Triazolic hsl inhibitors, methods of preparation and uses thereof

Abstract

The present application relates to the field of HSL inhibitors. Specifically, the present application relates to a class of HSL inhibitors containing triazole structure, the preparation method and the pharmaceutical compositions containing them, and their application in the preparation of treating diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity. Wherein, each substituent group is shown in the specification.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of HSL inhibitors. In particular, the present invention relates to a class of HSL inhibitors containing a triazole structure which produce a therapeutic effect on HSL inhibition, methods for their preparation, as well as pharmaceutical compositions containing them and their use in the treatment of diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity. BACKGROUND

[0002] The main physiological role of white adipose tissue (WAT) is to supply energy when needed by other tissues. In mammals, white adipose tissue is the main energy storage depot, accumulating energy in the form of triacylglycerol (TAG) when energy is in excess. However, unlike TAG synthesis, which also occurs at high levels in the liver for very low density lipoprotein (VLDL) production, lipolysis for providing fatty acids as energy sources for use by other organs is unique to adipocytes. The release of free fatty acids (FFA) from TAG occurs in an ordered and controlled manner (Annu. Rev. Med., 2002, 53, 319-336), which is stimulated by catecholamines and regulated by hormones such as insulin, glucagon and adrenaline.

[0003] The most important enzyme in WAT that is responsible for the hormone-regulated hydrolysis of triglycerides is hormone-sensitive lipase (HSL). This enzyme is also present in liver, skeletal muscle, skin and adrenal glands. In the basal state, it has minimal activity towards its substrate. Activation of protein kinase A in adipocytes by hormones leads to phosphorylation of HSL and the lipid droplet coat protein, perilipin. Phosphorylation of perilipin leads to its removal from the lipid droplet, and phosphorylated HSL migrates from the cytosol to the lipid droplet, where it catalyzes the hydrolysis of triglycerides.

[0004] Disorders in the regulation of adipocyte lipolysis leading to elevated circulating non- esterified fatty acids (NEFA) are associated with obesity and the complex of pathologies including the development of type 2 diabetes (Annu. Rev. Med., 2002, 53, 319-336). Obese or insulin resistant subjects have increased visceral adipose tissue stores. These stores contain elevated levels of HSL protein and exhibit elevated lipolytic activity due to their resistance to insulin-mediated inhibition of lipolysis. This leads to elevated plasma levels of free fatty acids, which further exacerbate insulin resistance due to the accumulation of triglycerides in tissues other than WAT, such as liver, muscle and membranes. The abnormal deposition of triglycerides has pathological consequences, such as increased glucose production in the liver, reduced insulin secretion by the pancreas, and reduced glucose uptake and fatty acid oxidation in skeletal muscle. Thus, elevated plasma levels of FFA due to increased HSL activity contribute to and worsen insulin resistance in obese and type 2 diabetic individuals. In addition, elevated FFA by adipose tissue are associated with increased production of the inflammatory cytokine TNF-α. TNF-α also impairs insulin signaling by activating serine kinases, such as JNK-1, which phosphorylate IRS-1, thereby inhibiting insulin signaling (Mol. Endocrinol., 2004, 18, 2024-2034). Thus, restoring excessive plasma FFA and triglyceride levels by inhibiting HSL would reduce the accumulation of triglycerides in tissues other than WAT, such as liver, muscle and membranes, leading to reduced hepatic glucose output, increased intramuscular fatty acid oxidation and improved β-cell function. Inflammatory cytokine production would also be reduced, leading to further reduction in FFA production and improved insulin signaling. Elevated FFA are also associated with increased cardiovascular risk, including atherosclerosis and myocardial dysfunction. It has been shown that long-term low-dose lipid infusion in healthy patients leads to markers of endothelial activation independent of their metabolic effects. Here, it has been shown that moderate lipid infusion elevates markers of endothelial activation ET-1, ICAM-1, VCAM-1. Furthermore, high lipolytic activity and elevated FFA lead to increased insulin resistance and hypertension in hypertensive rats (J. Physiol. Biochem., 2009, 65, 33-41).

