Pyrimidine derivative or pharmaceutically acceptable salt and application thereof

Abstract

The invention discloses a pyrimidine derivative or a pharmaceutically acceptable salt thereof. The general formula of the pyrimidine derivative is shown as a formula I; wherein n is 1 or 2, m is 0 or 1, and l is 1 or 2; x1 is selected from a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a halogen C1-C6 alkyl group, and a C3-C6 cycloalkyl group; in the formula, R3 is selected from H, halogen, nitryl, cyano, halogen C1-C6 alkyl and C1-C6 alkoxy; y1 and Y2 are independently selected from C, N, O and S; r1 and R2 are independently selected from H, halogen, nitryl, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, halogen C1-C6 alkyl, C1-C6 alkoxy, carbamoyl and ester group. The compound can inhibit the inducible nitric oxide synthase and is used for preparing a medicine for inhibiting the inducible nitric oxide synthase.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry. More specifically, this invention relates to a pyrimidine derivative or a pharmaceutically acceptable salt thereof and its applications. Background Technology

[0002] Nitric oxide (NO), as a gaseous endogenous biological messenger, participates in the regulation of various physiological functions such as nerve conduction and immune response, and is exclusively produced by the nitric oxide synthase (NOS) pathway. The NOS family comprises three members: neural NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). The first two are present and expressed under normal physiological conditions. iNOS, however, is not expressed under normal circumstances. When stimulated by inflammatory signals, the iNOS reductase domain transfers electrons provided by the co-substrate nicotinamide adenine dinucleotide phosphate (NADPH) to the oxygenase domain's (6R-)5,6,7,8-tetrahydro-L-biopterin (BH4) and heme iron via cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). This process oxidizes the substrate L-arginine (L-Arg) and molecular oxygen (O2) into L-citrulline (L-Cit) and NO. Excess NO reacts with superoxide radicals to generate various reactive nitrogen species (RNS), triggering inflammation and oxidative stress, and exacerbating the progression of diseases such as rheumatoid arthritis (RA), neurodegenerative diseases, tumors, diabetes, and inflammatory bowel disease. Studies have confirmed that RA patients not only have increased iNOS mRNA and protein expression in synovial cells, but also significantly higher levels of nitrates in urine, nitrites in serum, and 3-nitrotyrosine than the control group. This indicates that NO can synergistically activate matrix metalloproteinases with various cytokines, reduce the synthesis of proteoglycans and collagen, promote chondrocyte apoptosis, and destroy cartilage tissue. In other words, NO participates in the "synovial-immune-bone destruction" triple pathological network, accelerating the progression of RA. The use of NO production inhibitors or iNOS inhibitors has also been shown to have cartilage protection and damage repair effects, cleverly supplementing the "NO-dependent osteoclastosis" pathway that targeted synthetic antirheumatic drugs (tsDMARDs) and JAK inhibitors cannot cover. Therefore, the development of NO production inhibitors or iNOS inhibitors can provide new strategies for RA drug resistance or combination therapy. Summary of the Invention

[0003] One object of the present invention is to provide a pyrimidine derivative or a pharmaceutically acceptable salt thereof capable of inhibiting inducible nitric oxide synthase, and for use in the preparation of a medicament that inhibits inducible nitric oxide synthase.

[0004] In order to achieve these objects and other advantages of the present invention, according to one aspect of the present invention, the present invention provides pyrimidine derivatives or pharmaceutically acceptable salts thereof, the general formula of which is shown in Formula I; Where n is 1 or 2, m is 0 or 1, and l is 1 or 2; X1 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogenated C1-C6 alkyl, and C3-C6 cycloalkyl; X2 is R3 is selected from H, halogen, nitro, cyano, halogen C1-C6 alkyl, and C1-C6 alkoxy. Y1 and Y2 are independently selected from C, N, O and S; R1 and R2 are independently selected from H, halogen, nitro, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, halogen C1-C6 alkyl, C1-C6 alkoxy, carbamoyl, and ester.

