Diaminourea HSL inhibitors, preparation methods and uses

Abstract

This invention relates to the field of HSL inhibitors. Specifically, this invention relates to a class of HSL inhibitors containing a diaminourea structure, methods for their preparation, pharmaceutical compositions containing them, and their use in the preparation of treatments for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis, or obesity. The substituents are as shown in the specification.

Description

Technical Field

[0001] This invention relates to the field of HSL inhibitors. Specifically, this invention relates to a class of HSL inhibitors containing a diaminourea structure that exert a therapeutic effect by inhibiting HSL, methods for their preparation, pharmaceutical compositions containing them, and their use in the treatment of diabetes, metabolic syndrome, dyslipidemia, atherosclerosis, or obesity. Background Technology

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

[0003] The most important enzyme in adipocytes involved in the hormone-regulated hydrolysis of triglycerides in the cytokine (WAT) is hormone-sensitive lipase (HSL). This enzyme is also found in the liver, skeletal muscles, membranes, and adrenal glands. In its basal state, it exhibits minimal cleanliness towards its substrate. Hormonal stimulation of adipocytes activates protein kinase A, leading to phosphorylation of HSL and perilipin, the lipid droplet-coating protein. Phosphorylation of perilipin results in its desorption from the lipid droplets, and phosphorylated HSL migrates from the cytosol to the lipid droplets, where it catalyzes the hydrolysis of triglycerides.

[0004] Disruptions in the regulation of adipocyte lipolysis leading to elevated circulating unesterified fatty acids (NEFA) are associated with obesity and complex complications 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 proteins and exhibit enhanced lipolytic activity due to their resistance to insulin-mediated inhibition of lipolysis. This results in elevated plasma levels of free fatty acids, which further exacerbates insulin resistance, attributed to the accumulation of triglycerides in tissues other than NEFA, such as the liver, membranes, and muscles. Abnormal triglyceride deposition produces pathological effects, such as increased glucose production in the liver, decreased insulin secretion by the pancreas, and reduced glucose uptake and fatty acid oxidation in skeletal muscles. Therefore, elevated plasma FFA levels due to increased HSL activity contribute to and worsen insulin resistance in obese and type 2 diabetic individuals. Additionally, elevated FFA, via adipose tissue, is associated with increased production of the inflammatory cytokine TNF-α. TNF-α also disrupts insulin signaling by activating serine kinases, such as JNK-1, which phosphorylate IRS-1, thereby inhibiting insulin signaling (Mol. Endocrinol., 2004, 18, 2024-2034). Therefore, restoring excessively high plasma FFA and triglyceride levels by inhibiting HSL will reduce triglyceride accumulation in tissues other than WAT, such as the liver, muscle, and membranes, leading to reduced hepatic glucose output, increased intramuscular fatty acid oxidation, and improved β-cell function. Inflammatory cytokine production will also decrease, leading to further reductions in FFA production and improved insulin signaling. Elevated FFA is also associated with increased cardiovascular risk, including atherosclerosis and myocardial dysfunction. Long-term low-dose lipid infusion in healthy patients has been shown to lead to markers of endothelial activation independent of its metabolic effects. Here, moderate lipid infusion has been shown to increase markers of endothelial activation, including ET-1, ICAM-1, and 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 a major hormone-regulated lipase, it is known that in insulin-resistant states, insulin's ability to inhibit lipolysis is reduced, leading to increased FFA (fatty acid esters), or lipotoxicity. These fatty acids accumulate in the liver and cause increased TAG (tachycardia glycosides) production, which are encapsulated in secreted VLDL. Lipids also accumulate in the liver, leading to a fatty liver phenotype. Lipolysis is increased in diabetes and obesity, contributing to this phenotype. Therefore, reduced HSL activity will decrease the release of FFA into the bloodstream, thereby limiting the supply of FFA to the liver for TAG synthesis. Thus, HSL inhibitors could have beneficial effects in treating NAFLD (non-alcoholic fatty liver disease) and NASH (non-alcoholic steatohepatitis).

