2-chloro-n6-cyclopentyladenosine derivatives, processes for their preparation and use

Abstract

The application discloses a 2-chloro-N6-cyclopentyl adenosine derivative, a preparation method and application thereof, and belongs to the technical field of medicines. The derivative is connected with a phosphoric acid or sulfonic acid group through a stable C-P bond or C-S bond at the 5' position of 2-chloro-N6-cyclopentyl adenosine, the metabolic stability of the derivative is enhanced in the body, the derivative is not easy to hydrolyze, and central side effects such as drowsiness caused by 2-chloro-N6-cyclopentyl adenosine can be avoided. The application further provides a preparation method of the derivative, wherein 2-chloro-N6-cyclopentyl adenosine is used as a starting material, an acetone fork protection reaction is first carried out on the starting material in the presence of an acid catalyst to obtain an intermediate I, then the 5'-hydroxyl group of the intermediate I is activated and converted into an intermediate connected with a phosphorus-containing or sulfur-containing functional group on the 5' carbon, and finally a deprotection reaction is carried out to obtain the target compound. The compound disclosed by the application can be used for preparing medicines for treating liver diseases, in particular, metabolic dysfunction related fatty hepatitis, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a series of novel 2-chloro-N6-cyclopentyladenosine derivatives, their preparation methods, and the use of these compounds in the preparation of drugs for treating liver diseases (especially metabolic dysfunction-related steatohepatitis). Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a serious liver disease with a complex pathogenesis and limited treatment options. Adenosine and its derivatives, as important bioactive molecules, have shown great potential in the treatment of liver diseases. Adenosine derivatives can regulate hepatic metabolic function and improve steatosis and inflammatory responses by activating adenosine receptors.

[0003] Early studies have shown that C2,5′-disubstituted and N6,C2,5′-trisubstituted adenosine derivatives are effective adenosine receptor agonists and have therapeutic value in treating and preventing diseases affected by adenosine receptor agonists, such as cancer (Chinese Patent CN1259332C).

[0004] Chinese patent CN118845814A discloses that N6-cyclopentyl adenosine and its derivatives and salts can be used to prepare drugs for treating metabolic dysfunction-related steatotic liver disease (MASLD), MASH, liver injury and hyperlipidemia, and can effectively treat MASLD / MASH, reduce ALT / AST and hyperlipidemia.

[0005] However, while existing compounds with OP-linked bonds obtained by modifying the 5' position of 2-chloro-N6-cyclopentyladenosine exhibit significant anti-MASH activity, the OP bond is easily hydrolyzed in vivo, releasing the parent drug 2-chloro-N6-cyclopentyladenosine. 2-chloro-N6-cyclopentyladenosine has central nervous system side effects such as drowsiness in vivo, limiting its safety and efficacy in clinical applications. Therefore, there is an urgent need to design and synthesize compounds with more stable chemical bonds to improve efficacy while avoiding side effects caused by the release of the parent drug. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing 2-chloro-N6-cyclopentyladenosine derivatives, such as easy hydrolysis in vivo, poor stability, and central nervous system side effects like drowsiness. It provides a novel class of 2-chloro-N6-cyclopentyladenosine derivatives in which a phosphate or sulfonic acid group is linked at the 5' position of 2-chloro-N6-cyclopentyladenosine via a stable carbon-phosphorus (CP) or carbon-sulfur (CS) bond. These compounds are metabolically stable in vivo, not easily hydrolyzed, and can avoid the central nervous system side effects such as drowsiness associated with 2-chloro-N6-cyclopentyladenosine (CCPA) while maintaining high anti-MASH activity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a 2-chloro-N6-cyclopentyl adenosine derivative as shown in formula (I):

[0009] In equation (Ⅰ), m = 0, 1, 2, 3 or 4, n = 0, 1, 2, 3 or 4, and X in the connecting chain is selected from any one of bond, NH, O or S; In formula (I), R2 is selected from any one of phosphoric acid, sulfonic acid, phosphate or sulfonate groups, and R1 is selected from any one of hydroxyl, halogen, hydrogen or amino groups.

[0010] Preferably, in formula (Ⅰ), m = 0, n = 0, 1, 2, 3 or 4, R1 is selected from any one of hydrogen, hydroxyl, halogen or amino, and X in the connecting chain is a bond; R2 is selected from any one of phosphoric acid, sulfonic acid, phosphate or sulfonate group.

[0011] Preferably, in formula (Ⅰ), m = 0, 1, 2, 3 or 4; n = 0; X is selected from any one of NH, O, S; R1 is selected from any one of hydrogen, hydroxyl, halogen, amino; R2 is selected from any one of phosphoric acid, sulfonic acid, phosphate or sulfonate group.

[0012] Preferably, the phosphate or sulfonate is selected from any one of sodium salt, potassium salt, calcium salt or magnesium salt.

[0013] Preferably, the 2-chloro-N6-cyclopentyl adenosine derivative is selected from any one of the following formulas CO2-CO7: .