[0005] According to another aspect of the invention, the use of pyrimidine derivatives or pharmaceutically acceptable salts thereof for the preparation of medicaments that inhibit inducible nitric oxide synthase is also provided.

[0006] According to another aspect of the invention, the use of pyrimidine derivatives or pharmaceutically acceptable salts thereof for the preparation of medicaments for treating rheumatoid arthritis is also provided.

[0007] The present invention has at least the following beneficial effects: This invention features a novel structure that, through precise control of substituents and ring systems, significantly enhances the selective inhibitory activity of inducible nitric oxide synthase (iNOS). In vitro experiments show that some compounds exhibit IC50 values ​​for NO production and iNOS expression. 50 With a concentration of 1 μM or less, these compounds effectively block excessive NO production and inflammation and oxidative stress induced by reactive nitrogen species (RNS). They target the NO-dependent osteoclast pathway, filling a gap in existing antirheumatic drugs such as JAK inhibitors, achieving cartilage protection and repair, and effectively intervening in the synovial-immune-bone destruction pathological network of rheumatoid arthritis (RA). Compared to traditional arginine analogues and other inhibitors, these compounds have fewer side effects, providing a new strategy for RA drug-resistant patients and combination therapy. They also lay the foundation for the treatment of NO-related diseases such as neurodegenerative diseases and inflammatory bowel disease, showing broad clinical application prospects.

[0008] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0009] Figure 1 The Western blotting results of A38 and A39 on LPS-induced iNOS expression in RAW264.7 cells are shown. Detailed Implementation

[0010] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0011] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0012] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0013] Embodiments of this application provide pyrimidine derivatives or pharmaceutically acceptable salts thereof, the general formula of which is shown in Formula I; Where n is 1 or 2, m is 0 or 1, and l is 1 or 2; X1 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogenated C1-C6 alkyl, and C3-C6 cycloalkyl; X2 is... R3 is selected from H, halogen, nitro, cyano, halogen C1-C6 alkyl, C1-C6 alkoxy; Y1 and Y2 are independently selected from C, N, O and S; R1 and R2 are independently selected from H, halogen, nitro, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, halogen C1-C6 alkyl, C1-C6 alkoxy, carbamoyl, ester.

[0014] Among C1-C6 alkyl groups, methyl and ethyl are typical short-chain alkyl groups. Methyl, as a C1 alkyl group, has a simple molecular structure and is easy to introduce, allowing for moderate adjustment of the compound's hydrophobicity. Ethyl, as a C2 alkyl group, has a slightly longer carbon chain than methyl, allowing for fine-tuning of the binding space between the molecule and the target site without significantly increasing molecular volume. Among C2-C6 alkenyl groups, vinyl is a common C2 alkenyl group. Its unsaturated double bonds can introduce flexible sites into the compound structure and may enhance the binding ability with target proteins through π-π interactions. Among C2-C6 alkynyl groups, ethynyl, as a C2 alkynyl group, has a linear triple bond structure that can improve molecular rigidity, suitable for binding scenarios requiring specific conformations. Among halogenated C1-C6 alkyl groups, trifluoromethyl and chloromethyl are halogen-substituted C1 alkyl groups. The strong electronegativity of trifluoromethyl significantly alters the electron cloud distribution of the molecule, while chloromethyl retains the simple structure of methyl while introducing halogen atoms. 2-Fluoroethyl, as a halogen-substituted C2 alkyl group, has a slightly longer carbon chain, allowing for a smoother transfer of the electronic effects of halogen atoms. In C3-C6 cycloalkyl groups, cyclopropyl and cyclobutyl are commonly used cyclic structures. The three-membered ring structure of cyclopropyl has less steric hindrance, allowing the introduction of cyclic segments into the molecule without excessively affecting the overall conformation. The four-membered ring of cyclobutyl is slightly more flexible than that of cyclopropyl, and can be selected according to the spatial requirements of the target binding pocket.

