Method for synthesizing small-molecule inhibitor D359-0396

Abstract

The invention provides a method for synthesizing a small molecule inhibitor D359-0396. According to the invention, commercially available 6-methyl uracil is used as an initial raw material, and the small-molecule inhibitor D359-0396 is prepared through five steps of reactions (including methylation reaction, formylation reaction, bromination reaction, cyclization reaction with o-phenylenediamine and cyclization reaction with 4-isopropyl benzaldehyde). The synthesized target product has important biological activity, and has good development prospect and development value in the aspect of treating multiple sclerosis and sepsis. The synthesis method provided by the invention has the advantages of simple steps, mild reaction conditions and high synthesis efficiency, and has positive effects in the fields of biological medicines, medicine synthesis and the like.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis, specifically relating to a method for synthesizing the small molecule inhibitor D359-0396. Background Technology

[0002] The small molecule inhibitor D359-0396, with the structural formula shown in Formula 6, is systematically named 6-(4-isopropylphenyl)-7,9-dimethyl-6,7-dihydropyrimidino[4',5':3,4]pyrrolo[1,2-a]quinoxaline-8,10(5H,9H)-dione, and its CAS registration number is 1031977-31-7. In 2022, Xu Yun et al. evaluated the effects of D359-0396 on pyroptosis and IL-1β inflammatory factor secretion in human and mouse macrophages through a series of pharmacodynamic experiments, and verified the therapeutic effect of D359-0396 on the disease through mouse EAE and sepsis models (Xu Yun, Zhang Cunjin, Li Zihao. Application of a small molecule inhibitor in the preparation of drugs for treating multiple sclerosis and sepsis, Invention Patent, ZL2022104494255). Studies have shown that D359-0396 can significantly reduce pyroptosis in humans and mice and decrease the secretion of related inflammatory factors such as IL-1β. Simultaneously, D359-0396 can significantly slow the progression of EAE in mice and improve the survival rate of sepsis patients, indicating that D359-0396 is a small molecule inhibitor capable of combating multiple sclerosis and sepsis. Therefore, D359-0396 is a promising new inhibitory molecule for the treatment of multiple sclerosis and sepsis, possessing significant development potential and value.

[0003]

[0004] To date, only one patent has reported the biological activity of D359-0396 (Xu Yun, Zhang Cunjin, Li Zihao. Application of a small molecule inhibitor in the preparation of drugs for treating multiple sclerosis and sepsis, Invention Patent, ZL2022104494255), and no literature or patents have reported its chemical synthesis.

[0005] The present invention aims to provide a simple and efficient method for synthesizing the small molecule inhibitor D359-0396. Summary of the Invention

[0006] This invention employs the synthetic route shown in Reaction Formula 1, using 6-methyluracil, o-phenylenediamine, and 4-isopropylbenzaldehyde (as shown in Formula 1) as starting materials, and undergoes five steps to prepare the target product D359-0396 (as shown in Formula 6). All raw materials, reagents, and solvents used in this invention are readily available for purchase.

[0007]

[0008] The technical solution of the present invention is as follows:

[0009] Step 1: Using commercially available 6-methyluracil as shown in Formula 1 as the raw material, and commercially available dimethyl sulfate (Me2SO4) or iodomethane (MeI) as the methylating agent, react in acetone to synthesize 1,3,6-trimethyluracil as shown in Formula 2.

[0010] Step 2: 1,3,6-trimethyluracil as shown in Formula 2 undergoes a formylation reaction with phosphorus oxychloride (POCl3) and N,N-dimethylformamide (DMF) to generate the intermediate 1,3,6-trimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde as shown in Formula 3.

[0011] Step 3: The intermediate shown in Formula 3 undergoes a bromination reaction with a commercially available brominating reagent in chloroform solvent to generate the intermediate shown in Formula 4, 6-(bromomethyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde.

[0012] Step 4: The intermediate shown in Formula 4 reacts with commercially available o-phenylenediamine in an ethanol solvent to generate the intermediate shown in Formula 5, 6-(2-aminophenyl)1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione.

