WO2023000708A1 - Polysubstituted purine compound, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a polysubstituted purine compound as shown in formula (I), and a pharmaceutically acceptable salt thereof, a preparation method therefor and the use thereof. Also disclosed is that the compound has an obvious inhibitory effect on RNA adenosine deaminase 1 (ADAR1) and can be used for preventing and/or treating cancer or tumor-related diseases caused by abnormal activity of such enzymes, especially prostate cancer, leukemia, breast cancer, multiple myeloma, lung cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, pancreatic cancer, human glioma and other diseases.

Description

A kind of polysubstituted purine compound and its preparation method and application technical field

The invention relates to the field of medicinal chemistry, in particular to a multi-substituted purine compound and its preparation method and application.

Background technique

Adenosine deaminases acting on RNA enzymes (Adenosine deaminases acting on RNA enzymes, ADARs) are one of the members of the RNA editing enzyme family. It is converted into inosine nucleotides, which are mistakenly recognized as guanine nucleotides in organisms and participate in a series of biological processes such as transcription and translation. ADARs include three subtypes, ADAR1, ADAR2, and ADAR3, all of which contain two to three N-terminal double-stranded RNA (double stranded RNA, dsRNA) binding domains and a C-terminal conserved deaminase catalytic domain. ADAR1 and ADAR2 are distributed and expressed in many tissues and organs of the human body, and their structures and functions are currently being studied more. ADAR3 has no catalytic deamination activity and is mainly expressed in the central nervous system.

The human ADAR1 protein is expressed by the ADAR1 gene located in the q21 region of chromosome 1. The transcription of the ADAR1 gene is driven by two promoters, one constitutively expressed on the genome to produce p110 protein, and the other is an interferon-induced promoter, Promotes transcription to produce p150 protein, so p150 protein is interferon-inducible. Compared with the p110 protein, the p150 protein has an extra Z-DNA binding domain Zα, and only the Zα domain can bind Z-DNA/RNA.

ADAR1-mediated A-to-I editing events widely occur in cells in various tissues and organs. Because hypoxanthine nucleotides are mistakenly recognized as guanylic acid in biological processes, they occur in the A-to-I editing region of the mRNA coding region. to-I editing events may result in changes in protein amino acids, resulting in structurally and functionally mutated proteins. However, most A-to-I editing events occur in the introns and 3'-untranslated regions (3'-UTRs) of RNA, which affect the shearing, degradation and translation regulation of mRNA. The biological significance of editing events in coding regions is far from fully understood.

The expression and activity of ADAR1 are significantly upregulated in many cancers, and these A-to-I editing events mediated by ADAR1 are significantly different in normal tissues and cancer tissues, and are significantly negatively correlated with the clinical prognosis of cancer patients. relevant. In many cancer cell lines, such as lung cancer, liver cancer, breast cancer, multiple myeloma, gastric cancer, pancreatic cancer, etc., ADAR1 is knocked down by short-hairpin RNA, and ADAR1 is knocked down by CRISPER-CAS9 technology, After reducing the expression of ADAR1 protein, it was found that the proliferation activity, survival, metastasis and invasion ability of cells were all significantly reduced. This is mainly due to the fact that the unedited dsRNA will mistakenly cause the antiviral response of the cell, be recognized by MDA5 in the cell, cause the downstream MAVS to activate and secrete interferon, lead to an immune response, be recognized by PKR, cause translation inhibition, and lead to cell death , while the ADAR1 protein level and activity are excessively elevated in many cancers, dsRNA will not be recognized by MDA5 and PKR after being edited, resulting in cell death and immune response, thereby allowing cancer cells to survive and proliferate excessively. In addition, knockout of ADAR1 in melanoma enhances tumor sensitivity to radiotherapy and immunosuppressants such as PD-1 monoclonal antibody, and overcomes resistance to immunotherapy. This reveals ADAR1 to be a promising cancer target. However, no small molecule inhibitors directly acting on ADAR1 have been reported yet. Therefore, the discovery of the first small molecule inhibitor of ADAR1 is of great therapeutic interest.

Contents of the invention

Purpose of the invention: the purpose of the present invention is to provide a kind of polysubstituted purine compound and its preparation method; the present invention also provides the application of this kind of compound.

Technical scheme: a kind of multi-substituted purine compound of the present invention, such as the compound shown in general formula (I) or its pharmaceutically acceptable salt;

In the formula, R ₁ is selected from hydrogen, C ₁ -C ₃ alkyl;

R ₂ is selected from halogen, -SCH ₂ CH ₂ COOH;

R ₃ is selected from hydrogen, halogen, C ₁ -C ₃ alkyl;

R ₄ is selected from C ₁ -C ₃ alkyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

further,

R ₁ is selected from hydrogen, methyl;

R ₂ is selected from fluorine, -SCH ₂ CH ₂ COOH;

_R is selected from hydrogen, chlorine, methyl;

R ₄ is selected from C ₁ -C ₃ alkyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

further,

R ₁ is selected from hydrogen, methyl;

R ₂ is selected from fluorine, -SCH ₂ CH ₂ COOH;

_R is selected from hydrogen, chlorine, methyl;

R ₅选自以下基团： R ₄ is selected from methyl, propyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

further,

R ₁ is selected from hydrogen;

R ₂ is selected from fluorine;

_R is selected from hydrogen;

R ₅选自以下基团： R ₄ is selected from methyl, propyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

further,

R ₁ is selected from hydrogen;

R ₂ is selected from fluorine;

_R is selected from hydrogen;

R ₄ is selected from methyl or

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

further,

The compound is selected from I-1 to I-21:

further,

The pharmaceutically acceptable salt is an acid addition salt of the compound of general formula (I), wherein the acid used for salt formation includes inorganic acid and organic acid, and the inorganic acid includes hydrochloric acid, sulfuric acid, phosphoric acid and methanesulfonic acid, Organic acids include acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid and tartaric acid.