[0006] This invention discloses a class of HSL inhibitors containing a diaminourea structure, which can be used to prepare therapeutic drugs for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis, or obesity. Summary of the Invention

[0007] One object of the present invention is to provide an HSL inhibitor having general formula I.

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

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising a compound of general formula I as an active ingredient, and one or more pharmaceutically acceptable carriers, excipients or diluents, and its use in the treatment of diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity.

[0010] The present invention will now be described in detail in conjunction with its objectives.

[0011] The compounds of general formula I of this invention have the following structural formula:

[0012]

[0013] Among them, R 1 Selected from H, C1-C 10 Alkyl groups, F, Cl, Br, I, and R 3 O;R 2 Selected from H, C1-C 10 Alkyl group, C3-C 10 cycloalkyl groups, F, Cl, Br, I, NO2, CN and R 3 O; where R 3 Selected from C1-C 10 Alkyl and C3-C 10 cycloalkyl groups.

[0014] The following compounds of general formula I are preferred.

[0015] Among them, R 1 Selected from H, C1-C4 alkyl groups, F, Cl and R 3 O;R 2 Selected from H, C1-C4 alkyl groups, C3-C6 cycloalkyl groups, F, Cl, NO2, CN, and R. 3 O; where R 3 Selected from C1-C4 alkyl groups and C3-C6 cycloalkyl groups.

[0016] More preferred compounds having general formula I are as follows:

[0017]

[0018] The compound of general formula I described in this invention can be synthesized via the following route:

[0019]

[0020] Compound II is a commercially available industrial-grade reagent, and VII is a commercially available reagent or can be prepared using conventional methods.

[0021] Compound II was treated with sulfur phosgene in the presence of a base to give compound III; compound III was first reacted with formyl hydrazine, and the intermediate was treated with a base to give compound IV; compound IV was reacted with 2-haloisobutyrate to give compound V; compound V was reacted with thiourea under heating to give VI; compound VI was reacted with isothiocyanate VII to give compound I; wherein, X is selected from Cl, Br and I, R 4 Selected from C1-C 10 alkyl, R 1 and R 2 The definition is as described above.

[0022] The compound of Formula I described in this invention can be co-formed with one or more pharmaceutically acceptable carriers, excipients, or diluents to create a pharmaceutical composition. This pharmaceutical composition can be formulated into dosage forms such as solid oral preparations, liquid oral preparations, and injections. The solid and liquid oral preparations include: tablets, dispersible tablets, sugar-coated tablets, granules, dry powders, capsules, and solutions. The injections include: small injections, large-volume infusions, and lyophilized powder injections.

[0023] The pharmaceutically or food-grade excipients in the compositions of the present invention are selected from: fillers, disintegrants, lubricants, glidants, effervescent agents, flavoring agents, preservatives, coating materials, or other excipients.

[0024] The compositions of the present invention contain pharmaceutically or food-grade excipients. The fillers include one or more of lactose, sucrose, dextrin, starch, pregelatinized starch, mannitol, sorbitol, dicalcium phosphate, calcium sulfate, calcium carbonate, and microcrystalline cellulose; the binders include one or more of starch, sucrose, povidone, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose, polyethylene glycol, pharmaceutical grade ethanol, and water; the disintegrants include one or more of starch, croscarmellose, croscarmellose sodium, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, and effervescent disintegrants.

[0025] The compound of general formula I described in this invention has HSL inhibitory activity and can be used as an active ingredient in the preparation of therapeutic drugs for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis, or obesity. The activity of the compound of general formula I described in this invention was verified by in vitro HSL inhibition experiments. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the invention. Various modifications made by those skilled in the art based on the teachings of the present invention should be within the scope of protection claimed in the claims of this application.