[0014] Secondly, the present invention provides a method for preparing the above-mentioned 2-chloro-N6-cyclopentyl adenosine derivative, comprising the following steps: (1) 2-chloro-N6-cyclopentyl adenosine reacts with 2,2-dimethoxypropane in the presence of an acid catalyst to undergo an acetone-protected reaction to obtain intermediate I; (2) Activate the 5'-hydroxyl group of intermediate I obtained in step (1) and convert it into an intermediate with a phosphorus- or sulfur-containing functional group attached to the carbon at the 5' position; (3) The intermediate containing phosphoryl or sulfonyl groups obtained in step (2) is subjected to a deprotection reaction to obtain a compound as shown in general formula (I); Preferably, the method for activating the 5'-hydroxyl group of intermediate I and converting it into an intermediate with a phosphorus- or sulfur-containing functional group attached to the 5' carbon in step (2) is selected from any of the following: (a) Albuzov reaction pathway: After converting the 5' hydroxyl group into a leaving group, it undergoes an Albuzov reaction with a trivalent phosphorus compound to introduce a phosphoryl substituent; (b) Michael addition pathway: Under base catalysis, the 5' hydroxyl group or its activated form undergoes a conjugate addition reaction with α,β-unsaturated phosphonates; (c) Direct alkylation route: Under base catalysis, the 5' hydroxyl group undergoes an SN2 substitution reaction with a haloalkylphosphonate; (d) Carbonyl addition pathway: After oxidizing the 5' hydroxyl group to an aldehyde group, it undergoes a nucleophilic addition reaction with a phosphite; (e) Sulfation-oxidation pathway: The 5' hydroxyl group is converted into a leaving group, which is then substituted with a thiocarboxylate and oxidized with a peroxide to introduce a sulfonic acid group.

[0015] More preferably, wherein the (a) Albuzov reaction pathway comprises: converting the 5'-hydroxyl group to a halogen using a triphenylphosphine / halogen system, a sulfoxide halide, or a phosphohalogenated reagent; the halogenated reagent reacts with a compound selected from triphosphite (C... 1-6 Alkyl esters, triaryl phosphites, di(C) phosphites 1-6 Reactions of trivalent phosphorus compounds with alkyl esters, diaryl phosphites, or alkyl phosphinates; The Michael addition pathway (b) includes: the reaction of the 5'-hydroxyl group with diethyl vinylphosphonate, dimethyl vinylphosphonate or aryl-substituted vinylphosphonate in the presence of cesium carbonate, sodium hydride, potassium tert-butoxide or DBU; The (c) direct alkylation pathway includes: reacting the 5' hydroxyl group with diethoxyphosphorylmethyltrifluoromethanesulfonate, diethoxyphosphorylethyl bromide, or (diethoxyphosphoryl)propyl iodide in the presence of sodium hydride, cesium carbonate, or potassium tert-butoxide; The (d) carbonyl addition pathway includes: oxidizing the 5'-hydroxyl group to an aldehyde group using a Desmartin oxidant, PCC, or Swern reagent; and nucleophilic addition of the aldehyde group to diethyl phosphite, dimethyl phosphite, or diphenyl phosphite in the presence of potassium tert-butoxide, sodium hydride, or LDA. The (e) sulfidation-oxidation pathway includes: the 5'-hydroxyl group is activated by iodination or methanesulfonation, and then reacts with potassium thioacetate, thiourea or sodium hydrosulfide to generate a thio intermediate; the thio intermediate is oxidized to a sulfonic acid group by oxyacid, hydrogen peroxide or m-chloroperoxybenzoic acid.

[0016] Preferably, the leaving group is selected from iodine, bromine, chlorine, methanesulfonyloxy, p-toluenesulfonyloxy, or trifluoromethanesulfonyloxy.

[0017] Preferably, the deprotection in step (3) includes: first removing the alkyl protecting group on the phosphoryl group or sulfonyl group with trimethylbromosilane, and then removing the acetone fork protecting group with dilute acid.

[0018] Figure 1 This is a synthesis route diagram in some preferred embodiments.

[0019] Thirdly, the present invention provides the use of the above-mentioned 2-chloro-N6-cyclopentyladenosine derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver diseases, wherein the liver disease is preferably MASH.

[0020] Preferably, the 2-chloro-N6-cyclopentyl adenosine derivative is of formula C02:

[0021] The drug can significantly reduce liver weight and NAFLD activity score (NAS) in patients, effectively alleviating hepatic steatosis and overall pathological damage.

[0022] The drug can significantly downregulate key pro-inflammatory factors in liver tissue. Il-6 (Interleukin-6) and Il-1β (Interleukin-1β), chemokines ( Ccl2 (Monocyte chemokine 2) and Cxcl9 It reduced the expression of CXC motif chemokine 9 and α-SMA (α-smooth muscle actin), a key marker of liver fibrosis, and significantly reduced the levels of serum liver injury markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), while also decreasing serum IL-6 levels.

[0023] Preferably, the dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments, or films.

[0024] Fourthly, the present invention provides a pharmaceutical composition for treating metabolic dysfunction-related steatohepatitis, comprising a therapeutically effective amount of the 2-chloro-N6-cyclopentyladenosine derivative or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also possible and will not be elaborated here.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The compound provided by this invention introduces a stable CP or CS bond at the 5' position of 2-chloro-N6-cyclopentyladenosine, replacing the traditional easily hydrolyzed OP bond, which significantly enhances the metabolic stability of the compound in vivo. It will not be hydrolyzed into 2-chloro-N6-cyclopentyladenosine in vivo, thus avoiding the central nervous system side effects such as drowsiness caused by 2-chloro-N6-cyclopentyladenosine.

[0027] This invention provides a clear and efficient synthetic route that can yield the target product in high yield, offers good process flexibility, and is suitable for large-scale preparation.

[0028] In vitro activity tests showed that the compounds of the present invention (such as CO2) at a concentration of 1 μM had a better effect on improving triglyceride accumulation than the lead compound 2-chloro-N6-cyclopentyladenosine.