[0015] When R3 is H, the benzene ring remains unsubstituted, forming the basic structure of X2 and serving as a reference for other substitution forms. If R3 is a halogen, the meta-fluorine atom often appears in the meta position of the benzene ring. The small size and strong electronegativity of the fluorine atom regulate the electronic properties of the benzene ring without significantly increasing steric hindrance. When the ortho-chlorine atom is substituted in the ortho position, its atomic radius is moderate, allowing it to influence molecular activity through electronegativity without hindering the binding of the molecule to the target site due to excessive steric hindrance. When the para-bromine atom is in the para position, its electronegativity and size can create specific steric and electronic effects, adapting to the binding requirements of different targets. If R3 is a nitro group, the meta-nitro group is a common substituent in the meta position of the benzene ring. The strong electron-withdrawing property of the nitro group can significantly change the electron cloud density of the benzene ring, enhancing the polar interaction between the molecule and the target site. When the ortho-nitro group is in the ortho position, in addition to the electron-withdrawing effect, it may also influence the conformation of the molecule through steric hindrance, thereby optimizing the binding activity. If R3 is a cyano group, the meta-cyano and para-cyano groups introduced at the meta and para positions of the benzene ring, respectively, can form specific interactions with hydrogen bond donors in the target protein due to the strong electron-withdrawing properties and linear structure of the cyano group, thereby enhancing the binding affinity. If R3 is a halogenated C1-C6 alkyl group, the strong hydrophobicity and electronegativity of the trifluoromethyl group at the para position of the benzene ring can enhance the lipophilicity of the molecule while regulating the electronic properties of the benzene ring; the combination of the electronegativity of the chlorine atom and the hydrophobicity of the methyl group at the meta position can balance the polarity and lipophilicity of the molecule. If R3 is a C1-C6 alkoxy group, the introduction of a methoxy group at the para position of the benzene ring can increase the electron cloud density of the benzene ring due to the electron-donating effect of the methoxy group, which is suitable for binding scenarios requiring an electron-rich benzene ring structure; the electron-donating effect of the ethoxy group at the meta position is slightly weaker than that of the methoxy group, and can be finely adjusted according to electron requirements.