Further, the pharmaceutically acceptable salt is hydrochloride.

Further, a preparation method of multi-substituted purine compounds.

Furthermore, a pharmaceutical composition comprises the compound of general formula (I) or a pharmaceutically acceptable salt or isomer thereof, and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier refers to an excipient or diluent that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound.

Further, the application of a multi-substituted purine compound in the preparation of drugs for the prevention and/or treatment of cancer or tumor-related diseases, including prostate cancer, leukemia, breast cancer, multiple myeloma , lung cancer, gastric cancer, ovarian cancer, colon cancer, liver cancer, pancreatic cancer, and human glioma.

The compound of the general formula (I) or the pharmaceutically acceptable salt thereof in the present invention has ADAR1 target inhibitory activity and has a therapeutic effect on tumors.

Unless otherwise specified, the terms in the present invention generally have the following meanings.

The term "alkyl" means a straight or branched chain saturated hydrocarbon group having the stated number of carbon atoms.

The term "C ₁ -C ₃ alkyl" refers to a straight chain or branched chain saturated hydrocarbon group with 1-3 carbon atoms; C ₁ -C ₃ alkyl includes but not limited to methyl, ethyl, n-propyl, iso Propyl.

"P(O)" means "-P(O)-", specifically a phosphorus-oxygen double bond.

The term "halogen" is fluorine, chlorine, bromine or iodine; preferably fluorine, chlorine, bromine.

The invention also discloses the preparation method of the compound of general formula (I).

Beneficial effects: Compared with the prior art, the present invention discloses a compound represented by a new general formula (I), which has the ability to inhibit ADAR1 activity, and provides a druggable drug for targeting ADAR1 therapy to treat cancer Compound; this type of compound can inhibit cancer proliferation and metastasis at the same time, has good therapeutic effect, low toxicity, and is not easy to produce drug resistance, and can be used to treat cancer or tumor-related diseases; the invention also discloses a preparation method of the compound of general formula (I) .

Description of drawings

Fig. 1 is the result graph of the binding constant of the compound of the present invention and ADAR1 protein;

Fig. 2 is the result graph of the inhibitory ability of the compound of the present invention to ADAR1 deaminase activity;

Fig. 3 is a schematic diagram of the body weight change of mice in the acute toxicity assay of the present invention;

Fig. 4 is HE staining result figure in the acute toxicity assay of the present invention;

Fig. 5 is a graph showing the results of the present invention on prostate cancer tumor volume.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings. The following examples are only used to illustrate the technical solution of the present invention more clearly, but not to limit the protection scope of the present invention.

Example 1: (2R, 3R, 4S, 5S)-2-(6-amino-2-fluoro-9H-purin-9-yl)-5-(chloromethyl)tetrahydrofuran-3,4-diol ( I-1) synthesis:

At 0°C, 2-fluoroadenosine A-1 (5.70g, 20mmol) was dissolved in acetonitrile (80mL), pyridine (3.22mL, 40mmol) was added, and thionyl chloride (7.25mL , 100mmol), stirred for 4h and raised to room temperature, reacted overnight, TLC monitored that the raw materials were completely consumed, and the solvent was removed by rotary evaporation under reduced pressure, and methanol (120mL), water (12mL), and ammonia water (24mL) were added again, stirred for 0.5h, and reduced pressure Concentrated by rotary evaporation, the product was precipitated in the water phase, filtered to obtain a filter cake, redissolved in a small amount of methanol at 60°C, added dropwise with dichloromethane, cooled to precipitate a solid and suction filtered to obtain a filter cake, washed with cold methanol to obtain the product I-1 as a white solid (5.45 g, 90%). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.34(s,1H),7.92(s,2H),5.84(d,J=5.6Hz,1H),5.58(s,2H),4.67(t, J=5.4Hz, 1H), 4.26–4.16(m, 1H), 4.10(q, J=5.4, 5.0Hz, 1H), 3.99–3.81(m, 2H).

Example 2: 9-((3aR,4R,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuran[3, Synthesis of 4-d][1,3]bisoxazol-4-yl)-2-fluoro-9H-purin-6-amine (I-2):

Step 1, 2-fluoroadenosine (1.48g, 5mmol) was dissolved in anhydrous acetone (200mL) to form a suspension, anhydrous p-toluenesulfonic acid (4.31g, 25mmol) was added to form a clear solution, and dimethyl Oxypropane (1.04g, 10mmol), after the mixture was stirred at room temperature under nitrogen atmosphere for 4h, cold saturated NaHCO ₃ solution (100mL) was added to the above mixture; the volatiles were removed under reduced pressure, and the residue was dried; The solid was dissolved in acetone (400 mL), stirred for 1 h, then filtered; the volatiles were removed, and the crude product was purified by column chromatography to obtain white solid A2 (1.50 g, 95%); ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.33(s,1H),7.92(s,2H),6.03(d,J=2.9Hz,1H),5.29(dd,J=6.2,2.9Hz,1H),5.10(s,1H) ,4.93(dd,J＝6.2,2.6Hz,1H),4.27–4.10(m,1H),3.63–3.44(m,2H),1.54(s,3H),1.33(s,3H);

Step 2, 0°C, under the protection of _N2 , A2 (0.66g, 2mmol) was dissolved in 10mL DMF, imidazole (0.41g, 6mmol) and tert-butyldimethylsilyl chloride (0.45g, 3mmol) were added and reacted for 2h Afterwards, it was raised to room temperature and reacted overnight; TLC monitored that the raw material was completely consumed, treated with methanol (2.0 mL), stirred for 30 minutes, and removed the solvent under reduced pressure; the residue was purified by silica gel column chromatography to obtain a white product I-2 (0.84 g, 95%). ¹ H NMR (400MHz, Chloroform-d) δ8.02(s,1H),6.41(s,2H),6.10(d,J=2.7Hz,1H),5.20(dd,J=6.1,2.7Hz,1H ),4.95(dd,J=6.2,2.6Hz,1H),4.40(q,J=3.9Hz,1H),3.99–3.70(m,2H),1.63(s,3H),1.40(s,3H) ,0.87(s,9H),0.04(s,6H).