[0029] In vivo MASH model experiments confirmed that the representative compound CO2 significantly improved the MASH effect: CO2 significantly reduced liver weight and liver activity score (NAS) in model animals, effectively alleviating hepatic steatosis and overall pathological damage. It also significantly reduced serum levels of liver injury markers ALT, AST, and ALP, confirming its definite hepatoprotective effect. Simultaneously, CO2 significantly downregulated key pro-inflammatory factors in liver tissue. Il-1β , Il-6 and chemokines Ccl2 , Cxcl9 The expression of its mRNA was inhibited, inflammatory signaling pathways and the recruitment of immune cells to the liver were suppressed, and the expression of α-SMA, a key marker of liver fibrosis, was significantly reduced, suggesting that it can effectively inhibit the occurrence and development of liver fibrosis by suppressing the activation of hepatic stellate cells.

[0030] Furthermore, no significant central nervous system side effects such as drowsiness were observed during the treatment process (the spontaneous activity of mice was not inhibited in the open field experiment), which initially demonstrated its good safety profile.

[0031] In summary, the compounds of this invention achieve simultaneous and effective intervention on the core pathological features of MASH—steatodegeneration, inflammation, liver damage, and fibrosis—and possess both high potency and low central toxicity, providing important candidate molecules for the development of a new generation of safe and effective MASH therapeutics. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the synthetic route of the compound described in this invention.

[0033] Figure 2Animal-level pharmacodynamic experiment results: A shows the experimental grouping and flowchart; B shows the H&E staining results of liver tissue; C shows the liver weight; D shows the NAS score results; E shows the Western blot electrophoresis image of α-SMA; F shows the relative expression level of α-SMA; G and J represent... Il-1β , Il-6 , Ccl2 , Cxcl9 The mRNA expression levels were denoted as α; K and N were the serum ALT, AST, ALP, and IL-6 levels, respectively.

[0034] Figure 3 The results of the mouse open field experiment are shown in Figure A, where A is a schematic diagram of the experimental grouping and process, and B is the mouse open field movement trajectory. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0036] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0037] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0038] Unless otherwise specified, all experimental materials and reagents used in the following examples are available from commercially available sources.

[0039] Table 1 shows the source information of the main raw materials and reagents in the examples: Table 1. Information on the sources of main raw materials and reagents in the embodiments.

[0040] Example 1: Preparation of 2-chloro-N6-cyclopentyladenosine The reaction pathway is as follows:

[0041] (1) 2,6-Dichloro-9H-purine (5 g, 26.6 mmol) was dissolved in 10 mL HMDS, and (NH4)2SO4 (0.7 g, 5.29 mmol) was added. The reaction was carried out at 130 °C for 3 hours under nitrogen protection. The reaction solution was concentrated, dissolved in 10 mL dry acetonitrile, and tetraacetylribose (9.2 g, 29.1 mmol) was added. TMSOTf (7.2 mL, 39.67 mmol) was added dropwise at 0 °C and stirred for 2 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution, diluted with ethyl acetate, and the reaction solution was transferred to a 250 mL separatory funnel. The solution was washed with saturated sodium chloride aqueous solution (50 mL × 2), dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The solution was purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V = 2:1) to obtain C-1 (7.35 g) white solid, with a yield of 62.1%.

[0042] 1 H NMR (400 MHz, Chloroform-d): δ 8.29 (s, 1H), 6.21 (d, J = 5.5 Hz,1H), 5.79 (t, J = 5.6 Hz, 1H), 5.58-5.56 (m, 1H), 4.49-4.47 (m, 1H), 4.41-4.41 (m, 2H), 2.17 (s, 3H), 2.14 (s, 3H), 2.09 (s, 3H). (2) C-1 (5 g, 11.2 mmol) was dissolved in 20 mL of methanol, and cyclopentylamine (1.1 mL, 12.32 mmol) and triethylamine (1.7 mL, 12.32 mmol) were added. The mixture was stirred overnight at room temperature. After the reaction was confirmed by TLC, a methanol solution of 7M amine (5 mL) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography using dichloromethane:methanol (V:V=15:1) to obtain CCPA (3.4 g) as a white solid, with a yield of 82.1%.

[0043] Example 2 Preparation of Intermediate I The reaction pathway is as follows:

[0044] CCPA (3 g, 8.13 mmol) was dissolved in 10 mL of acetone, and p-toluenesulfonic acid (7.9 g, 24.38 mmol) and 2,2-dimethoxypropane (12.5 mL, 101.5 mmol) were added. The reaction was carried out at room temperature, and the reaction was detected by TLC. The reaction solution was diluted with saturated sodium chloride solution and carefully quenched with saturated sodium bicarbonate solution. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to give intermediate I (3.25 g), a pale yellow solid, with a yield of 98.1%.

[0045] 1 H NMR (400 MHz, DMSO-d6): δ 8.35-8.32 (m, 2H), 6.06 (d, J = 2.6 Hz,1H), 5.29-5.26 (m, 1H), 5.08-5.05 (m, 1H), 4.94-4.92 (m, 1H), 4.22-4.18 (m,1H), 3.57-3.49 (m, 2H), 1.98-1.89 (m, 2H), 1.75-1.65 (m, 2H), 1.62-1.49 (m,7H), 1.33 (s, 3H). Example 3 Synthesis of CO2 The reaction pathway is as follows:

[0046] (a) Intermediate I (0.4 g, 0.98 mmol), imidazole (0.21 g, 3.1 mmol), and triphenylphosphine (0.54 g, 1.96 mmol) were dissolved in dry benzene (10 mL). After stirring for ten minutes, iodine (0.5 g, 1.96 mmol) was added, and the mixture was reacted at 80 °C for 1 hour. The reaction was stopped by TLC. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate and water, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA, V:V = 5:1) to obtain C-3a (0.42 g) as a yellow solid with a yield of 82.1%.

