CN115322226A - A kind of covalent targeting arsenic inhibitor and preparation method and application thereof - Google Patents

Abstract

The invention discloses a covalent targeting arsenic inhibitor, a preparation method and application thereof, wherein the structural formula is

The covalent targeting arsenic inhibitor designed by the invention has a targeting group and trivalent arsenic (As) ^III ) A reaction group, a targeting group is (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-amine group (Targeting group), capable of highly specifically Targeting Bruton's Tyrosine Kinase (BTK), trivalent arsenic (As) ^III ) The reactive group can be covalently combined with BTK cysteine residue (Cys 481) with high affinity, effectively inhibits a BTK-mediated B Cell Receptor (BCR) signal channel, leads to Ramos cell death, and has better in-vivo anti-tumor effectTumor proliferation activity.

Description

A kind of covalent target arsenic inhibitor and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a covalent targeting arsenic inhibitor and its preparation method and application.

Background technique

Arsenic (Arsenic, As) is widely known for its toxicity, but on the other hand, humans began to use inorganic arsenic compounds (realgar, realgar, and arsenic trioxide, etc.) as early as 2,000 years ago to treat carbuncles, cancerous ulcers, cancer and other diseases. Fowler's solution (1% potassium arsenite) was a common drug used to treat syphilis, leukemia, skin cancer, and other ailments in the 19th and early 20th centuries. The first chemotherapy drug in the modern sense, arsenamine, was also an arsenic drug. Especially arsenic (As ₂ O ₃ ) is famous for its excellent therapeutic effect on acute promyelocytic leukemia (APL).

The carcinogenic and anticancer properties of arsenic reflect its unique biological characteristics. The reason is that arsenic compounds and their metabolites change the conformation and function of proteins by combining with the sulfhydryl groups in protein cysteine, affecting the arsenic-binding protein. Physiological activity, causing arsenic to cause cancer or to fight cancer. The specific mechanism of As ₂ O ₃ to treat APL is that arsenic directly binds to the cysteine residue in the zinc finger motif of PML, changes the conformation of PML, induces PML-RARα oligomerization, ubiquitination, and degradation in the proteasome , eventually leading to the degradation of oncoproteins and apoptosis of APL cells. However, the systemic toxicity and low bioavailability of As ₂ O ₃ make its therapeutic effect on solid tumors and other cancers insignificant. Organic arsenic is easier to molecular design and chemical modification than inorganic arsenic, and it is an important way to develop arsenic-based drugs. Organoarsenic drugs with different chemical structures, such as Melarsoprol (Melarsoprol), MER1 (S-dimethylarsylsulfosuccinic acid), PAzPAO (phenylarsenic azide oxide), GSAO (4-(N- (S-glutathione acetyl) amino) phenyl arsenous acid) and Darinaparsin (dimethyl arsenyl glutathione) have been developed for anti-tumor or treatment of other diseases. However, the ever-developing small-molecule arsenic drugs have always been less toxic to cancer cells, and their IC ₅₀ is still at the micromolar level, and the Kd value between arsenic and thiol is at the micromolar level, with low affinity, making the existing arsenic drugs Unable to meet the needs of clinical medication. On the other hand, the existing small-molecule arsenic drugs have no specific targeting functional groups and randomly bind to cysteine, a protein widely present in cells, but cannot selectively bind to target oncoproteins. Therefore, the strategy of developing covalently targeted arsenic inhibitors with high selectivity and high affinity binding to specific oncoproteins not only provides new methods and ideas for the development of new arsenic-based anticancer drugs, but is also extremely necessary.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art and provide a covalent targeting arsenic inhibitor.

Another object of the present invention is to provide a method for preparing the above-mentioned covalent targeting arsenic inhibitor.

Another object of the present invention is to provide the application of the above-mentioned covalent targeting arsenic inhibitor.

Technical scheme of the present invention is as follows:

其中，R为

A kind of covalent target arsenic inhibitor, its structural formula is

Among them, R is

In a preferred embodiment of the present invention, said R is

The preparation method of the above-mentioned covalent targeting arsenic inhibitor comprises the following steps:

(1) Organic arsenic ligand, (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine- 4-Amine, EDC and NHS were dissolved in DMF, and triethylamine was added under ice bath to adjust the pH to 7.5-8.4, then the temperature was naturally raised to room temperature and reacted for 3-5h; the organic arsenic ligands were arsenoacetic acid, arsenopropionic acid- ethanedithiol or arsenobutyrate-ethanedithiol;

(2) Purifying the material obtained in step (1) with a silica gel column to obtain the covalently targeted arsenic inhibitor.