[0005] Since HSL is the major hormone regulated lipase, it is known that in insulin resistant states, the ability of insulin to suppress lipolysis is reduced and increased FFA, i.e. lipotoxicity, is promoted. These fatty acids accumulate in the liver and cause increased TAG production, which is packaged into secreted VLDL. There is also accumulation of lipids in the liver, leading to a fatty liver phenotype. Lipolysis is increased in diabetes and obesity and contributes to this phenotype. Thus, a reduction in HSL activity would reduce the release of FFA into the blood, thereby limiting the supply of FFA to the liver for TAG synthesis. Therefore, HSL inhibitors can have a beneficial effect in the treatment of NAFLD (non-alcoholic fatty liver disease) and NASH (non-alcoholic steatohepatitis).

[0006] The present application discloses a class of HSL inhibitors containing triazole structure, which can be used for preparing therapeutic drugs for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity, etc. SUMMARY

[0007] An object of the present application is to provide a HSL inhibitor having the general formula I.

[0008] Another object of the present application is to provide a method for preparing the compound having the general formula I.

[0009] Still another object of the present application is to provide a pharmaceutical composition containing the compound having the general formula I as an effective ingredient, and one or more pharmaceutically acceptable carriers, excipients or diluents, and its application in treating diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity, etc.

[0010] The present application will now be described in detail in connection with the objects of the present application.

[0011] The compound having the general formula (I) of the present application has the following structural formula:

[0012]

[0013] wherein R 1 is selected from H, C1-C 10 alkyl, F, Cl, Br, I, NO2, CN and R 3 O; R 2 is selected from H, C1-C 10 alkyl, C3-C 10 cycloalkyl; wherein R 3 is selected from C1-C 10 alkyl and C3-C 10 cycloalkyl.

[0014] The following compound of general formula (I) is preferred,

[0015] wherein R1 selected from H, C1-C4 alkyl, F, Cl, NO2, CN and R 3 O; R 2 selected from H, C1-C4 alkyl, C3-C6 cycloalkyl; wherein R 3 selected from C1-C4 alkyl and C3-C6 cycloalkyl.

[0016] More preferred compounds of formula (I) are as follows,

[0017]

[0018] The compounds of formula (I) according to the present application can be synthesized by the following route:

[0019]

[0020] Compound II is a commercially available reagent of technical grade, and VI is commercially available or can be prepared according to conventional methods.

[0021] Compound II is treated with thiophosgene in the presence of a base to obtain compound III; compound III is first reacted with formylhydrazine, and the obtained intermediate is treated with a base to obtain compound IV; compound IV is reacted with a haloacetate to obtain compound V; compound V is heated to react with compound VI to obtain I; wherein X is selected from Cl, Br and I, R 4 selected from C1-C 10 alkyl, R 1 and R 2 are as defined above.

[0022] The compounds of formula I according to the present application can be formulated into pharmaceutical compositions with one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical compositions can be formulated into solid oral preparations, liquid oral preparations, injections and the like. The solid and liquid oral preparations include tablets, dispersible tablets, sugar-coated tablets, granules, dry powder, capsules and solutions. The injections include small needles, large infusions, freeze-dried powder needles and the like.

[0023] The compositions according to the present application, the pharmaceutically or dietetically acceptable adjuvants are selected from fillers, disintegrants, lubricants, glidants, effervescent agents, flavoring agents, preservatives, coating materials or other excipients.

[0024] The composition of the present application comprises pharmaceutically or dietetically acceptable excipients. The filler comprises a combination of one or more of lactose, sucrose, dextrin, starch, pregelatinized starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, microcrystalline cellulose; the binder comprises a combination of one or more of starch, sucrose, povidone, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, medicinal ethanol, water; and the disintegrant comprises a combination of one or more of starch, crospovidone, sodium croscarmellose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, effervescent disintegrant.

[0025] The compound of the general formula I has HSL inhibiting effect and can be used as an effective ingredient for preparing a therapeutic drug for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity. The activity of the compound of the general formula I is verified by in vitro HSL inhibition experiment. DETAILED DESCRIPTION

[0026] The present application is further illustrated by the following examples. It should be understood that the following examples are intended to be illustrative only and are not intended to limit the present application. Various modifications within the scope of the claims will be apparent to those of ordinary skill in the art based on this disclosure.