[0027] Example 1: Synthesis of 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 and stirred under ice-water bath cooling. Et3N (3.04 g, 30 mmol) was added, followed by the slow dropwise addition of a solution (5 mL) of phosgene (1.26 g, 11 mol) dissolved in dry dichloromethane. After the addition was complete, the reaction mixture was stirred overnight at room temperature, and TLC showed that the reaction was complete.

[0031] The reaction mixture was carefully poured into ice water (200 mL), stirred, and extracted with CH₂Cl₂ (50 mL × 3). The extracts were combined and washed successively with 1% dilute hydrochloric acid (200 mL) and 5% brine (100 mL), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound III-I, 1.53 g (79% yield). 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. Then, formyl hydrazine (0.42 g, 7 mmol) was added, and the mixture was stirred overnight at room temperature. TLC showed the reaction was complete at this point. Solid K₂CO₃ (1.38 g, 10 mmol) was then added to the reaction mixture, and the temperature was raised to 50-60 °C. The mixture was stirred for 5 hours, and TLC showed the reaction was complete.

[0034] The reaction mixture was carefully poured into ice water (200 mL), stirred, and the pH was adjusted to 5-6 with dilute hydrochloric acid. Extraction was performed with CH₂Cl₂ (50 mL × 3). The extracts were combined, washed with 5% brine (100 mL), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound IV-I, 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), stirred, and then methyl 2-bromoisobutyrate (0.91 g, 5 mmol) was added. The resulting reaction compound was stirred overnight at room temperature, and TLC showed that the reaction was complete.

[0037] The reaction mixture was carefully poured into ice water (200 mL), stirred, and extracted with CH₂Cl₂ (50 mL × 3). The extracts were combined, washed with 5% brine (100 mL), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound VI, 1.51 g (90% yield). ESI-MS, m / z = 336 ([M+H)). + ).

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

[0039] Compound V-1 (1.01 g, 3 mmol) and diaminourea (0.27 g, 3 mmol) were dissolved in 1,4-dioxane (15 mL), and the mixture was then heated to reflux until the reaction was complete (usually 10 hours).

[0040] The reaction mixture was carefully poured into ice water (200 mL), stirred, and extracted with CH₂Cl₂ (50 mL × 3). The extracts were combined, washed with 5% brine (100 mL), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound II, 0.91 g (77% yield). ESI-MS, m / z = 394 ([M+H)). + ).

[0041] Step E. Synthesis of Compound I-1

[0042] Compound VI-1 (0.39 g, 1 mmol) and phenyl isothiocyanate VII-1 (0.14 g, 1 mmol) were dissolved in 1,4-dioxane (5 mL) and stirred at room temperature until the reaction was complete (usually 5 hours).

[0043] The reaction mixture was carefully poured into ice water (200 mL), stirred, and extracted with CH₂Cl₂ (50 mL × 3). The extracts were combined, washed with 5% brine (100 mL), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to give compound II, 0.47 g (yield 89%). ESI-MS, m / z = 529 ([M+H)). + ).

[0044] Example 2-12

[0045] Following the procedure in Example 1, the compounds listed in the table below were synthesized.

[0046]

[0047]

[0048] Example 13

[0049]

[0050]

[0051] The active ingredients, pregelatinized starch, and microcrystalline cellulose are sieved, thoroughly mixed, and then polyvinylpyrrolidone solution is added and mixed to form a soft mass. This mass is then sieved, wet granules are formed, and dried at 50-60°C. Sodium carboxymethyl starch, magnesium stearate, and talc are pre-sieved and then added to the granules and compressed into tablets.

[0052] Example 14

[0053] Components Dosage / capsule Example 1 Sample 100mg microcrystalline cellulose 30mg Pregelatinized starch 20mg Polyvinylpyrrolidone 3mg magnesium stearate 2mg talcum powder 1mg

[0054] The active ingredients, pregelatinized starch, and microcrystalline cellulose are sieved, thoroughly mixed, and then polyvinylpyrrolidone solution is added and mixed to form a soft mass. This mass is then sieved again to form wet granules, which are dried at 50-60°C. Magnesium stearate and talc are pre-sieved and then added to the granules. The mixture is then encapsulated to obtain the final product.