Example 3: ((3aR,4R,6R,6aR)-6-(6-amino-2-fluoro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuran[3,4-d] Synthesis of [1,3]bisoxazol-4-yl)methyl acetate (I-3):

A2 (0.66g, 2mmol) was dissolved in 10mL DMF, triethylamine (0.83mL, 6mmol), acetic anhydride (0.21mL, 2.2mmol) and DMAP (0.05g, 0.4mmol) were added and reacted at room temperature overnight. The complete consumption of raw materials was monitored by TLC, the reaction was quenched with saturated aqueous NH ₄ Cl (5 mL), the mixture was extracted with ethyl acetate (3×30 mL), the combined organic layers were washed with saturated aqueous NaCl, dried over anhydrous Na ₂ SO ₄ , filtered and Concentrate under reduced pressure and purify by silica gel column chromatography to obtain compound I-3 (0.66 g, 90%). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.28(s, 1H), 7.92(d, J=26.2Hz, 2H), 6.11(d, J=2.4Hz, 1H), 5.41(dd, J= 6.2,2.5Hz,1H),5.00(dd,J＝6.2,3.3Hz,1H),4.45–4.30(m,1H),4.30–4.08(m,2H),1.96(s,3H),1.54(s ,3H), 1.34(s,3H).

Example 4: ((3aR,4R,6R,6aR)-6-(6-amino-2-fluoro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuran[3,4-d] Synthesis of [1,3]bisoxazol-4-yl)methyl benzoate (I-4):

The method is the same as that of I-3, and the yield is 92%. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.26(s,1H),7.87(dd,J＝8.3,1.4Hz,4H),7.69–7.59(m,1H),7.48(t,J＝7.8 Hz, 2H), 6.14(d, J=2.4Hz, 1H), 5.47(dd, J=6.3, 2.4Hz, 1H), 5.14(dd, J=6.3, 3.0Hz, 1H), 4.59–4.47(m ,2H), 4.48–4.38(m,1H), 1.56(s,3H), 1.36(s,3H).

Example 5: ((3aR,4R,6R,6aR)-6-(6-amino-2-fluoro-9H-purin-9-yl)-2,2-dimethyltetrahydrofuran[3,4-d] Synthesis of [1,3]bisoxazol-4-yl)methyl 4-(trifluoromethyl)benzenesulfonate (I-5):

The method is the same as that of I-3, and the yield is 89%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.23(s, 1H), 8.06–7.83(m, 6H), 6.10(d, J=2.3Hz, 1H), 5.30(dd, J=6.3, 2.3 Hz, 1H), 4.93 (dd, J=6.3, 3.3Hz, 1H), 4.47–4.31 (m, 3H), 1.51 (s, 3H), 1.29 (s, 3H).

Example 6: Methyl ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)acetate Synthesis of (I-6):

Take compound I-3 (0.37g, 1mmol) and add it to a 10mL single-necked bottle, add 5mL trifluoroacetic acid: water = 4:1 dropwise under ice bath conditions, bring it to room temperature and react overnight; TLC monitors that the raw materials are completely consumed, saturated NaHCO ₃ The aqueous solution was used to neutralize the reaction system, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography to obtain the product I-6 (0.31 g, 96%). ¹ H NMR (400MHz, Methanol-d ₄ ) δ8.18(s, 1H), 5.91(d, J=4.5Hz, 1H), 4.72(t, J=4.9Hz, 1H), 4.45–4.27(m, 3H), 4.22(td, J=5.1, 3.6Hz, 1H), 2.06(s, 3H).

Example 7: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)benzoic acid Synthesis of esters:

The method is the same as that of I-6, and the yield is 94%. ¹ H NMR (300MHz,DMSO-d ₆ )δ8.29(s,1H),8.05–7.76(m,4H),7.72–7.63(m,1H),7.52(t,J=7.7Hz,2H), 5.86(d, J=4.7Hz, 1H), 5.78–5.22(m, 2H), 4.68(t, J=5.0Hz, 1H), 4.59(dd, J=12.0, 3.7Hz, 1H), 4.47(dd , J=12.0, 5.5Hz, 1H), 4.41(t, J=5.1Hz, 1H), 4.27–4.19(m, 1H).

Example 8: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)4-( Synthesis of methyl trifluoromethyl)benzenesulfonate (I-8):

The method is the same as that of I-6, and the yield is 93%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.24(s, 1H), 8.06(d, J=8.2Hz, 2H), 7.92(d, J=8.2Hz, 4H), 5.76(d, J= 5.1Hz, 1H), 5.72–5.33(m, 2H), 4.55(t, J=5.1Hz, 1H), 4.48–4.40(m, 2H), 4.18(t, J=4.7Hz, 1H), 4.12– 4.06(m,1H).