[0013] Step 5: The intermediate shown in Formula 5 is reacted with commercially available 4-isopropylbenzaldehyde in a dry tetrahydrofuran solvent under acid catalysis to generate the target product D359-0396 shown in Formula 6, namely 6-(4-isopropylphenyl)-7,9-dimethyl-6,7-dihydropyrimidino[4',5':3,4]pyrrolo[1,2-a]quinoxaline-8,10(5H,9H)-dione.

[0014] In the above preparation method, in step 1, the commercially available methylating agent used is dimethyl sulfate, iodomethane, or a mixture thereof; the amount of the methylating agent used is 2 to 10 molar equivalents relative to 6-methyluracil represented by Formula 1, preferably 3.5 molar equivalents; potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, or a mixture thereof need to be added to the reaction system as a base; the amount of the base used is 1 to 10 molar equivalents relative to 6-methyluracil represented by Formula 1, preferably 3.5 molar equivalents; the reaction is generally carried out in acetone solvent.

[0015] In the preparation method described above, in step 2, N,N-dimethylformamide (DMF) is both a solvent and a reactant; the amount of phosphorus oxychloride used is 1 to 10 molar equivalents relative to the intermediate shown in Formula 2, preferably 2.5 molar equivalents.

[0016] In the preparation method described above, in step 3, the commercially available brominating reagent used can be one or a mixture of several of the following: liquid bromine, bromosuccinimide, 1,3-dibromo-5,5-dimethylhydantoin, 1,3,5-tribromo-1,3,5-thiazine-2,4,6-trione, tetrabutylammonium tribromide, tetramethylammonium tribromide, and tribromopyridinium salt, preferably liquid bromine; the amount of brominating reagent used relative to the intermediate shown in Formula 3 is 1 to 10 molar equivalents, preferably 1.5 molar equivalents; the bromination reaction is generally carried out in chloroform solvent.

[0017] In the preparation method described above, in step 4, the amount of o-phenylenediamine used relative to the intermediate shown in Formula 4 is 1 to 10 molar equivalents, preferably 2.2 molar equivalents; ethanol is preferably used as the solvent.

[0018] In the preparation method described above, step 5 requires the addition of an acid as a catalyst. The acid used is one or a mixture of several of boron trifluoride, aluminum trichloride, tin tetrachloride, titanium tetrachloride, ferric chloride, zinc dichloride, and p-toluenesulfonic acid. The amount of acid used relative to the intermediate shown in Formula 5 is 0.1 to 10 molar equivalents, and the amount of 4-isopropylbenzaldehyde used is 1 to 10 molar equivalents, preferably 1.2 molar equivalents.

[0019] In the preparation method described above, in step 5, the solvent for the reaction is preferably dry tetrahydrofuran; to promote the smooth occurrence of the reaction, benzotriazole also needs to be added to the reaction system; relative to the intermediate shown in Formula 5, the amount of benzotriazole is 0.5 to 10 molar equivalents, preferably 1 molar equivalent.

[0020] Advantages and positive effects of the present invention:

[0021] The small molecule inhibitor D359-0396 (CAS Registry No. 1031977-31-7) prepared by this invention has therapeutic effects on multiple sclerosis and sepsis. This invention provides for the first time a synthetic method for the small molecule inhibitor D359-0396, which has a short synthetic route, high synthetic efficiency, simple operation, and is suitable for large-scale preparation, thus possessing high application value. Attached Figure Description

[0022] Figure 1 The 1H NMR spectrum (400 MHz, DMSO-d6) of 6-(2-aminophenyl)1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione.

[0023] Figure 2 The carbon NMR spectrum of 6-(2-aminophenyl)1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione (101 MHz, DMSO-d6).

[0024] Figure 3 The 1H NMR spectrum (400MHz, CDCl3) of the small molecule inhibitor D359-0396.

[0025] Figure 4 The carbon NMR spectrum (101 MHz, CDCl3) of the small molecule inhibitor D359-0396. Detailed Implementation

[0026] The present invention will be further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein.