[0016] For R1 and R2, H is the most basic substitution form. When R1 or R2 is H, no other substituents are attached to the corresponding site, which reduces steric hindrance and makes the effects of other substituents more prominent. Among halogen-based R1 or R2, chlorine and bromine atoms are commonly chosen. Chlorine atoms have a moderate atomic radius and electronegativity, which can introduce halogen effects while avoiding excessive steric hindrance; bromine atoms have a slightly larger atomic radius and electronegativity similar to chlorine atoms, making them suitable for binding sites requiring slightly larger space. When nitro is used as R1 or R2, its strong electron-withdrawing property can significantly alter the electron distribution of the molecule, enhancing the interaction between the molecule and positive sites in the target protein, especially in binding scenarios requiring strong polar interactions. When cyano is used as R1 or R2, the linear cyano structure can penetrate deep into the narrow binding pocket of the target protein, while its strong electron-withdrawing property can optimize the electronic properties of the molecule and improve binding specificity. In C1-C6 alkyl groups, the methyl group is the most commonly used short-chain alkyl group, which can moderately increase the hydrophobicity of the molecule and has little steric hindrance, hardly affecting the overall conformation of the molecule. The ethyl group, compared to the methyl group, has a slightly longer carbon chain, allowing for more flexible adjustment of hydrophobicity. In C2-C6 alkenyl groups, the propynyl group, as a C3 alkenyl group, contains a double bond structure that can increase the degree of unsaturation of the molecule, potentially enhancing binding to the target site through π-π stacking. In C2-C6 alkynyl groups, the propynyl group, as a C3 alkynyl group, has a rigid triple bond structure that can fix the local conformation of the molecule, suitable for targets requiring specific conformations for binding. In C3-C6 cycloalkyl groups, the cyclopropyl group's three-membered ring structure can introduce cyclic segments into the molecule, increasing structural diversity, while the small size of the cyclopropyl group does not excessively hinder binding. Among the R1 or R2 groups of halogenated C1-C6 alkyl groups, trifluoromethyl's strong hydrophobicity and electronegativity significantly enhance the lipophilicity and electronic effects of the molecule, making it suitable for targets with high lipophilicity requirements. Pentafluoroethyl, compared to trifluoromethyl, has more halogen substitutions, resulting in stronger hydrophobicity and electronegativity, and can be selected according to specific needs. Among the R1 or R2 groups of C1-C6 alkoxy groups, methoxy and ethoxy are common electron-donating groups. Methoxy has a stronger electron-donating effect, while ethoxy is slightly weaker; the electronic properties of the molecule can be finely tuned by the difference between the two. Among the R1 or R2 groups of carbamoyl groups, formamido and acetamiprido are commonly used carbamoyl forms. Formamido is composed of an amino group and a methyl carbonyl group, while acetamiprido is composed of an amino group and an ethyl carbonyl group. Both can form hydrogen bonds through the amino and carbonyl groups, enhancing the hydrogen bonding interaction between the molecule and the target. In the R1 or R2 groups of ester groups, the methyl ester group and the ethyl ester group are typical ester group structures. The methyl ester group is formed by the carboxyl group and methanol, and the ethyl ester group is formed by the carboxyl group and ethanol. The presence of the ester group can increase the polarity of the molecule, and at the same time, it may release the active group through hydrolysis by esterase in vivo, thereby improving the bioavailability of the drug.

[0017] The value of n is 1 or 2, mainly corresponding to the structural type of the core ring system in the molecule (such as nitrogen-containing heterocycles like piperidine and pyrrolidine rings). When n=1, the corresponding ring system is usually a six-membered nitrogen-containing heterocycle, such as the piperidine ring. This type of six-membered ring structure has moderate flexibility, with carbon and nitrogen atoms evenly distributed on the ring. This allows the molecule to slightly adjust its conformation to fit the spatial morphology of the binding pocket when binding to the target site. At the same time, the stability of the six-membered ring also ensures that the molecule is not easily deformed in bulk, maintaining the active conformation. When n=2, the corresponding ring system is mostly a five-membered nitrogen-containing heterocycle, such as the pyrrolidine ring. Compared with the six-membered ring, the five-membered ring is more rigid, and the bond angles between atoms in the ring are smaller. This structural feature is suitable for scenarios where the target binding pocket space is relatively compact. It can form a precise interaction with the target through a fixed conformation, reducing the decrease in binding affinity caused by conformational changes. At the same time, the small volume of the five-membered ring can also reduce the overall steric hindrance of the molecule, facilitating entry into narrow binding regions. The value of m is 0 or 1, representing the number of spacer atoms between X2 (benzene ring) and adjacent nitrogen atoms in the molecule. When m=0, the X2 benzene ring is directly connected to the nitrogen atom without any additional spacer atoms. This connection method makes the benzene ring closer to the core structure of the molecule, and the substituents on the benzene ring (such as the nitro group corresponding to R3, halogens, etc.) can interact more directly with the amino acid residues of the target protein. For example, the nitro group at the meta position of the benzene ring can directly form a polar interaction with the hydrogen bond donor of the target, reducing the attenuation of the interaction strength caused by the spacer atoms. At the same time, the direct connection can also make the molecular structure more compact and reduce the overall steric hindrance. When m=1, there will be a spacer atom between the X2 benzene ring and the nitrogen atom. The most common spacer atom is CH2 (methylene). The introduction of methylene can provide a certain degree of flexibility to the benzene ring, allowing the benzene ring to rotate slightly around the methylene bond, thereby better adapting to different spatial orientations in the target binding pocket. Especially when there is a certain angle of indentation in the target binding region, the flexibility of methylene can help the benzene ring adjust its position to ensure that the substituents on the ring form effective binding with the target. At the same time, methylene can also moderately increase the overall length of the molecule without significantly changing the molecular polarity, adapting to a slightly longer binding channel.