Example 9: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridine-3 -Synthesis of methyl sulfonate (I-9):

The method is the same as the synthesis of I-6, and the yield is 96%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.99(dd, J=2.5,0.8Hz,1H),8.83(dd,J=4.8,1.6Hz,1H),8.25(ddd,J=8.1,2.5 ,1.6Hz,1H),8.20(s,1H),7.90(s,2H),7.58(ddd,J＝8.1,4.9,0.9Hz,1H),5.76(d,J＝5.2Hz,1H),5.62 (d, J = 5.8Hz, 1H), 5.46 (d, J = 5.3Hz, 1H), 4.54 (q, J = 5.3Hz, 1H), 4.42 (qd, J = 11.0, 5.0Hz, 2H), 4.17 (q, J = 4.9 Hz, 1H), 4.11–4.05 (m, 1H).

Example 10: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl 4 -Synthesis of oxo-4-(phenylamino)butanoic acid (I-10):

Step 1: Dissolve succinic anhydride (0.50g, 5mmol) and aniline (0.47g, 5mmol) in toluene, heat to 110°C and reflux for 2h, cool and suction filter, wash with ether, collect the filter cake and dry to obtain product B1 (0.92g ,95%); ¹ H NMR (300MHz, DMSO-d ₆ ) (300MHz,) δ12.13,9.95,7.58(d,J=8.0Hz),7.28(t,J=7.8Hz),7.01(t, J=7.4Hz), 2.62–2.52(m);

Step 2, A2 (0.66g, 2mmol) was dissolved in 10mL DMF, and B1 (0.58g, 3mmol), dicyclohexylcarbodiimide (0.83g, 4mmol), 4-dimethylaminopyridine (48mg, 0.4 mmol), reacted overnight at room temperature; TLC monitored the complete consumption of starting materials, and quenched with saturated aqueous NH ₄ Cl (5 mL); the mixture was extracted with dichloromethane (3×30 mL), and the combined organic layers were washed with saturated aqueous NaCl, anhydrous NaCl ₂ SO ₄ dried, filtered and concentrated under reduced pressure, purified by silica gel column chromatography to obtain compound B2 (0.90g, 90%); ¹ H NMR (400MHz, DMSO-d ₆ ) δ9.98(s, 1H), 8.31(s ,1H),7.93(d,J=28.4Hz,2H),7.56(d,J=8.0Hz,2H),7.27(t,J=7.7Hz,2H),7.01(t,J=7.3Hz,1H ), 6.09(d, J=2.6Hz, 1H), 5.38(dd, J=6.2, 2.6Hz, 1H), 4.99(dd, J=6.2, 3.3Hz, 1H), 4.37(q, J=3.9Hz ,1H),4.31–4.12(m,2H),2.59(s,4H),1.53(s,3H),1.31(s,3H);

Step 3: Take compound B2 (0.50 g, 1 mmol) and add it to a 10 mL single-necked bottle, add 5 mL of trifluoroacetic acid: water = 4:1 dropwise under ice bath conditions, bring it to room temperature and react overnight; TLC monitors that the raw materials are completely consumed and saturated The reaction system was neutralized with NaHCO ₃ aqueous solution, the solvent was removed by rotary evaporation under reduced pressure, and purified by silica gel column chromatography to obtain the product I-10 (0.43 g, 93%). ¹ H NMR (400MHz,DMSO-d ₆ )δ9.98(s,1H),8.33(s,1H),7.89(d,J=29.7Hz,2H),7.56(d,J=7.6Hz,2H) ,7.28(t,J=7.9Hz,2H),7.01(t,J=7.4Hz,1H),5.81(d,J=5.0Hz,1H),5.61(s,1H),5.41(s,1H) ,4.57(t,J=5.1Hz,1H),4.34(dd,J=11.9,3.7Hz,1H),4.26–4.15(m,2H),4.14–4.05(m,1H),2.62(t,J = 3.9Hz, 4H).

Example 11: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl 4 -Synthesis of (benzylamino)-4-oxobutyrate (I-11):

The method is the same as that of I-10, and the yield is 94%. ¹ H NMR (400MHz, Methanol-d ₄ )δ8.21(s,1H),7.35–7.14(m,5H),5.90(d,J=4.6Hz,1H),4.70(t,J=4.9Hz, 1H), 4.44–4.33 (m, 5H), 4.23 (q, J=4.8Hz, 1H), 2.72–2.64 (m, 2H), 2.59–2.52 (m, 2H).

Example 12: ((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)4-amino Synthesis of methyl butyrate (I-12):

The method is the same as that of I-10, and the yield is 90%. ¹ H NMR (400MHz, Methanol-d ₄ )δ8.18(s,1H),5.90(d,J＝4.4Hz,1H),4.77–4.72(m,1H),4.49–4.32(m,3H), 4.27–4.17 (m, 1H), 3.00–2.92 (m, 2H), 2.51 (t, J=7.1 Hz, 2H), 1.92 (p, J=7.2 Hz, 2H).