[0055] Example 15

[0056] Components Dosage / 50mL Example 3 Sample 10mg Citric acid 100mg NaOH Appropriate amount (to adjust pH to 4.0-5.0) distilled water 50mL

[0057] First, add distilled water and citric acid to distilled water, stir to dissolve, then add the sample, gently heat to dissolve, adjust the pH to 4.0-5.0, add 0.2 g of activated carbon, stir at room temperature for 20 minutes, filter, measure the concentration of the filtrate, dispense 5 ml per ampoule, sterilize at high temperature for 30 minutes to obtain the injection solution.

[0058] Example 16

[0059]

[0060]

[0061] Preparation process: The active pharmaceutical ingredient and excipients are passed through a 100-mesh sieve separately and thoroughly mixed. Then, the prescribed amount of excipients and active pharmaceutical ingredient are weighed and thoroughly mixed. A binder is added to form a soft mass, which is then granulated through a 14-mesh sieve, dried at 55℃, granulated again through a 12-mesh sieve, and finally packaged after the bag weight is determined.

[0062] Example 17

[0063] Components Dosage Example 5 Sample 2.0g Polosham 1.0g Sodium hydroxide 0.2g Citric acid QS Mannitol 26.0g lactose 23.0g Water for Injection 100mL

[0064] Preparation process: Take 80 mL of water for injection, add the active pharmaceutical ingredient, mannitol, lactose, and poloxamer, and stir until dissolved. Adjust the pH to 7.0-9.0 with 1 mol / L citric acid, and add water to 100 mL. Add 0.5 g of activated carbon, stir at 30 °C for 20 minutes to remove the activated carbon, and filter sterilize using a microporous membrane. Dispense the filtrate into 1 mL vials, pre-freeze for 2 hours, then freeze-dry under reduced pressure for 12 hours until the sample temperature reaches room temperature, and then dry for another 5 hours to obtain a white, loose, blocky substance. Seal the vials to obtain the final product.

[0065] Example 18: In vitro HSL inhibition analysis of the compound

[0066] 1) Cloning. cDNA was prepared from commercial human brain polyA+ RNA and used as a template in overlap PCR to generate cDNA with 3'-His... 6A labeled full-length human HSL ORF was generated. This full-length incoherent was cloned into the pFast-BAC vector, and the DNA sequences of several individual clones were examined. DNA from the correct full-length clone with a 3'-His... 6 The labeled DNA was used to transform E. coli strain DH10BAC. The resulting baculosome DNA was used to produce the original baculovirus strain for protein production. The sequence encoding HSL conforms to Swissprot entry Q05469 and has an additional C-terminal His. 6 -mark.