[0027] Step 1: Synthesis of 1,3,6-trimethylpyrimidine-2,4(1H,3H)-dione 2

[0028] To a solution of commercially available 6-methyluracil (3.0 g, 23.8 mmol, 1.0 equiv.) in acetone (60 mL), K₂CO₃ (11.5 g, 83.3 mmol, 3.5 equiv.) was added, and the resulting suspension was stirred vigorously at room temperature. After 30 minutes, dimethyl sulfate (7.9 mL, 83.3 mmol, 3.5 equiv.) was added, and the reaction mixture was heated at 60 °C for 3 h, with the reaction progress monitored by TLC. The resulting suspension was filtered, concentrated by rotary evaporation to give a crude product, which was recrystallized from ethyl acetate to give 1,3,6-trimethyluracil 2 (3.5 g, 98%) as a pale yellow solid.

[0029] Replacing dimethyl sulfate with the same molar equivalent of iodomethane yields the same product, with a yield of 95%.

[0030] Synthesis of step 21,3,6-trimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde 3

[0031] Under ice bath conditions, POCl3 (1.5 mL, 9.73 mmol, 2.5 equiv.) was added dropwise to a stirred solution of 1,3,6-trimethyluracil (0.6 g, 3.9 mmol, 1.0 equiv.) in anhydrous DMF (2 mL). After the addition was complete, the system was heated to 80 °C and stirred for 2 h, and the reaction progress was monitored by TLC. After the starting material was consumed, the system was placed in an ice-water bath, and an appropriate amount of water was added to quench the reaction. When the system was no longer exothermic, 50 mL of water was added to the system, and the mixture was extracted three times with dichloromethane (3 × 20 mL). The organic phases were combined and washed once with saturated brine. The product was purified by column chromatography to obtain a white crystalline product (355 mg, 30%). Step 3:

[0032] Synthesis of 6-(bromomethyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde

[0033] 1,3,6-Trimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde (1.5 g, 8.2 mmol) was dissolved in chloroform (5 mL) and placed in an ice-water bath. Liquid bromine (1.32 g, 0.42 mL, 1.1 equiv.) was dissolved in chloroform (5 mL) and then transferred to a dropping funnel and slowly added dropwise to the system. After the addition was complete, the system was brought to room temperature and stirred for 10 min to remove the solvent and excess liquid bromine. The crude product was purified by column chromatography to give a white oily product (1.25 g, 59%).

[0034] Replacing liquid bromine with the same molar equivalent of brominated succinimide, 1,3-dibromo-5,5-dimethylhydantoin, 1,3,5-tribromo-1,3,5-thiazine-2,4,6-trione, tetrabutylammonium tribromide, tetramethylammonium tribromide, or tribromopyridinium salt yields the same product in yields between 20% and 50%.

[0035] Synthesis of step 46-(2-aminophenyl)1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione 5

[0036] 6-(bromomethyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxaldehyde (1.2 g, 4.58 mmol, 1 equiv.) was dissolved in 10 mL of ethanol. o-Phenylenediamine (1.09 g, 10.08 mmol, 2.2 equiv.) was added at room temperature. The mixture was stirred at room temperature for half an hour, then refluxed for half an hour. After cooling to room temperature, the mixture was filtered, the filter cake was washed with ethanol, and dried. A pale yellow solid (0.9 g, 80%) was obtained.

[0037] 1 H NMR (400MHz, DMSO-d6) δ7.52(d,J=2.2Hz,1H),7.15(ddd,J=8.4,7.2,1.2Hz,1H),7.10(dd,J=7.6,1.2Hz,1H),6.94 (d,J=2.2Hz,1H),6.87(dd,J=8.0,1.2Hz,1H),6.65(td,J=7.6,1.6Hz,1H),5.07(s,2H),3.31(s,3H),3.22(s,3H). 13 C NMR (101MHz, DMSO-d6)δ

[0038] 159.7, 151.5, 143.7, 129.7, 129.5, 127.2, 125.3, 121.6, 116.7, 116.6, 106.1, 105.2, 32.2, 27.9. Step 56: Synthesis of (4-isopropylphenyl)-7,9-dimethyl-6,7-dihydropyrimidino[4',5':3,4]pyrrolo[1,2-a]quinoxaline-8,10-(5H,9H)-dione 6