Example 13: (((5-(6-Amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl )-L-alanine methyl ester (I-13) synthesis:

Step 1, under the protection of N ₂ at -78°C, C1 (2.95g, 14mmol) and C2 (1.95g, 14mmol) were dissolved in anhydrous dichloromethane (40mL), and anhydrous triethylamine (2.83g , 28mmol), slowly rise to room temperature, continue to react for 24h, TLC monitors that the raw material is completely consumed, removes the solvent by rotary evaporation under reduced pressure, redissolves in 40mL anhydrous ether and filters, collects the filtrate, and concentrates by rotary evaporation to obtain crude oil C3, directly without purification use;

Step 2: Dissolve A2 (0.65g, 2mmol) and N-methylimidazole (0.49g, 6mmol) in 20mL of THF, and add dropwise a solution of C3 (0.89g, 3.2mmol) in THF (5mL) at -78°C , stirred for 2h, slowly raised to room temperature to continue the reaction for 24h, quenched with methanol, removed the solvent by rotary evaporation under reduced pressure, and purified by silica gel column chromatography to obtain a pair of diastereoisomers C4 (0.93g, 82%); ¹ H NMR (400MHz,Chloroform-d)δ7.94(d,J＝4.2Hz,1H),7.32–7.23(m,2H),7.20–7.08(m,3H),6.56(s,2H),6.04(dd, J=9.1,2.7Hz,1H),5.28(dd,J=6.3,2.5Hz,0.5H),5.09(dd,J=6.3,2.8Hz,0.5H),5.04(dd,J=6.3,3.3Hz ,0.5H),4.94(dd,J＝6.3,2.9Hz,0.5H),4.55–4.43(m,1H),4.40–4.27(m,2H),4.14–3.93(m,1H),3.67(d ,J=1.2Hz,3H),1.60(d,J=4.4Hz,3H),1.36(d,J=13.4Hz,3H),1.33–1.28(m,3H);

Step 3: Take compound C4 (0.57g, 1mmol) and add it to a 10mL single-necked bottle, add 5mL trifluoroacetic acid: water = 4:1 dropwise under ice bath conditions, bring it to room temperature and react overnight; TLC monitors that the raw materials are completely consumed and saturated The reaction system was neutralized with NaHCO ₃ aqueous solution, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography to obtain the product, a pair of diastereoisomers I-13 (0.47 g, 90%). ¹ H NMR (400MHz, Methanol-d ₄ ) δ8.18 (d, J=13.7Hz, 1H), 7.44–6.92 (m, 5H), 5.93 (dd, J=4.8, 3.2Hz, 1H), 4.63( dt,J=15.8,5.0Hz,1H),4.48–4.18(m,4H),4.03–3.78(m,1H),3.64(d,J=2.0Hz,3H),1.32–1.29(m,1.5H ), 1.23 (dd, J=7.2, 1.3Hz, 1.5H).

Example 14: (2R,3R,4S,5R)-2-(2-fluoro-6-(methylamino)-9H-purin-9-yl)-5-(methoxymethyl)tetrahydrofuran-3 , the synthesis of 4-diol (I-14):

Step 1: Add A2 (0.65g, 2mmol) in anhydrous DMF (10mL) solution dropwise to sodium hydride (0.16g, 4mmol) in anhydrous DMF (10mL) solution at 0°C, rise to room temperature and react for 2h Finally, iodomethane (0.57g, 4mmol) was added dropwise to react overnight at room temperature, quenched with saturated NH ₄ Cl aqueous solution (5 mL); the mixture was extracted with dichloromethane (3×30 mL), and the combined organic layers were washed with saturated NaCl aqueous solution, Dry over anhydrous Na ₂ SO ₄ , filter and concentrate under reduced pressure, and purify by silica gel column chromatography to obtain compound D1 (0.52g, 73%); ¹ H NMR (400MHz, Chloroform-d) δ7.70(s, 1H), 5.78 (d,J=4.9Hz,1H),5.65(dd,J=11.6,2.0Hz,1H),5.23–5.16(m,1H),5.12(dd,J=6.0,1.4Hz,1H),4.50( d, J＝1.7Hz, 1H), 3.98(dt, J＝12.8, 1.8Hz, 1H), 3.92–3.61(m, 4H), 3.27(s, 3H), 1.64(s, 3H), 1.38(s ,3H);

Step 2: Add compound C4 (0.57 g, 1 mmol) into a 10 mL single-necked bottle, add 5 mL of trifluoroacetic acid: water = 4:1 dropwise under ice-bath conditions, and bring it to room temperature for overnight reaction. The complete consumption of raw materials was monitored by TLC, the reaction system was neutralized with saturated NaHCO ₃ aqueous solution, the solvent was removed by rotary evaporation under reduced pressure, and purified by silica gel column chromatography to obtain the product I-14 (0.29 g, 91%). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.38(s, 1H), 5.81(d, J=5.7Hz, 1H), 4.49(t, J=5.3Hz, 1H), 4.15–4.11(m, 1H), 3.94 (q, J = 3.9Hz, 1H), 3.78–3.61 (m, 4H), 3.55 (dd, J = 12.0, 3.9Hz, 1H), 3.17 (s, 3H).

Example 15: 3-((((2S,3S,4R,5R)-5-(6-amino-2-((2-carboxyethyl)thio)-9H-purin-9-yl)-3 , the synthesis of 4-dihydroxytetrahydrofuran-2-yl) methyl) thio) propionic acid (I-15):

Under N ₂ atmosphere, 3-mercaptopropionic acid (1.11g, 10.5mmol) was dissolved in 1M NaOH aqueous solution (18mL), added I-1 (0.91g, 3mmol), heated to 105°C, reflux reaction overnight, TLC monitoring raw material consumption Completely, the pH was adjusted to 5-6 with acetic acid, the solid precipitated out, suction filtered, washed with water and cold methanol, and dried to obtain the product as a white solid (1.14 g, 83%). ¹ H NMR (400MHz,DMSO-d ₆ )δ8.24(s,1H),7.41(s,2H),5.81(d,J=5.9Hz,1H),4.76(t,J=5.5Hz,1H) ,4.21–4.06(m,1H),4.04–3.96(m,1H),3.32–3.13(m,2H),2.95–2.79(m,2H),2.69(t,J＝6.4Hz,4H),2.50 –2.41(m,2H).