[0047] 1 H NMR (400 MHz, Chloroform- d): δ 7.80 (s, 1H), 6.04-6.03 (m, 1H), 5.35-5.33 (m, 1H), 5.09-5.07 (m, 1H), 4.58-4.51 (m, 1H), 4.41-4.37 (m, 1H), 3.55-3.51 (m, 1H), 3.29-3.26 (m, 1H), 2.14-2.07 (m, 2H), 1.75-1.63 (m, 4H), 1.58 (s, 3H), 1.54-1.46 (m, 2H), 1.37 (s, 3H). (b) Under nitrogen protection, diethyl phosphite (0.09 g, 0.64 mmol) was dissolved in dry DMF (10 mL), cesium carbonate (0.56 g, 1.71 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Then, a DMF solution of intermediate Ia (0.3 g, 0.57 mmol) was added, and the mixture was stirred at room temperature for 24 hours. The reaction was stopped by LTC detection. The reaction solution was poured into 100 mL of water, stirred for 15 min, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with dichloromethane:methanol (V:V = 50:1) to obtain C-3a-1 as a white solid of 0.17 g, with a yield of 62.2%.

[0048] ESI-MS: 530.4 [M+H] + , 552.3 [M+Na] + . 1 H NMR (400 MHz, Chloroform- d ): δ 7.86 (s, 1H), 5.99-5.98 (m, 1H), 5.41-5.39 (m, 1H), 5.13-5.12 (m, 1H), 4.62-4.55 (m, 2H), 4.07-3.99 (m, 4H), 2.45-2.22 (m, 2H), 2.16-2.09 (m, 2H), 1.78-1.72 (m, 2H), 1.70-1.63 (m, 2H), 1.58 (s, 3H), 1.55-1.46 (m, 2H), 1.37 (s, 3H), 1.26-1.21 (m, 6H). (c) Under nitrogen protection, C-3a-1 (0.2 g, 0.38 mmol) was dissolved in dry acetonitrile (5 mL), TEA (0.38 g, 3.8 mmol) was added, and TMS-Br (0.29 g, 1.89 mmol) was added at 0 °C. The reaction was stirred overnight at room temperature. The reactants were concentrated under reduced pressure, and the resulting residue was dissolved in 1,4-dioxane (4 mL). 0.1 mol / L H2SO4 (3 mL) was added, and the reaction was carried out at 60 °C for 4 hours. The mixture was then concentrated under reduced pressure, and the resulting residue was purified by reversed-phase semi-preparative high-performance liquid chromatography (RP-HPLC) using a ZORBAX SB-C18 column (5 μm 9.4 × 250 mm). The separation conditions were: gradient elution of the mobile phase, consisting of 0.1% TFA in water and acetonitrile in a ratio of 70:30 to 10:90 (v / v), and a flow rate of 1.5%. The fraction was collected at a rate of mL / min, then frozen and lyophilized to obtain the target compound CO2 as a white solid, with a yield of 23.1%.

[0049] The chemical name of CO2 is: {[(2S, 3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl]methyl}phosphonic acid, which... 1 H NMR and 13 The C NMR data are attributed as follows: 1 H NMR (600 MHz, DMSO- d 6): δ 8.41 (s, 1H), 8.31-8.30 (m, 1H), 5.79 (d, J = 5.6 Hz, 1H), 4.67-4.65 (m, 1H), 4.45-4.39 (m, 1H), 4.20-4.19 (m, 1H), 4.16-4.13 (m, 1H), 2.21-2.14 (m, 1H), 2.02-1.99 (m, 1H), 1.97-1.91 (m, 2H), 1.71-1.69 (m, 2H), 1.62-1.52 (m, 4H). 13 C NMR (151 MHz, DMSO- d 6) δ 154.1,152.7, 149.1, 139.4, 117.9, 86.5, 79.5, 73.1, 72.3, 51.1, 32.5, 31.7, 31.2,22.9(2C). Example 4 Synthesis of CO3 The reaction route is as follows:

[0050] (a) Intermediate I (0.2 g, 0.49 mmol) was added to diethyl vinylphosphonate (2 mL), followed by cesium carbonate (0.03 g, 0.098 mmol). The mixture was heated to 50 °C and reacted for 16 hours. The reaction was stopped by LTC detection. The mixture was cooled to room temperature, diluted with ethyl acetate (10 mL), filtered, and the reaction solution was concentrated. The solution was purified by silica gel column chromatography using dichloromethane:methanol (V:V = 70:1) to obtain C-3b as a white solid, 0.14 g, with a yield of 50.1%.

[0051] (b) The C-3b obtained in step a was deprotected in a manner similar to that in Example 3 to synthesize the title compound CO3.

[0052] The chemical name of CO3 is (2-{[(2R, 3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl]methoxy}ethyl)phosphonic acid, and its 1H NMR and 13C NMR data are assigned as follows: 1 H NMR (400 MHz, DMSO-d6): δ 8.39 (s, 1H), 8.35-8.29 (m, 1H), 5.83(d, J = 5.1 Hz, 1H), 4.50-4.48 (m, 1H), 4.43-4.38 (m, 1H), 4.15-4.12 (m, 1H), 4.03-4.01 (m, 1H), 3.67-3.56 (m, 4H), 1.96-1.85 (m, 4H), 1.74-1.68 (m, 2H), 1.60-1.52 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 155.1, 153.8, 150.0, 139.7,118.8, 87.8, 83.6, 74.2, 70.7, 70.3, 66.6, 52.2, 32.3, 32.3, 30.1, 23.9(2C). Example 5 Synthesis of CO4 The reaction pathway is as follows:

[0053] (a) Intermediate I (0.2 g, 0.49 mmol) was dissolved in dry THF (10 mL). NaH (0.04 g, 0.98 mmol) was slowly added at 0 °C and stirred for 30 min. Then, a THF solution of (diethoxyphosphoryl)methyltrifluoromethanesulfonate (0.18 g, 0.55 mmol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 12 hours. The reaction was detected by TLC to indicate completion. The reaction was quenched by adding saturated ammonium chloride solution (10 mL). The mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with dichloromethane:methanol (V:V = 50:1) to obtain C-3c as a white solid, 0.14 g, with a yield of 51.5%.