[0027] Synthesis of Example 1 compound I-1

[0028]

[0029] Step A. Synthesis of compound III-1

[0030] Compound II-1 (1.51 g, 10 mmol) was dissolved in 20 mL of dry dichloromethane, stirred under ice water bath cooling, Et3N (3.04 g, 30 mmol) was added, and then a solution (5 mL) of thiophosgene (1.26 g, 11 mol) dissolved in dry dichloromethane was slowly added dropwise. After the dropwise addition was completed, the reaction mixture was stirred at room temperature overnight, and TLC showed that the reaction was completed.

[0031] The reaction mixture was carefully poured into ice water (200 mL), stirred, extracted with CH2Cl2(50 mL x 3), the combined extract was washed with 1% dilute hydrochloric acid (200 mL) and 5% salt water (100 mL) in turn, and dried over anhydrous sodium sulfate. The filter was removed by suction filtration, and the filtrate was evaporated on a rotary evaporator. The residue was purified by silica gel column chromatography to obtain compound III-I, 1.53 g (yield 79%). ESI-MS, m / z = 194 ([M+H] + ).

[0032] Step B. Synthesis of compound IV-1

[0033] Compound III-1 (1.35 g, 7 mmol) was dissolved in DMF (20 mL) and stirred at room temperature. Formylhydrazide (0.42 g, 7 mmol) was added and the reaction was stirred at room temperature overnight. TLC showed the reaction was complete. Solid K2CO3 (1.38 g, 10 mmol) was added and the reaction was stirred at 50-60 °C for 5 h. TLC showed the reaction was complete.

[0034] The reaction mixture was carefully poured into ice water (200 mL) and stirred. The pH was adjusted to 5-6 with dilute hydrochloric acid and extracted with CH2Cl2(50 mL x 3). The combined extracts were washed with 5% brine (100 mL) and dried over anhydrous sodium sulfate. The drying agent was removed by suction filtration and the filtrate was evaporated on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound IV-1, 1.19 g (72% yield). ESI-MS, m / z = 236 ([M+H] + ).

[0035] Step C. Synthesis of compound V-1

[0036] Compound IV-1 (0.94 g, 4 mmol) and solid K2CO3 (1.66 g, 12 mmol) were added to DMF (20 mL) and stirred. Ethyl bromoacetate (0.84 g, 5 mmol) was added and the resulting reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete.

[0037] The reaction mixture was carefully poured into ice water (200 mL) and stirred. The pH was adjusted to 5-6 with dilute hydrochloric acid and extracted with CH2Cl2(50 mL x 3). The combined extracts were washed with 5% brine (100 mL) and dried over anhydrous sodium sulfate. The drying agent was removed by suction filtration and the filtrate was evaporated on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound V-1, 1.18 g (92% yield). ESI-MS, m / z = 322 ([M+H] + ).

[0038] Step D. Synthesis of compound I-1

[0039] Compound V-1 (0.96 g, 3 mmol) and compound VI-1 (0.67 g, 3 mmol) were dissolved in toluene (15 mL) and then heated to reflux until the reaction was complete (typically 5 h).

[0040] The reaction mixture was carefully poured into ice water (200 mL), stirred, extracted with CH2Cl2(50 mL x 3), combined extracts were washed with 5% brine (100 mL), dried over anhydrous sodium sulfate. The drying agent was removed by suction filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified using silica gel column chromatography to obtain compound I-I, 1.34 g (yield 90%). ESI-MS, m / z = 498 ([M+H] + ).

[0041] Examples 2-12

[0042] The compounds listed in the following table were synthesized according to the procedure of Reference Example 1.

[0043]

[0044]

[0045] Example 13

[0046]

[0047]

[0048] The active ingredient, pregelatinized starch and microcrystalline cellulose were sieved and mixed well, the polyvinylpyrrolidone solution was added, mixed, soft material was prepared, sieved, wet granules were prepared, dried at 50-60°C, sodium carboxymethyl starch, magnesium stearate and talc were sieved in advance, then added to the above granules to press into tablets.

[0049] Example 14

[0050] Ingredient Amount per tablet Example 1 sample 100 mg Microcrystalline cellulose 30 mg Pre-gelatinized starch 20 mg Polyvinyl pyrrolidone 3 mg Magnesium stearate 2 mg Talc 1 mg

[0051] The active ingredient, pregelatinized starch and microcrystalline cellulose were sieved and mixed well, the polyvinylpyrrolidone solution was added, mixed, soft material was prepared, sieved, wet granules were prepared, dried at 50-60°C, sodium carboxymethyl starch, magnesium stearate and talc were sieved in advance, then added to the above granules to press into tablets.