[0018] In the l=1 scenario, the thiophene ring, as a typical five-membered heterocycle, has two key atoms constituting Y1 and Y2, both of which are carbon atoms. The aromaticity of the thiophene ring can also enhance the hydrophobic interaction between the molecule and the target. The furan ring is also a five-membered heterocycle, with Y1 being an oxygen atom and Y2 being an adjacent carbon atom. The presence of the oxygen atom can increase the polarity of the molecule and may form additional hydrogen bonds. The pyrazole ring, as a five-membered heterocycle containing two nitrogen atoms, has Y1 and Y2 corresponding to the two nitrogen atoms on the ring. The presence of the two nitrogen atoms can provide multiple hydrogen bond binding sites for the molecule, enhancing its binding ability to the target. When l=2, the benzene ring is the most common six-membered aromatic ring, with any two key atoms on its ring serving as Y1 and Y2 being carbon atoms. The aromaticity and structural stability of the benzene ring can provide a stable hydrophobic core for the molecule, facilitating binding to the target.

[0019] The embodiments of this application provide the application of pyrimidine derivatives or pharmaceutically acceptable salts thereof for the preparation of drugs that inhibit inducible nitric oxide synthase. For example, oral tablets, using the pyrimidine derivatives in the above embodiments and employing their pharmaceutically acceptable salt forms (such as hydrochloride) to improve water solubility, facilitate in vivo absorption, and effectively block iNOS-mediated NO generation. Excipient composition: Contains fillers (such as lactose, to improve tablet formability and taste), binders (such as microcrystalline cellulose, to enhance tablet hardness), and lubricants (such as magnesium stearate, to prevent sticking during compression). All excipients meet pharmaceutical standards and have high safety. Preparation steps: 1. Place the active ingredient and excipients in a mixer and mix at a uniform speed until the powder is homogeneous; 2. Add an appropriate amount of purified water to form a soft mass, which is then granulated by sieving; 3. Dry the wet granules at a suitable temperature and then granulate; 4. Add the lubricant and mix again; 5. Compress the mixed granules into tablets using a tablet press to obtain the final product.

[0020] This application provides embodiments for the use of pyrimidine derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating rheumatoid arthritis. For example, a topical gel, using a pyrimidine derivative selected from the above embodiments and in its pharmaceutically acceptable salt form (such as mesylate), enhances skin permeability and better targets local joint lesions. Excipient composition: Contains a gel matrix, humectant, pH adjuster, and purified water. The excipients are mild and non-irritating, suitable for skin application. Preparation steps: 1. Add the gel matrix to purified water, stir, and allow to swell fully to form a transparent gel matrix; 2. Add the humectant and preservative to the matrix and stir until dissolved; 3. Add the active ingredient and stir until homogeneous; 4. Add the pH adjuster to a suitable pH and stir until the gel is fine and free of particles; 5. Add purified water to the specified volume, stir evenly, and dispense to obtain the final product. The gel can directly act on the diseased joint, achieving a high local drug concentration. It can specifically inhibit the iNOS activity of synovial cells, reduce cartilage damage, and avoid the gastrointestinal side effects of oral medications.

[0021] The following is a description of a specific embodiment.

[0022] Tables 1-11 provide specific pyrimidine derivatives.

[0023] Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Example 1: Preparation method of some pyrimidine derivatives To facilitate the description of the synthetic routes and methods in the examples later, the abbreviations of the raw materials or reagents used are listed in the table below.