[0054] ESI-MS: 560.3 [M+H]+, 582.3 [M+Na]+. (b) C-3c was deprotected in a manner similar to that in Example 3 to obtain the title compound CO4.

[0055] The chemical name of C04 is: ({[(2R, 3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl]methoxy}methyl)phosphonic acid, which... 1 H NMR and 13 The C NMR data are attributed as follows: 1 H NMR (400 MHz, DMSO-d6): δ 8.41 (s, 1H), 8.36-8.29 (m, 1H), 5.84 (d, J = 5.9 Hz, 1H), 4.54-4.51 (m, 1H), 4.45-4.39 (m, 1H), 4.12-4.10 (m, 1H),4.05-4.02 (m, 1H), 3.74-3.68 (m, 2H), 3.62-3.60 (m, 2H), 2.01-1.88 (m, 2H),1.75-1.67 (m, 2H), 1.59-1.52 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 155.1,153.8, 150.2, 139.8, 118.6, 87.4, 84.1, 74.2, 73.2, 73.1, 71.2, 68.0, 66.9,52.1, 32.3, 30.2, 23.9(2C). Example 6 Synthesis of C05 The reaction route is as follows: (a) Compound C-3a (0.5 g, 0.96 mmol) prepared in Example 3 was dissolved in anhydrous DMF (10 mL), potassium thioacetate (0.28 g, 2.4 mmol) was added, and the reaction was stirred at 80 °C for 1 hour. The reaction was detected by TLC and the reaction was completed. The reactants were poured into 100 mL of water, stirred at room temperature for 20 min, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, the organic phase was concentrated, and purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V=4:1) to obtain 0.43 g of white solid C-3e, with a yield of 95.2%.

[0056] ESI-MS: 468.2 [M+H] + . (b) Compound C-3e (0.3 g, 0.64 mmol) was dissolved in anhydrous MeOH (10 mL), sodium methoxide (0.08 g, 1.6 mmol) was added, and the mixture was reacted overnight at room temperature. The reaction was detected by TLC and the reaction solution was concentrated under reduced pressure. The solution was purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V=1:1) to obtain 0.19 g of yellow oily substance C-3e-1, with a yield of 70.1%.

[0057] 1 H NMR (400 MHz, Chloroform- d ) δ 7.79 (s, 1H), 5.99-5.98 (m, 1H), 5.39-5.37 (m, 1H), 5.08-5.06 (m, 1H), 4.61-4.48 (m, 2H), 3.13-2.97 (m, 2H), 2.15-2.09 (m, 2H), 1.78-1.66 (m, 4H), 1.60 (s, 3H), 1.57-1.49 (m, 2H), 1.38(s, 3H), 1.27-1.24 (d, J = 14.2 Hz, 1H). (c) Under nitrogen protection, 5 mL of 30% H2O2 was dissolved in 7 mL of 88% formic acid in an ice-water bath at 0 °C and stirred for 1 hour at 0 °C to obtain performic acid. A 3 mL solution of C-3e-1 (0.2 g, 0.47 mmol) formic acid was slowly added dropwise to the performic acid solution, and the mixture was allowed to react at room temperature for 24 hours. The reaction was stopped by TLC, concentrated under reduced pressure, and the residue was purified by reversed-phase semi-preparative high-performance liquid chromatography (RP-HPLC) using a ZORBAX SB-C18 column (5 μm 9.4 × 250 mm). The separation conditions were: gradient elution of the mobile phase with a water and acetonitrile system containing 0.1% TFA in a ratio of 74:26 to 10:90 (v / v), and a flow rate of 1.5 mL / min. The desired fraction was collected, frozen, and lyophilized to obtain 103.5 mg of white solid CO5, with a yield of 50.8%.

[0058] C05 has the chemical name: {[(2R, 3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl]methoxy}sulfonic acid. 1 H NMR and 13 The C NMR data are attributed as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 8.51 (s, 1H), 5.78 (d, J = 6.0 Hz, 1H),4.67-4.65 (m, 1H), 4.41-4.33 (m, 2H), 4.25-4.22 (m, 1H), 3.09-3.05 (m, 1H),2.87-2.84 (m, 1H), 1.99-1.90 (m, 2H), 1.71-1.68 (m, 2H), 1.59-1.51 (m, 4H). 13 CNMR (151 MHz, DMSO- d 6) δ 154.6, 154.0, 149.9, 140.4, 117.9, 87.4, 81.9, 73.4,73.2, 55.1, 52.2, 33.4, 32.3, 23.9(2C). Example 7 Synthesis of C06 The reaction route is as follows:

[0059] (a) Intermediate I (0.2 g, 0.49 mmol) was dissolved in anhydrous DCM (10 mL), and Dysmartin oxidant (0.32 g, 0.73 mmol) was added in portions. The reaction was carried out at room temperature for 3 hours. The reaction was stopped by TLC. The reaction was quenched with sodium thiosulfate aqueous solution (10 mL) and sodium bicarbonate aqueous solution (10 mL). The mixture was extracted three times with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V=1:1) to give 0.16 g of white solid C4, yield 80.1%.