Example 16: (2R,3R,4S,5R)-2-(6-amino-2-fluoro-8-methyl-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3, Synthesis of 4-diol (I-16):

Step 1: Dissolve A1 (0.57g, 2mmol) and imidazole (0.82g, 12mmol) in anhydrous DMF (20mL), add TBSCl (1.2g, 8mmol), raise the temperature to 50°C overnight, and monitor the complete consumption of raw materials by TLC , quenched with saturated aqueous NH ₄ Cl (5 mL); the mixture was extracted with dichloromethane (3×30 mL), the combined organic layers were washed with saturated aqueous NaCl, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, silica gel Purified by column chromatography to obtain compound I-12 (1.17g, 93%); ¹ H NMR (400MHz, Chloroform-d) δ8.12 (s, 1H), 6.14 (s, 2H), 5.93 (d, J=5.1 Hz, 1H), 4.63(t, J=4.7Hz, 1H), 4.31(t, J=4.0Hz, 1H), 4.13(q, J=3.7Hz, 1H), 4.03(dd, J=11.3, 4.3 Hz,1H),3.78(dd,J＝11.4,2.9Hz,1H),0.94(d,J＝9.2Hz,18H),0.81(s,9H),0.12(dd,J＝13.6,2.9Hz,12H ),-0.02(s,3H),-0.20(s,3H);

Step 2: Dissolve compound E1 (0.94g, 1.5mmol) in anhydrous THF (10mL) at -78°C, slowly add lithium diisopropylamide (7.5mmol) dropwise, after stirring for 3h, add dropwise again Iodomethane (0.64g, 4.5mmol), reacted for 2h, TLC monitored the complete consumption of raw materials, quenched with saturated NH ₄ Cl aqueous solution (5mL); extracted the mixture with dichloromethane (3×25mL), combined the organic layers with saturated NaCl aqueous solution Washed, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, purified by silica gel column chromatography to obtain compound E2 (0.72g, 75%); ¹ H NMR (300MHz, Chloroform-d) δ5.93 (s, 2H) ,5.72(d,J=6.1Hz,1H),5.30(dd,J=6.1,4.4Hz,1H),4.47(dd,J=4.4,2.2Hz,1H),4.07(dd,J=4.4,2.2 Hz, 2H), 3.78–3.67(m, 1H), 2.58(s, 3H), 0.95(s, 9H), 0.85(s, 9H), 0.78(s, 9H), 0.15(d, J=1.1Hz ,6H),0.06–-0.01(m,6H),-0.05(s,3H),-0.35(s,3H);

Step 3, compound E2 (0.64g, 1mmol) was dissolved in anhydrous THF (10mL), a THF solution of tetrabutylammonium fluoride (4mmol) was added dropwise at 0°C, raised to room temperature and reacted for 3h, TLC was used to monitor the consumption of raw materials Completely, the solvent was removed by rotary evaporation under reduced pressure, extracted with dichloromethane (3×15mL), the combined organic layers were washed with saturated NaCl aqueous solution, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the compound I-16 (0.27 g, 90%). ¹ H NMR (400MHz, Methanol-d ₄ )δ5.85(d, J=7.1Hz, 1H), 4.88(s, 1H), 4.32(dd, J=5.3, 2.0Hz, 1H), 4.14(q, J=2.7Hz, 1H), 3.87(dd, J=12.6, 2.7Hz, 1H), 3.72(dd, J=12.5, 3.1Hz, 1H), 2.60(s, 3H).

Example 17: (2R,3R,4S,5R)-2-(6-Amino-8-chloro-2-fluoro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4 - Synthesis of diol (I-17):

Dissolve A (0.30g, 1mmol) in 5mL DMF, add N-chlorosuccinimide (0.19g, 1.4mmol), heat up to 55°C for 3h, TLC monitors that the raw materials are completely consumed, dichloromethane (3 × 15 mL), the combined organic layers were washed with saturated NaCl aqueous solution, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound I-17 (0.30 g, 95%). ¹ H NMR (300MHz, DMSO-d ₆ )δ8.09(d, J=29.7Hz, 2H), 5.79(d, J=6.4Hz, 1H), 5.02–4.95(m, 1H), 4.19(dd, J=5.2, 2.9Hz, 1H), 3.93(q, J=4.9Hz, 1H), 3.58(ddd, J=44.2, 11.9, 5.1Hz, 2H).

Example 18: (2R,3R,4S,5S)-2-(6-Amino-8-chloro-2-fluoro-9H-purin-9-yl)-5-(chloromethyl)tetrahydrofuran-3,4 - Synthesis of diol (I-18):

The method is the same as that of I-17, and the yield is 94%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.08(d, J=30.2Hz, 2H), 5.83(d, J=5.5Hz, 1H), 5.66(d, J=5.8Hz, 1H), 5.55 (d,J=5.3Hz,1H),5.04(q,J=5.4Hz,1H),4.33(q,J=4.7Hz,1H),4.14–4.02(m,1H),3.99–3.75(m, 2H).

Example 19: Synthesis of 2-fluoro-9-methyl-9H-purin-6-amine (I-19):

Dissolve 2-fluoroadenine (0.15g, 1mmol) in DMF (5mL), add cesium carbonate (0.39g, 1.2mmol) and methyl iodide (0.17g, 1.2mmol); heat the reaction mixture to 60°C for 24h , and quenched with water (10 mL); the precipitate was filtered and dried in vacuo to give the product I-19 as a white powder (0.15 g, 91%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.08 (s, 1H), 7.77 (s, 2H), 3.68 (s, 3H).