[0024] Table 12 Reagents and Abbreviations General formula for reaction equation 1: Reagents and conditions: a) Pd(PPh3)4, boric acid derivatives, etc., K2CO3, 1,4-dioxane / water, reflux; b) HX-NHBoc, DIEA, DMF, 120°C; c) HCl, EA, room temperature; d) benzyl bromide reagent, DIEA, DMF, room temperature; e) acyl chloride reagent, DIEA, DMF, room temperature.

[0025] Implementation Case A01: At room temperature, intermediate 1a (1.0 eq), boric acid derivative (1.1 eq), Pd(PPh3)4 (0.05 eq), and K2CO3 (3.0 eq) were added to a three-necked flask. After purging the air in the flask multiple times with N2, a mixed solvent of 1,4-dioxane / H2O (7 / 3, v / v) was injected into the flask using a syringe. The mixture was heated under reflux for approximately 5 h. Insoluble matter was removed by filtration with diatomaceous earth, and the filtrate was collected and removed under reduced pressure. The filtrate was extracted with EtOAc and water, and the oil layer was collected. The oil layer was then washed three times with saturated brine, dried over anhydrous Na2SO4, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and product 2a was purified using a normal-phase silica gel column chromatography.

[0026] At room temperature, intermediate 2a (1.0 eq), HX-NHBoc (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask, along with DMF solvent. The mixture was heated at 120°C for 3 h. The filtrate was extracted with EtOAc and water, and the oil layer was collected. The oil layer was then washed three times with saturated brine, dried over anhydrous Na2SO4, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and product 3a was purified using a normal-phase silica gel column chromatography.

[0027] At room temperature, intermediate 3a (1.0 eq) was added to a three-necked flask, followed by EA solvent containing saturated HCl. The reaction was carried out at room temperature for 5 h. The solvent was evaporated and the product was dried under vacuum to obtain product 4a.

[0028] At room temperature, intermediate 4a (1.0 eq), m-nitrobenzyl bromide (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask. DMF solvent was added, and the reaction was carried out at room temperature for 2 h. Acetyl chloride reagent (1.2 eq) was then added, and the reaction was carried out at room temperature for 1 h. The filtrate was extracted with DCM and water, the oil layer was collected, and the oil layer was washed three times with saturated brine. The oil layer was dried over anhydrous Na₂SO₄, the liquid was collected by filtration, the oil layer was evaporated to dryness, and the product A01 was purified using a normal-phase silica gel column chromatography.

[0029] Implementation Case A02: At room temperature, intermediate 4a (1.0 eq), m-nitrobenzyl bromide (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask. DMF solvent was added, and the reaction was carried out at room temperature for 2 h. Acryloyl chloride reagent (1.2 eq) was then added, and the reaction was carried out at room temperature for 1 h. The filtrate was extracted with DCM and water, the oil layer was collected, and the oil layer was washed three times with saturated brine. The oil layer was dried over anhydrous Na₂SO₄, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and the product A₂ was purified using a normal-phase silica gel column chromatography.

[0030] Implementation Case A34: At room temperature, intermediate 4a (1.0 eq), 3-chlorobenzyl bromide (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask. DMF solvent was added, and the reaction was carried out at room temperature for 2 h. Then, 3-chloropropionyl chloride reagent (1.2 eq) was added, and the reaction was carried out at room temperature for 1 h. The filtrate was extracted with DCM and water, the oil layer was collected, and the oil layer was washed three times with saturated brine. The oil layer was dried over anhydrous Na₂SO₄, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and the product A34 was purified using a normal-phase silica gel column chromatography.

[0031] General equation for reaction 2: Reagents and conditions: a) Dichloropyrimidine derivatives, aminopyrazole derivatives, etc., K2CO3, 1,4-dioxane, 80°C; b) HX-NHBoc, DIEA, DMF, 120°C; c) HCl, EA, room temperature; d) Benzyl bromide reagent, DIEA, DMF, room temperature; e) Acyl chloride reagent, DIEA, DMF, room temperature.