[0052] Example 15

[0053] Ingredient Amount per 50 mL Example 3 sample 10 mg Citric acid 100 mg NaOH q.s. (to pH 4.0-5.0) Distilled water 50 mL

[0054] In distilled water, distilled water and citric acid were added first, stirred and dissolved, then the sample was added, dissolved with slight heating, the pH value was adjusted to 4.0-5.0, 0.2 g of activated carbon was added, stirred at room temperature for 20 minutes, filtered, the filtrate was determined for solution concentration, 5 ml per ampoule was dispensed, high temperature sterilization for 30 minutes, and an injection solution was obtained.

[0055] Example 16

[0056]

[0057]

[0058] Preparation process: the main medicine and excipients are passed through 100 mesh sieve, mixed thoroughly, then the prescription amount of excipients and main medicine are weighed and mixed thoroughly. The soft material is prepared by adding binder, granulated by 14 mesh sieve, dried at 55°C, and the granules are sized by 12 mesh sieve. The bag weight is determined and the product is packed.

[0059] Example 17

[0060] Ingredient Amount Example 5 sample 2.0g Poloxamer 1.0g Sodium hydroxide 0.2g Citric acid q.s. Mannitol 26.0g Lactose 23.0g Water for injection 100 mL

[0061] Preparation process: 80 mL of water for injection is taken, the main medicine, mannitol, lactose and poloxamer are stirred to dissolve, then 1 mol / L citric acid is added to adjust the pH to 7.0-9.0, and water is added to 100 mL. 0.5 g of activated carbon is added, stirred at 30°C for 20 minutes, decarbonized, filtered with a microporous filter to remove bacteria, the filtrate is divided into 1 mL per vial, pre-frozen for 2 hours, then dried under reduced pressure for 12 hours while frozen, after the sample temperature reaches room temperature, dried for another 5 hours, to obtain white loose block-shaped product, which is sealed to obtain the product.

[0062] Example 18 in vitro inhibition of HSL assay

[0063] 1) Cloning. cDNA was prepared from commercial human brain polyA+ RNA and used as a template in overlapping PCR to generate a full-length human HSL ORF with a 3'-His 6 tag. This full-length insert was cloned into the pFast-BAC vector and the DNA sequence of several individual clones was verified. DNA from the correct full-length clone with the 3'-His 6 tag was used to transform E. coli strain DH10BAC. The resulting bacmid DNA was used to generate a stock of baculovirus for protein production at high titer. The sequence encoding HSL corresponds to Swissprot entry Q05469 with an additional C-terminal His 6 -tag.

[0064] 2) Protein purification. 5.5 L of High 5 cells expressing human full-length HSL-His 6 were grown for 48 hours with 25 μM E-64. Cell number: 1.78 x 1011 10cells / hour, 90% viability. Thaw the cells. Resuspend the cells on ice in a pH 8.0 base buffer containing the following: 10% glycerol, 25 mM Tris-Cl, 300 mM NaCl, 10 mM imidazole, 10 mM 2-mercaptoethanol, 2 μg / mL pepstatin, 2 μg / mL leupeptin, 2 μg / mL antipain in a final volume of 475 mL with 3.75 × 10 7 Cells were sanitized at 3 × 30-second intervals, and Lubrol PX was added to a final concentration of 0.2%, followed by stirring at 4°C for 15 minutes and centrifugation at 25,000 g for 60 minutes at 4°C. The soluble protein was mixed with 60 mL of pre-washed and equilibrated Ni-NTA agarose (Qiagen 30210), followed by end-over-end tumbling at 4°C for 45 minutes, centrifugation at 1000 rpm for 5 minutes, and the resin was allowed to settle for 5 minutes. The supernatant was removed, and the resin was washed in a centrifuge vessel with 5 volumes of base buffer containing 0.2% Lubrol PX. Centrifugation was repeated, and the supernatant discarded. The resin was poured onto a 0.8 μm membrane in a disposable filter unit (Nalge 450-0080) and washed with 5 volumes of base buffer containing 0.2% Lubrol PX. It was then washed with 30 volumes of base buffer containing 60 mM imidazole at pH 7.5 at 4°C. With 25mMTris-Cl, 300mM NaCl, 200mM imidazoles, 10mM 2-mercaptoethanol of 4 ℃, pH 7.5 5 volumes, by 4 ℃ with buffer, resin is turned upside down and rolled 30 minutes with protein elution.Resin is captured on 0.2 μ m membrane disposable filter unit (Millipore SCGP U02RE) and eluate is collected in reservoir.Use 30k MWCO centrifugal filter device (Sartorius Vivascience Vivacell 100, VC1022) that eluate is concentrated to 20ml.Then it is dialyzed overnight at 4 ℃ twice relative to 10% glycerol, 25mM Tris-Cl, 300mM NaCl, 0.2mM EDTA, 0.2mM DTT of 4 ℃, pH 7.5 2L.Use 0.22um disposable filter unit (Millipore SCGP00525) to filter protein.Utilize 280=0.67cm -1 mg -1 The protein concentration was calculated from the absorbance at 280 nm. The yield was 201 mg in total. The protein was stored at -80°C.