[0060] ESI-MS: 408.2 [M+H] + . (b) Under nitrogen protection, methyltriphenylphosphine bromide (0.77 g, 2.16 mmol) was added to dry tetrahydrofuran (10 mL). The system temperature was then lowered to 0 °C, and a 2 M solution of sodium bis(trimethylsilylamino)amino in tetrahydrofuran (1.1 mL) was slowly added dropwise while stirring for 1 hour. Subsequently, a solution of C-4 (0.3 g, 0.74 mmol) in dry tetrahydrofuran (10 mL) was added dropwise while maintaining the temperature at 0 °C. The mixture was then brought to room temperature and stirred overnight. After the reaction was completed by TLC, the reaction was extinguished with saturated ammonium chloride solution, extracted three times with ethyl acetate, and the organic layers were combined and washed with saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V = 3:1) to give 0.19 g of white solid C-4a, yield 63.7%.

[0061] ESI-MS: 406.3 [M+H] + , 428.3 [M+Na] + . (c) Under nitrogen protection, compound C-4a (0.2 g, 0.49 mmol) was dissolved in dry THF (5 mL), cooled to 0 °C, and 9-BBN (0.5 M in THF, 2.3 mL) was added dropwise. The mixture was stirred at room temperature for 1 hour. Then, saturated sodium bicarbonate solution (3 mL) was added dropwise at 0 °C, followed by hydrogen peroxide (2 mL). The mixture was stirred at 0 °C for 20 min, and then the mixture was transferred to room temperature for 1 hour. After the reaction was completed as detected by TLC, the mixture was extracted three times with ethyl acetate. The organic layers were combined and washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography on petroleum ether:ethyl acetate (V:V = 1:1) to give 10.15 g of white solid C-4a, yield 71.7%.

[0062] ESI-MS: 422.1 [MH] + . (df) The title compound C06 was synthesized from C-4a-1 by a method similar to step ac in Example 3.

[0063] C07 has the chemical name: {2-[(2R, 3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl]ethyl}phosphonic acid, which... 1 H NMR and 13 The C NMR data are attributed as follows: 1 H NMR (400 MHz, DMSO- d 6) :δ 8.23 ​​(s, 1H), 5.79 (d, J = 4.9 Hz, 1H),4.54-4.46 (m, 2H), 4.05 (t, J = 5.2 Hz, 1H), 3.93-3.88 (m, 1H), 2.00-1.87 (m,4H), 1.74-1.69 (m, 2H), 1.64-1.53 ​​(m, 6H). 13 C NMR (151 MHz, DMSO- d 6) δ 155.2,153.9, 150.1, 140.2, 118.9, 88.2, 84.3, 73.8, 72.3, 51.7, 33.5, 32.3, 27.3,24.7, 23.9(2C). Example 8 Synthesis of C07 The reaction route is as follows:

[0064] (a) Under nitrogen protection, compound C-4a (0.2 g, 0.49 mmol) synthesized in Example 7 was dissolved in dry THF (5 mL), and diethyl phosphite (0.1 g, 0.72 mmol) and potassium tert-butoxide (0.16 g, 1.44 mmol) were added. The reaction was carried out at room temperature for 3 hours. After the reaction was detected by TLC, the reaction was quenched with saturated ammonium chloride solution, extracted three times with ethyl acetate, the organic layers were combined and washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with petroleum ether:ethyl acetate (V:V=1:1) to give 0.14 g of white solid C-4b, yield 52.8%.

[0065] ESI-MS: 546.1 [M+H] + . 1H NMR (400 MHz, DMSO- d 6) δ 8.34-8.33 (m, 2H), 6.30-6.18 (m, 1H), 6.07(d, J = 3.0 Hz, 1H), 5.32-5.30 (m, 1H), 5.17-5.15 (m, 1H), 4.45-43.4 (m, 2H), 4.05-3.89 (m, 5H), 1.98-1.91 (m, 2H), 1.71-1.68 (m, 2H), 1.60-1.52 (m, 7H),1.33 (s, 3H), 1.18 (t, J = 7.0 Hz, 3H), 1.10 (t, J = 7.0 Hz, 3H). (b) C-4b was deprotected in a manner similar to that in Example 3 to obtain the target compound CO7.

[0066] C07 has the chemical name: {[(3S, 4R, 5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxyoxacyclopentan-2-yl](hydroxy)methyl}phosphonic acid. 1 H NMR and 13 The C NMR data are attributed as follows: 1 H NMR (600 MHz, DMSO- d 6) :δ 8.31 (s, 1H), 5.79 (d, J = 7.0 Hz, 1H),4.50-4.48 (m, 1H), 4.40-4.39 (m, 2H), 4.26-4.25 (m, 1H), 3.83-3.81 (m, 1H),1.97-1.90 (m, 2H), 1.71-1.67 (m, 2H), 1.59-1.53 ​​(m, 4H). 13 C NMR (151 MHz, DMSO- d 6) δ 155.0, 153.6, 149.9, 140.1, 118.8, 87.1, 74.9, 70.1(2C), 68.5,52.1, 33.4, 32.2, 23.9(2C). Experiment 1: Inhibition of Triglyceride Accumulation in Hepatocytes Cell source and culture: Normal mouse hepatocytes AMI-12 were seeded into 12-well cell culture plates and cultured in DMEMF12 medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0067] Cell model establishment and drug treatment: When the cell confluence reaches 50-60%, the following grouping treatments are performed (each group has 3 replicates): ① Solvent control group: Contains only culture medium and corresponding drug solvent; ② Model group: Glycerol (20 mM) + glucose (4.5 g / L) were added to the culture medium to establish a hepatocyte lipid accumulation model; ③ Derivative intervention group: Based on the modeling, target derivatives CO2, CO3, CO4, CO5, CO6 or CO7 (administered concentration 1 μM) were added respectively. ④ Positive control group: Based on the modeling, the control compound CCPA (administered concentration 1 μM) was added.