Example 20: Synthesis of 2-fluoro-9-propyl-9H-purin-6-amine (I-20):

The method is the same as that of I-19, and the yield is 93%. ¹ H NMR (300MHz, DMSO-d ₆ )δ8.14(s,1H),7.76(s,2H),4.04(t,J=7.1Hz,2H),1.79(h,J=7.3Hz,2H) , 0.84 (t, J=7.4Hz, 3H).

Example 21: Synthesis of ((6-amino-2-fluoro-9H-purin-9-yl)methyl)phosphonic acid diethyl ester (I-21):

The method is the same as that of I-19, and the yield is 81%. ¹ H NMR (400MHz, Methanol-d ₄ ) δ8.05(d, J=0.8Hz, 1H), 4.70(d, J=11.9Hz, 2H), 4.26–4.09(m, 4H), 1.27(t, J = 7.1 Hz, 6H).

3. Biological evaluation experiment:

(1) Determination of the inhibitory effect on cancer cell proliferation in vitro:

Compounds are effective against triple-negative breast cancer MDA-MB-231 cell line, multiple myeloma RPMI8226 cell line, acute myeloid leukemia (HL60, K562, NB4) cell line, gastric cancer cell line HGC-27, liver cancer HepG2 cell line, human neural Glioma (U251) cell line, non-small cell lung cancer A549 cell line, prostate cancer (PC-3, Du-145, LNcap, Vcap, 22Rv1) cell line, human embryonic kidney cell HEK293, human normal prostate epithelial cell RWPE1, etc. The 16 cell proliferation inhibitory effects were tested by the following method.

Experimental steps:

According to the CCK-8 method, the inhibitory effect of the compound on the proliferation of various cancer cells was determined, and the half inhibitory concentration IC ₅₀ value of the compound's inhibitory cell proliferation activity was obtained;

1) Cells in the logarithmic growth phase were seeded in a 96-well plate at 5,000-20,000 cells/well, and cultured at 37°C and 5% CO ₂ for 12-24 hours;

2) Add 100 μL of a gradiently diluted solution of the compound to be tested at different concentrations to the culture plate, and incubate the culture plate at 37° C. in a 5% CO ₂ incubator for 72 hours;

3) 4 hours before the end of the incubation, add 10 μL of CCK-8 solution (5 mg/mL) to each well. After the incubation, the OD ₄₅₀ was measured with a microplate reader, and the inhibition rate = (OD value of the control group - OD value of the experimental group)/OD value of the control group × 100%;

4) After obtaining the data, GraphPad Prism 8.0 fits to obtain IC ₅₀ .

As can be seen from the experimental results, the example compound of the present invention has a strong inhibitory effect on the prostate cancer Du-145 cell line, and the specific results are shown in Table 1; in addition, the compound (I-1) of Example 1 is also effective on other various cancers The cells have a strong inhibitory effect. The measured _IC50 values are shown in Table 2; (HL60, K562, NB4) cell line, gastric cancer cell line HGC-27, liver cancer HepG2 cell line, human glioma (U251) cell line, non-small cell lung cancer A549 cell line and prostate cancer (PC-3, Du- 145, LNcap, Vcap, 22Rv1) cell lines have a strong proliferation inhibitory effect, but only a weak proliferation inhibitory effect on normal cells RWPE1 and HEK293 cells.

Table 1 Inhibitory effect of compounds on the proliferation activity of prostate cancer Du-145 cells

Example 100m inhibition rate (%) (IC ₅₀ /m) Example 100m inhibition rate (%) (IC ₅₀ /m)

1 >99(0.144) 12 >99(0.298) 2 96.27 13 >99(0.731) 3 0 14 >99(14.030) 4 78.19 15 0 5 0 16 0 6 >99(0.136) 17 0 7 >99(0.319) 18 14.51 8 >99(1.105) 19 >99(2.897) 9 >99(1.401) 20 23.95 10 >99(1.117) twenty one 6.23 11 >99(0.065) the the

Table 2 IC ₅₀ of Compound (I-1) Inhibiting the Proliferation Activity of Various Cancer Cells

(2), ADAR1 protein binding analysis (MST experiment):

Carry out compound and ADAR1 affinity determination by MST micro-thermophoresis instrument (Monolith NT.115); Calculate the binding constant KD of embodiment 1 compound I-1 and ADAR1 protein through three repeated experiments to be 7.24 ± 2.88 μ M (see figure 1), showing that the example compounds of the present invention have a better combination with ADAR1 protein.

(3), ADAR1 deaminase activity detection method:

In this experiment, Abcam’s Adenosine Deaminase (ADA) Activity Assay Kit (cat.ab204695) was used for detection; the difference in fluorescence values at two time points in the linear growth interval can be used to calculate the deactivation of ADAR1 by compound I-1 of Example 1 to be tested. The inhibitory ability IC ₅₀ of ammonia enzyme activity was 0.87 μM (see FIG. 2 ), showing that the example compounds of the present invention have strong inhibitory effect on the deamination activity of ADAR1 protein.

(4) Determination of acute toxicity of compounds:

Test animals: ICR mice; 18-22g; female; 70 in total;

Group dose setting: Preliminary experiments show that the tested drug has certain toxicity. The intraperitoneal injection of 500mg/kg dose of the drug can cause 4/4 mice to die, while the intraperitoneal injection of 100mg/kg dose can cause 0/4 mice to die. On the basis of the pre-experiment, the dose setting of the formal test of the drug is shown in Table 3:

Table 3 group dose settings

Laboratory environment: room temperature 24±2°C, relative humidity 60-70%. Observation indicators: The drug (compound I-1 prepared in Example 1) was administered by intraperitoneal injection twice at the above dose, once in the morning and afternoon, and the poisoning symptoms and death conditions of mice in each group were recorded, and the dead animals were autopsied; the observation period for 14 days. The results showed that compound I-1 had certain toxicity to mice, and it would cause death of mice after administration at a higher dose; the LD ₅₀ value of intraperitoneal injection was 186.5641 (160.9764-216.2190) mg/kg. No obvious abnormality was found in the autopsy of the dead animals.