[0065] 3) Human hormone sensitive lipase (HSL) enzyme inhibition assay. HSL enzyme activity is measured by a colorimetric assay using 2,3-dimercapto-l-propanol tributyric acid ester as a substrate. Typically, 1.5 mM of 2,3-dimercapto-l-propanol tributyric acid ester (DMPT) in 100 mM MOPS, pH 7.2, 0.2 mg / mL fatty acid free BSA is prepared by sonicating to a homogeneous suspension at 4°C. Test compounds (2 mM stock in DMSO) are serially diluted 3-fold in DMSO. Compound solutions are diluted 24-fold in the solution containing 1.5 mM DMPT and added to a 384-well microplate (Corning Costar) at 18 uL per well. Human HSL (15 ug / mL) is added to each well at 12 uL and the reaction mixture is incubated at 37°C for 20 minutes. 6 uL of 12 mM dithio-bis(2-nitrobenzoic acid) (DTNB) in DMSO (plus 1.2% SDS and 0.6% Triton X-100) is added and the mixture is incubated at room temperature for 15 minutes. Product generation is monitored by reading the absorbance at 405 nm on an Envision Reader (PerkinElmer Life and Analytical Sciences). IC50values are calculated accordingly. 50 .

[0066] The test results are shown in the following table.

[0067] Compound EC 50 (nM) Compound EC 50 (nM) Example 1 compound 33 Example 7 compound 44 Example 2 compound 148 Example 8 compound 31 Example 3 compound 490 Example 9 compound 98 Example 4 compound 933 Example 10 compound 41 Example 5 compound 15 Example 11 compound 37 Example 6 compound 9.8 Example 12 compound 11

[0068] As can be seen from the results in the above table, the compounds of the present application have strong inhibitory effect on HSL and can be used as therapeutic drugs for the preparation of diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity.

Claims

1. A compound of general formula (I), in, R 1 Selected from H, C1-C 10 Alkyl, F, Cl, Br, I, NO2, CN and R 3 O; R 2 Selected from H, C1-C 10 Alkyl, C3-C 10 Cycloalkyl; wherein R 3 Selected from C1-C 10 Alkyl and C3-C 10 of a cycloalkyl group.

2. The compound of general formula (I) as defined in claim 1, wherein R 1 selected from C1-C4 alkyl, F, Cl, NO2, CN and R 3 O; R 2 is selected from C1-C4 alkyl, C3-C6 cycloalkyl; wherein R 3 Selected from C1-C4 alkyl and C3-C6 cycloalkyl.

3. The following compounds: 。 4. Use of the compound of general formula (I) as defined in any one of claims 1 to 2 in the preparation of a drug for treating HSL enzyme inhibitors, wherein: The application is: for preparing medicines for treating diabetes, dyslipidemia, atherosclerosis and obesity.

5. A pharmaceutical composition comprising a compound of general formula (I) as defined in any one of claims 1 to 2, and a suitable carrier or excipient.

6. The pharmaceutical composition according to claim 5, wherein The composition is a solid oral preparation, a liquid oral preparation or an injection.

7. The pharmaceutical composition according to claim 6, wherein the solid oral preparation or liquid oral preparation comprises: Dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, granules, oral solutions; the injections include water injection, freeze-dried powder injection, large infusion, and small infusion.

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