value cannot be detected; the compound of formula I has obvious inhibitory activity on SGLT-2, and the IC50 value for SGLT-2 is 89.1μM. The compound of formula I has highly selective SGLT-2 inhibitory activity.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, 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.

3. The pharmaceutical composition according to claim 2, which is any one of an injection or an oral preparation; Preferably, the oral preparation is any one of a mixture, a tincture, a pill, a pellet, a powder, a granule, a tablet, a micropill, a microcapsule, a capsule, a soft capsule, a suspension and a syrup.

4. The pharmaceutical composition according to claim 2 or 3, wherein the carrier is selected from any one or more substances in the group consisting of a binder, a filler, a disintegrant, a lubricant, a glidant, a surfactant, a wetting agent, an antioxidant, a plasticizer, a sweetener, a colorant and a coating agent.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 2 to 4, in the preparation of a SGLT-2 selective inhibitory drug.

6. The use according to claim 5, characterized in that The drug is used to treat diabetes, diabetic nephropathy and / or cardiovascular and cerebrovascular diseases; Preferably, the diabetes is type 2 diabetes, and the cardiovascular and cerebrovascular disease is heart failure; Further preferably, the heart failure is heart failure with reduced ejection fraction, heart failure with intermediate ejection fraction, or heart failure with preserved ejection fraction.

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