[0032] At room temperature, intermediate 5a (1.0 eq), aminopyrazole derivative (1.2 eq), and K₂CO₃ (5.0 eq) were added to a round-bottom flask. Solvent 1,4-dioxane was added, and the reaction was carried out at 80°C for 2 h. The filtrate was extracted with EtOAc and water, the oil layer was collected, and the oil layer was washed three times with saturated brine. The oil layer was dried over anhydrous Na₂SO₄, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and product 6a was purified using a normal-phase silica gel column chromatography.

[0033] At room temperature, intermediate 6a (1.0 eq), HX-NHBoc (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask, along with solvent DMF. The mixture was heated at 120 °C for 3 h. The filtrate was extracted with EtOAc and water, and the oil layer was collected. The oil layer was then washed three times with saturated brine, dried over anhydrous Na2SO4, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and product 7a was purified using a normal-phase silica gel column chromatography.

[0034] At room temperature, intermediate 7a (1.0 eq) was added to a three-necked flask, followed by EA solvent containing saturated HCl. The reaction was carried out at room temperature for 5 h. The solvent was evaporated and the product was dried under vacuum to obtain product 8a.

[0035] At room temperature, intermediate 8a (1.0 eq), m-nitrobenzyl bromide (1.0 eq), and DIEA (3.0 eq) were added to a round-bottom flask. DMF solvent was added, and the reaction was carried out at room temperature for 2 h. Acryloyl chloride reagent (1.2 eq) was then added, and the reaction was carried out at room temperature for 1 h. The filtrate was extracted with DCM and water, the oil layer was collected, and the oil layer was washed three times with saturated brine. The oil layer was dried over anhydrous Na₂SO₄, filtered, and the liquid was collected. The oil layer was then evaporated to dryness, and the product A38 was purified using a normal-phase silica gel column chromatography.

[0036] Table 13-19 shows the structural identification data for some of the compounds.

[0037] Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Example 2: In vitro NO generation inhibition experiment RAW 264.7 macrophages were divided into groups of 1.0 × 10⁻⁶. 5 Seeds were planted at a density of 1 / 2 in each well of a 96-well plate and incubated overnight at 37°C with 5% CO2. LPS (1 μg / mL) and gradient concentrations of the analyte were administered, and after 24 h of incubation, NO concentration was measured at 550 nM using a microplate reader according to the NO kit instructions, and the NO inhibition rate was calculated. IC50 was fitted using GraphPad Prism 8.0 software. 50 value.

[0038] The table below provides the average IC50 values ​​for the target compound in terms of NO generation inhibition. 50 Range, where "A" represents IC 50 Values ​​less than 1µM, "B" indicates IC 50 Values ​​are between 1 µM and 10 µM, where "C" indicates IC. 50 Values ​​between 10 µM and 20 µM, where "D" indicates IC 50 Value greater than 20 µM.

[0039] Table 20 Example 3: In vitro iNOS expression inhibition experiment RAW264.7 cell suspension was prepared at 3 × 10⁻⁶ cells per well. 6 Cells were seeded at a concentration of 1 / mL in 6 cm culture dishes and incubated overnight. Except for the control group and the model group (LPS, 1 μg / mL), which received 1 mL of complete culture medium, the drug-treated groups received 1 mL of different concentrations of drug solution per dish and were incubated for 2 h. After drug incubation, except for the control group, which received 1 mL of complete culture medium, the other groups received 1 mL of complete culture medium containing LPS and were incubated for 24 h. Cells were collected, centrifuged, and the supernatant was discarded. Cell lysis buffer was added, and proteins were extracted. Western blotting was used to detect the effect of the compounds on iNOS expression, and ImageJ software was used for grayscale analysis.