[0039] Add a magnetic flask, benzotriazole (440 mg, 3.7 mmol, 1.0 equiv.), and aluminum trichloride (50 mg, 0.37 mmol, 0.1 equiv.) to a dry 50 mL three-necked flask, and seal the flask. Purge with nitrogen three times, dissolve the flask in dry tetrahydrofuran using a syringe, and then add 4-isopropylbenzaldehyde (657 mg, 4.4 mmol, 1.2 equiv.). Place the flask in an ice-water bath, and add 6-(2-aminophenyl)-1,3-dimethyl-1,6-dihydro-2H-pyrrolo[3,4-d]pyrimidine-2,4(3H)-dione (1.0 g, 3.7 mmol, 1 equiv.) as shown in Formula 5. Stir for 10 min in an ice-water bath, then return to room temperature and stir for 1 h. Monitor the reaction progress by TLC. After the reaction is complete, quench the reaction with an appropriate amount of water, extract three times with dichloromethane, combine the organic phases, and wash with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography and recrystallized to give a white solid (1.25 g, 85%).

[0040] Replacing aluminum trichloride with the same molar equivalents of boron trifluoride, tin tetrachloride, titanium tetrachloride, ferric chloride, zinc dichloride, or p-toluenesulfonic acid yields the same product, with yields ranging from 10% to 60%.

[0041] 1 H NMR (400MHz, CDCl3) δ7.92(s,1H),7.44(dd,J=8.0,1.4Hz,1H),7.09(d,J=7.8Hz,2H),7.01(t,J=7.8Hz,1H),6.93(d,J=7.8Hz,2H),6.86(t,J= 7.8Hz,1H),6.66(dd,J=7.8,1.6Hz,1H),5.97(s,1H),4.45(s,1H),3.44(s,3H),3.40(s,3H),2.82(hept,J=7.0Hz,1H),1.17(d,J=7.0Hz,6H). 13C NMR(101MHz,CDCl3)δ159.9,152.1,148.9,140.3,134.3,127.3,126.9,126.0,124.1,123.5,119.8,116.7,115.4,113.1,110.1,107.9,53.9,33.7,32.2,28.0,23.8,23.8。

Claims

1. A method for synthesizing the small molecule inhibitor D359-0396, characterized in that, Starting with commercially available 6-methyluracil as shown in Formula 1, the small molecule inhibitor D359-0396, as shown in Formula 6, was synthesized through methylation (step 1), formylation (step 2), bromination (step 3), cyclization with o-phenylenediamine (step 4), and cyclization with 4-isopropylbenzaldehyde (step 5).

2. The method according to claim 1, characterized in that, Commercially available 6-methyluracil, o-phenylenediamine, and 4-isopropylbenzaldehyde, as shown in Formula 1, are used as raw materials.

3. The method according to claim 1, characterized in that, In step 1, the methylating agent used is dimethyl sulfate, iodomethane, or a mixture thereof, and the base used is one or a mixture of potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide.

4. The method according to claim 1, characterized in that, In step 2, phosphorus oxychloride (POCl3) and N,N-dimethylformamide (DMF) are used as formylation reagents.

5. The method according to claim 1, characterized in that, In step 3, the commercially available brominating reagent used is one or a mixture of several of the following: liquid bromine, bromosuccinimide, 1,3-dibromo-5,5-dimethylhydantoin, 1,3,5-tribromo-1,3,5-thiazine-2,4,6-trione, tetrabutylammonium tribromide, tetramethylammonium tribromide, and tribromopyridinium salt, preferably liquid bromine.

6. The method according to claim 1, characterized in that, In step 4, ethanol is used as the solvent.

7. The method according to claim 1, characterized in that, In step 5, benzotriazole and an acid catalyst need to be added. The acid catalyst is one or a mixture of several of boron trifluoride, aluminum trichloride, tin tetrachloride, titanium tetrachloride, ferric chloride, zinc dichloride, and p-toluenesulfonic acid.

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