[0068] Indicator Testing: Cells were collected 48 h after drug intervention. Using a commercially available triglyceride assay kit, cells were lysed and intracellular TG levels were measured according to the manufacturer's instructions. TG levels in the CCPA group were used as 100% normalization; TG levels of other CCPA derivatives were calculated using CCPA as the standard. The calculation formula is as follows: TG% = CCPA TG value / Derivative TG value × 100%.

[0069] The experimental results are shown in Table 2. It can be seen that derivatives CO2, CO3, CO4, CO5, CO6 and CO7 can all reduce the triglyceride (TG) content in cells. Among them, CO2 has better activity than CCPA.

[0070] Table 2 Effects of compounds CO2-CO7 on triglycerides (TG)

[0071] Experimental Example 2: MASH Animal Model Experiment Experimental Methods: Thirty-six male C57BL / 6J mice were selected and, after acclimatization, were subjected to a CD-HFD (choline-deficient high-fat diet) intervention for 6 weeks. The mice were divided into the following groups: ① Normal diet group, ② CD-HFD diet group, ③ CD-HFD diet + CCPA group, ④ CD-HFD diet + low-dose CO2 group, and ⑤ CD-HFD diet + high-dose CO2 group. After 3 weeks of dietary intervention, while continuing the corresponding diet, groups ③, ④, and ⑤ were given CCPA (4 mg / kg), low-dose CO2 (2.5 mg / kg), and high-dose CO2 (5 mg / kg), respectively, for a total of 6 weeks. The experimental grouping and procedure are detailed below. Figure 2 A.

[0072] At the end of the experiment, mice were fasted overnight but allowed free access to water. They were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and blood was collected from the orbital fossa. Samples were collected from the liver, epididymal fat, subcutaneous fat, and brown fat. Small pieces of liver lobe tissue were fixed in 4% neutral paraformaldehyde fixative for histopathological examination. Whole blood was incubated at room temperature for 30 min, then centrifuged at 4°C, 4000 rpm for 15 min, and the supernatant serum was collected. The remaining samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for later use. The collected blood and tissue samples were tested for the following indicators: (1) Weighing the liver (2) Detection of serum ALT, AST, ALP and IL-6 levels Serum ALT, AST, ALP, and IL-6 levels were detected using either an enzymatic method or an ELISA. Refer to the kit instructions for specific methods.

[0073] (3) Liver histopathological analysis Liver samples fixed in 4% paraformaldehyde were dehydrated, paraffin-embedded, sectioned, and stained with H&E for pathological evaluation of hepatic steatosis. NAS scores were calculated based on hepatic steatosis, inflammasomes, and ballooning degeneration.

[0074] (4) Detection of gene and protein expression in liver tissue 15-20 mg of liver tissue was collected, total RNA was extracted, and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect liver tissue inflammation-related factors. Il-6 and Il-1β Chemokines ( Ccl2 and Cxcl9 Information on the primers used for detection is shown in Table 3.

[0075] Table 3 Primer List

[0076] Liver proteins were extracted, and the content of α-SMA protein, a marker protein of myofibroblasts, in liver tissue was detected by Western blot.

[0077] The experimental results are shown in Figure 2 The results showed that the compound (CO2) provided by this invention can significantly reduce liver weight and NAS index (C02). Figure 2 C and 2D), inflammatory factors ( Il-1β and Il-6 ), chemokines ( Ccl2 and Cxcl9 mRNA expression level of ) Figure 2 G-2J indicates that it has a strong anti-inflammatory effect and can inhibit the recruitment and activation of immune cells in the liver. CO2 can significantly reduce the levels of liver injury markers ALT, AST and ALP in serum, while also reducing serum IL-6 levels (G-2J). Figure 2 K-2N has been shown to effectively protect hepatocyte function and alleviate systemic inflammation. CO2 can significantly reduce the expression of α-SMA, a key marker of liver fibrosis. Figure 2 (E-2F), suggesting that it can exert an anti-liver fibrosis effect by inhibiting the activation of hepatic stellate cells. The above results indicate that the compound CO2 of this invention can improve multiple pathological manifestations of MASH, including steatosis, inflammation, liver damage, and fibrosis.

[0078] Experimental Example 3: Mouse Open Field Experiment Experimental Methods: Eight-week-old male SPF-grade C57BL / 6 mice, weighing 20±2 g, were randomly assigned to groups after 7 days of acclimatization. Open field tests were conducted in a 40 cm × 40 cm × 40 cm cube-shaped open box with black inner walls, the bottom of which was divided into 16 equal compartments. The experiment was conducted from 8:00 to 12:00 in a soundproof room with 50 lx illumination. One hour before the experiment, the animals were moved to the testing room and allowed to rest. Mice in the normal group, CCPA group, and CO2 group were intraperitoneally injected with saline, CCPA (4 mg / kg), and CO2 (4 mg / kg), respectively. They were then gently placed in the center of the box and allowed to explore freely for 5 minutes. The Smart 3.0 video tracking system recorded their movement trajectories in real time. See the experimental grouping and flowchart below. Figure 3 A.