[0040] Figure 1 The Western blotting results for the expression of iNOS in LPS-induced RAW264.7 cells by A38 and A39 were obtained, with 42kDa β-actin as an internal control (stable band intensity to ensure experimental reliability). The core trend was clear: compared with the blank control group, the gray value of the iNOS (130kDa) band in the LPS model group was significantly increased; and as the concentration of A38 and A39 increased from 1μM to 20μM, the gray value of the iNOS band in both groups gradually decreased in a concentration-dependent manner, indicating that the compounds can inhibit iNOS protein expression in a dose-dependent manner.

[0041] This trend is highly correlated with in vitro NO generation inhibition experiments: Table 20 data show the IC50 inhibition rate of NO generation for A38 and A39. 50 All are class A (<1 μM). The concentration-dependent inhibition of iNOS expression shown in the figure precisely explains the molecular mechanism of reduced NO production in in vitro experiments—blocking NO synthesis by downregulating iNOS protein expression. Structurally, the highly efficient inhibitory effect of A38 and A39 stems from the precise three-ring linked core design. Both use a "pyrimidine ring-nitrogenous heterocycle-pyrazole ring" as the core framework, forming a stable conformation through the rigid connection of the three rings, providing a structural basis for targeting iNOS binding.

[0042] In summary, the pyrimidine derivatives and their pharmaceutically acceptable salts disclosed in this invention utilize a tricyclic structure of "pyrimidine ring-nitrogenous heterocycle-pyrazole ring" as the core, coupled with precisely designed substituents (such as meta-nitro groups, halogens, etc.), to construct a molecular structure highly adapted to the iNOS target. In vitro experiments have confirmed that several pyrimidine derivatives exhibit excellent inhibitory effects on NO production and iNOS expression. Some compounds (such as A38, A39, etc.) have IC50 values ​​less than or approximately equal to 1 μM, effectively blocking iNOS-mediated excessive NO synthesis and inflammation and oxidative stress induced by reactive nitrogen substances. From a disease application perspective, their mechanism of action is highly consistent with the "synovium-immunity-bone destruction" triple pathological network of rheumatoid arthritis (RA). By targeting the "NO-dependent osteoclastosis" pathway, they fill the gap in the efficacy of existing antirheumatic drugs such as JAK inhibitors, achieving cartilage protection and damage repair, and providing a new strategy for combination therapy in RA treatment, especially for drug-resistant patients. Given the association between iNOS overactivation and various diseases such as neurodegenerative diseases, inflammatory bowel disease, and tumors, these compounds also lay the foundation for the treatment of various iNOS-related diseases.

[0043] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A pyrimidine derivative or a pharmaceutically acceptable salt thereof, characterized in that, The general formula of the pyrimidine derivative is shown in Formula I; Where n is 1 or 2, m is 0 or 1, and l is 1 or 2; X1 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogenated C1-C6 alkyl, and C3-C6 cycloalkyl; X2 is R3 is selected from H, halogen, nitro, cyano, halogen C1-C6 alkyl, and C1-C6 alkoxy. Y1 and Y2 are independently selected from C, N, O and S; R1 and R2 are independently selected from H, halogen, nitro, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, halogen C1-C6 alkyl, C1-C6 alkoxy, carbamoyl, and ester.

2. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, When l is 1, and Y1 is C, O, and S, Y2 is C; When l is 1 and Y1 is N, Y2 is N; When l is 2, Y1 and Y2 are C.

3. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, X1 is selected from C1-C2 alkyl, C2 alkenyl, and halogenated C1-C2 alkyl.

4. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, R3 is a halogen, nitro, cyano, or trifluoromethyl group substituted at the ortho, meta, or para positions.

5. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, R1 or R2 can be halogen, nitro, cyano, methyl, trifluoromethyl, carbamoyl, or ester.

6. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that, The structural formula of the pyrimidine derivative is selected from: 。 7. The pyrimidine derivative or a pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that, The structural formula of the pyrimidine derivative is selected from: 。 8. The use of the pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, Used to prepare drugs that inhibit inducible nitric oxide synthase.

9. The use of the pyrimidine derivative or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, Used to prepare drugs for the treatment of rheumatoid arthritis.

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