[0067] 2) Protein purification. 5.5L, expressing full-length human HSL-His protein. 6 High 5 cells, after 48 hours, contained 25 μM E-64. Cell count: 1.78 × 10⁻⁶. 10 Cells / hour, 90% viability. Thaw the cells. On ice, suspend the cells in an alkaline buffer at pH 8.0 containing the following at 4°C: 10% glycerol, 25 mM Tris-Cl, 300 mM NaCl, 10 mM imidazole, 10 mM 2-mercaptoethanol, 2 μg / mL pepsin inhibitor, 2 μg / mL leucine, 2 μg / mL analgesic, for a final volume of 475 mL with 3.75 × 10⁻⁶ cells / hour. 7Cells were collected at 1 / mL, sanitized at 3 × 30 sec, and Lubrol PX was added to a final concentration of 0.2%. The mixture was then stirred at 4°C for 15 min and centrifuged at 25000g for 60 min at 4°C. Soluble proteins were mixed with 60 mL of pre-washed and equilibrated Ni-NTA agarose (Qiagen 30210), inverted for 45 min at 4°C, centrifuged at 1000 rpm for 5 min, and allowed to settle for 5 min. The supernatant was removed, and the resin was washed in a centrifuge container with 5 volumes of alkaline buffer containing 0.2% Lubrol PX. The mixture was centrifuged again, and the supernatant was discarded. The resin was poured onto a 0.8 μm membrane in a disposable filter unit (Nalge450-0080) and washed with 5 volumes of alkaline buffer containing 0.2% Lubrol PX. It was then washed at 4°C with 30 volumes of alkaline buffer containing 60 mM imidazole at pH 7.5. Proteins were eluted using 5 volumes of 25 mM Tris-Cl, 300 mM NaCl, 200 mM imidazole, and 10 mM 2-mercaptoethanol at 4°C and pH 7.5 by inverting and rolling the resin in buffer for 30 minutes at 4°C. The resin was trapped on a 0.2 μm disposable membrane filter unit (Millipore SCGP U02RE), and the eluent was collected in a reservoir. The eluent was concentrated to 20 mL using a 30 kJ MWCO centrifuge filter (Sartorius Vivascience Vivacell 100, VC1022). It was then dialyzed twice overnight at 4°C at 4°C using 2 L of 10% glycerol, 25 mM Tris-Cl, 300 mM NaCl, 0.2 mM EDTA, and 0.2 mM DTT. Proteins were filtered using a 0.22 μm disposable filter unit (Millipore SCGP00525). The eluent was collected using 280 = 0.67 cm⁻¹. -1 mg -1 Protein concentration was calculated from absorbance at 280 nm. The total yield was 201 mg. The protein was stored at -80°C.

[0068] 3) Human Hormone-Sensitive Lipase (HSL) Inhibition Assay. HSL enzyme activity was measured colorimetrically using 2,3-dimercapto-1-propanol tributyrate as a substrate. Typically, 1.5 mM 2,3-dimercapto-1-propanol tributyrate (DMPT) was prepared by sonication to a homogeneous suspension at 4°C in 100 mM MOPS, pH 7.2, and 0.2 mg / mL fatty acid-free BSA. The test compound (2 mM stock solution in DMSO) was serially diluted 3-fold in DMSO. The compound solution was diluted 24-fold in a solution containing 1.5 mM MPT and added in 18 μL per well to a 384-well microplate (Corning Costar). 12 μL of human HSL (15 μg / mL) was added to each well, and the reaction mixture was incubated at 37°C for 20 min. 6 μL of 12 mM dithiobis(2-nitrobenzoic acid) (DTNB) in DMSO (with 1.2% SDS and 0.6% Triton X-100) was added, and the mixture was incubated at room temperature for 15 minutes. Product formation was monitored by reading the absorbance at 405 nm on an Envision Reader (PerkinElmer Life and Analytic Sciences). The IC50 was calculated accordingly. 50 .

[0069] The test results are shown in the table below.

[0070]

[0071]

[0072] As can be seen from the results in the table above, the compounds of the present invention have a strong inhibitory effect on HSL and can be used as therapeutic drugs for diabetes, metabolic syndrome, dyslipidemia, atherosclerosis or obesity.

Claims

1. Compounds having the general formula (I), in, R 1 Selected from C1-C 10 Alkyl groups, F, Cl, Br, I, and R 3 O;R 2 Selected from C1-C 10 Alkyl group, C3-C 10 cycloalkyl groups, F, Cl, Br, I, NO2, CN and R 3 O; where R 3 Selected from C1-C 10 Alkyl and C3-C 10 cycloalkyl groups.

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

3. The following compounds: 。 4. The use of the compound of general formula (I) as defined in any one of claims 1-2 in the preparation of a medicament for the treatment of diabetes, dyslipidemia, atherosclerosis or obesity by way of its use as an HSL inhibitor.

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

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

7. The pharmaceutical composition of claim 6, wherein the solid oral formulation or liquid oral formulation comprises: Dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, granules, and oral solutions; the injectables include water for injection, lyophilized powder for injection, large-volume infusions, and small-volume infusions.

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