The body weight change is shown in Figure 3. Compared with the control group, no obvious toxic reaction was seen.

The results of HE staining are shown in Figure 4. The compound (I-1) prepared in Example 1 has no obvious toxicity to the heart, liver, spleen, lung, kidney and other major organs.

(5) Determination of compound anti-prostate cancer (Du-145) activity:

Medicine is the compound (I-1) that embodiment 1 makes; Cell strain is human prostate cancer Du-145 cell; Experimental animal is SPF grade BALB/c nude mouse; Experimental animal is SPF grade BALB/c nude mouse Rats; male; 8 rats in the model group and 12 rats in the experimental group, a total of 32 rats; drug dosage settings are shown in Table 4.

Table 4 Drug dosage configuration

Experimental method: Collect cultured human prostate cancer Du ^- 145 cell suspension at a concentration of 1X10 cells/mL, inoculate 0.1 mL of each cell subcutaneously in the right axilla of nude mice; measure the diameter of the transplanted tumor with a vernier caliper, After 23 days of inoculation, when the tumor grows to ^70-100mm3 , the animals are randomly divided into groups; at the same time, the nude mice in each group start to be administered. The dosage regimen is shown in the group and dosage regimen. Using the method of measuring the diameter of the tumor, observe the samples dynamically antitumor effect of the product. Immediately after the experiment, the nude mice were sacrificed, and the tumor mass was surgically removed and weighed.

The experimental results are shown in Table 5: test drug embodiment 1 has obvious inhibitory effect on tumor growth, and the inhibitory effect of high dose group (20mg/kg) is better than low dose group (10mg/kg), the inhibitory effect of high and low dose group Tumor rates were 83.2% and 68.5%. Compared with the control group, the dosing group had no significant effect on the body weight of the animals. The specific tumor growth curve is shown in Figure 5.

Therefore, the test drug (I-1) prepared in Example 1 has obvious inhibitory effect on the growth of human prostate cancer Du-145 xenograft tumor in nude mice, has no obvious effect on the body weight of animals, and can be used as an ADAR1 target-related disease candidate therapeutic compounds.

Table 5 Effects of test samples on tumor growth of human prostate cancer cell Du-145 xenografted tumor in nude mice

Compared with the model control group, ^* P<0.05, ^** P<0.01

Compared with the model control group, ^* P<0.05, ^** P<0.01

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (10)

A polysubstituted purine compound, characterized in that it is a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof;

In the formula, R ₁ is selected from hydrogen, C ₁ -C ₃ alkyl; R ₂ is selected from halogen, -SCH ₂ CH ₂ COOH; R ₃ is selected from hydrogen, halogen, C ₁ -C ₃ alkyl; R ₄ is selected from C ₁ -C ₃ alkyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

A kind of polysubstituted purine compound according to claim 1, is characterized in that, R ₁ is selected from hydrogen, methyl; R ₂ is selected from fluorine, -SCH ₂ CH ₂ COOH; _R is selected from hydrogen, chlorine, methyl; R ₄ is selected from C ₁ -C ₃ alkyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

A kind of polysubstituted purine compound according to claim 1, is characterized in that, R ₁ is selected from hydrogen, methyl; R ₂ is selected from fluorine, -SCH ₂ CH ₂ COOH; _R is selected from hydrogen, chlorine, methyl; R ₄ is selected from methyl, propyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

A kind of polysubstituted purine compound according to claim 1, is characterized in that, R ₁ is selected from hydrogen; R ₂ is selected from fluorine; _R is selected from hydrogen; R ₄ is selected from methyl, propyl, -CH ₂ P(O)(OCH ₂ CH ₃ ) ₂ ,

R _is selected from the following groups:

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

A kind of polysubstituted purine compound according to claim 1, is characterized in that, R ₁ is selected from hydrogen; R ₂ is selected from fluorine; _R is selected from hydrogen; R ₄ is selected from methyl or

R ₆ is selected from hydroxyl, halogen, -SCH ₂ CH ₂ COOH or the following groups:

A kind of polysubstituted purine compound according to claim 1, is characterized in that, described compound is selected from I-1 to I-21:

A multi-substituted purine compound according to claim 1, wherein the pharmaceutically acceptable salt is an acid addition salt of a compound of general formula (I), wherein the acid used to form a salt includes an inorganic acid And organic acid, described inorganic acid comprises hydrochloric acid, sulfuric acid, phosphoric acid and methanesulfonic acid, and organic acid comprises acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, Cinnamic Acid, Citric Acid, Fumaric Acid, Camphoric Acid, Digluconic Acid, Aspartic Acid and Tartaric Acid. A preparation method of the multi-substituted purine compound according to claim 1. A pharmaceutical composition, characterized in that it comprises the compound of general formula (I) of claim 1 or a pharmaceutically acceptable salt or isomer thereof, and a pharmaceutically acceptable carrier. The use of a multi-substituted purine compound according to claim 1 in the preparation of medicines for the prevention and/or treatment of cancer or tumor-related diseases, which include prostate cancer, leukemia, breast cancer, Multiple myeloma, lung cancer, gastric cancer, ovarian cancer, colon cancer, liver cancer, pancreatic cancer, and human glioma.

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