[0079] The experimental results are shown in Figure 3 B. The results showed that in the open field experiment, mice in the CCPA group experienced drowsiness and their spontaneous activity was significantly inhibited, while mice in the CO2 group did not experience drowsiness and their spontaneous activity was not significantly inhibited. This indicates that CCPA has significant central nervous system side effects such as drowsiness, while the compound (CO2) provided by this invention has virtually no central nervous system side effects such as drowsiness.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The 2-chloro-N6-cyclopentyl adenosine derivative shown in formula (Ⅰ): In equation (Ⅰ), m = 0, 1, 2, 3 or 4, n = 0, 1, 2, 3 or 4, and X in the connecting chain is selected from any one of bond, NH, O or S; In formula (I), R2 is selected from any one of phosphoric acid, sulfonic acid, phosphate or sulfonate groups, and R1 is selected from any one of hydroxyl, halogen, hydrogen or amino groups.

2. The 2-chloro-N6-cyclopentyladenosine derivative according to claim 1, characterized in that, The 2-chloroN6-cyclopentyladenosine derivative is selected from any one of the following formulas CO2-CO7: 。 3. A method for preparing the 2-chloro-N6-cyclopentyladenosine derivative according to any one of claims 1-2, characterized in that... Includes the following steps: (1) 2-chloro-N6-cyclopentyl adenosine reacts with 2,2-dimethoxypropane in the presence of an acid catalyst to undergo an acetone-protected reaction to obtain intermediate I; (2) Activate the 5'-hydroxyl group of intermediate I obtained in step (1) and convert it into an intermediate with a phosphorus- or sulfur-containing functional group attached to the carbon at the 5' position; (3) The intermediate with phosphorus or sulfur functional groups attached to the 5' carbon obtained in step (2) is subjected to a deprotection reaction to obtain a compound as shown in general formula (I).

4. The preparation method according to claim 3, characterized in that, The method for activating the 5'-hydroxyl group of intermediate I and converting it into an intermediate with a phosphorus- or sulfur-containing functional group attached to the 5' carbon in step (2) is selected from any of the following: (a) Albuzov reaction pathway: After converting the 5' hydroxyl group into a leaving group, it undergoes an Albuzov reaction with a trivalent phosphorus compound to introduce a phosphoryl substituent; (b) Michael addition pathway: Under base catalysis, the 5' hydroxyl group or its activated form undergoes a conjugate addition reaction with α,β-unsaturated phosphonates; (c) Direct alkylation route: Under base catalysis, the 5' hydroxyl group undergoes an SN2 substitution reaction with a haloalkylphosphonate; (d) Carbonyl addition pathway: After oxidizing the 5' hydroxyl group to an aldehyde group, it undergoes a nucleophilic addition reaction with a phosphite; (e) Sulfation-oxidation pathway: The 5' hydroxyl group is converted into a leaving group, which is then substituted with a thiocarboxylate and oxidized with a peroxide to introduce a sulfonic acid group.

5. The method according to claim 4, wherein the (a) Albuzov reaction pathway comprises: The 5'-hydroxyl group is converted to a halogenated product using a triphenylphosphine / halogen system, sulfoxide, or phosphohalogenated reagent; the halogenated product is then reacted with a compound selected from triphenylphosphine (C... 1-6 Alkyl esters, triaryl phosphites, di(C) phosphites 1-6 Reactions of trivalent phosphorus compounds with alkyl esters, diaryl phosphites, or alkyl phosphinates; The Michael addition pathway (b) includes: the reaction of the 5'-hydroxyl group with diethyl vinylphosphonate, dimethyl vinylphosphonate or aryl-substituted vinylphosphonate in the presence of cesium carbonate, sodium hydride, potassium tert-butoxide or DBU; The (c) direct alkylation pathway includes: reacting the 5'-hydroxyl group with diethoxyphosphorylmethyltrifluoromethanesulfonate, diethoxyphosphorylethyl bromide, or (diethoxyphosphoryl)propyl iodine in the presence of sodium hydride, cesium carbonate, or potassium tert-butoxide; The (d) carbonyl addition pathway includes: oxidizing the 5'-hydroxyl group to an aldehyde group using a Desmartin oxidant, PCC, or Swern reagent; and nucleophilic addition of the aldehyde group to diethyl phosphite, dimethyl phosphite, or diphenyl phosphite in the presence of potassium tert-butoxide, sodium hydride, or LDA. The (e) sulfidation-oxidation pathway includes: the 5'-hydroxyl group is activated by iodination or methanesulfonation, and then reacts with potassium thioacetate, thiourea or sodium hydrosulfide to generate a thio intermediate; the thio intermediate is oxidized to a sulfonic acid group by oxyacid, hydrogen peroxide or m-chloroperoxybenzoic acid.

6. The method according to claim 4, characterized in that, The leaving group is selected from iodine, bromine, chlorine, methanesulfonyloxy, p-toluenesulfonyloxy, or trifluoromethanesulfonyloxy.

7. The method according to any one of claims 3-5, characterized in that, The deprotection process in step (3) includes: first removing the alkyl protecting group on the phosphoryl or sulfonyl group with trimethylbromosilane, and then removing the acetone-containing protecting group with dilute acid.

8. Use of any 2-chloro-N6-cyclopentyladenosine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the preparation of a medicament for treating liver diseases.

9. The application according to claim 8, characterized in that, The liver disease mentioned is metabolic dysfunction-related fatty liver disease.

10. A pharmaceutical composition for treating metabolic dysfunction-associated steatohepatitis, comprising a therapeutically effective amount of the 2-chloro-N6-cyclopentyladenosine derivative or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

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