In a preferred embodiment of the present invention, the organic arsenic ligand is arsenoacetic acid, arsenoacetic acid, (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)- The molar ratio of 1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:0.25:2:1.2.

In a preferred embodiment of the present invention, the organic arsenic ligand is arsenopropionic acid-ethanedithiol, arsenopropionic acid-ethanedithiol, (R)-3-(4-phenoxyphenyl) The molar ratio of -1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:1:2:1.2.

In a preferred embodiment of the present invention, the organic arsenic ligand is arsenobutyric acid-ethanedithiol, arsenobutyric acid-ethanedithiol, (R)-3-(4-phenoxyphenyl) The molar ratio of -1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:1:2:1.2.

In a preferred embodiment of the present invention, in the silica gel column purification, n-hexane and ethyl acetate are mobile phases.

Application of the above-mentioned covalent targeting arsenic inhibitor in the preparation of antitumor drugs.

An antitumor drug, the active ingredient of which includes the above-mentioned covalent targeting arsenic inhibitor.

In a preferred embodiment of the present invention, its active ingredient is the above-mentioned covalent targeting arsenic inhibitor.

The beneficial effects of the present invention are: the covalent targeted arsenic inhibitor of the present invention has a targeting group and a trivalent arsenic (As ^III ) reactive group, and the covalent targeted arsenic inhibitor can highly specifically target BTK , and covalently binds to BTK cysteine residue (Cys481) with high affinity, effectively inhibits BTK-mediated B cell receptor (BCR) signaling pathway, leading to the death of Ramos cells, and has good anti-tumor proliferation activity in vivo .

Description of drawings

Fig. 1 is a structural diagram of the organic arsenic ligand and the covalent targeting arsenic inhibitor in Example 1 of the present invention.

Fig. 2 is the graph of the experimental results in Example 2 of the present invention, the dose-effect curve of inhibition of BTK kinase activity by arsenic-based compounds and Ibrutinib.

Fig. 3 is a diagram of the experimental results in Example 3 of the present invention, evaluating the covalent binding ability of the covalently targeted arsenic inhibitor to BTK in Ramos cells by competitive space-occupying fluorescent labeling. After PCI-33380 labeled Ramos cells, use gel electrophoresis and gel fluorescence scanning to detect the electrophoresis results of the probe label (about 78kDa, the molecular weight of BTK), and the band was confirmed to be BTK by Western blot.

Fig. 4 is that I-As-1 in the embodiment of the present invention 4, I-As-2, I-As-3 and I-As-V are to the BTK-mediated BCR signaling pathway induced by Anti-IgM stimulation in Ramos cells Inhibition results, Western blot experiments were labeled with corresponding antibodies.

Fig. 5 is the result figure that detects the proteome reactivity of I-As-1 and Ibrutinib in Ramos cells by gel electrophoresis and gel fluorescence scanning in Example 5 of the present invention, and the arrow indicates the main bar of the probe label Band (about 78kDa, expected molecular weight of BTK), which was confirmed to be BTK by Western blot.

Fig. 6 is the experimental result figure in the embodiment 6 of the present invention, wherein, (A) the dose-effect curve of the anti-proliferation activity of (A) covalent target arsenic inhibitor and Ibrutinib to Ramos cell, (B) Ramos cell to different concentrations of I-As -1, I-As-2, I-As-3, I-As-V and Ibrutinib uptake results.

Figure 7 is a graph showing the inhibition results of I-As-1 in Example 7 of the present invention on mouse transplanted tumors, wherein (A) I-As-1 can more effectively prevent tumor growth in mouse transplanted tumor model experiments , (B) The body weight of the mice changed over time during the study, with mice in each group gaining slightly by the end of the study.

Fig. 8 is a graph showing the biodistribution results of I-As-1 in Example 7 of the present invention in mouse heart, liver, spleen, lung, kidney, tumor and blood.

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

其中：The chemical structural formula of the covalent targeting arsenic inhibitor involved in the present invention is

in:

时，为(R)-3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3，4-d]嘧啶-4-砷乙酸(I-As-V)；when

, as (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenoacetic acid (I -As-V);

时，为(R)-3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3，4-d]嘧啶-4-砷乙酸-乙二硫醇(I-As-1)；when

When (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenoacetic acid-ethyl Dithiol (I-As-1);

时，为(R)-3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3，4-d]嘧啶-4-砷丙酸-乙二硫醇(I-As.2)；when

When (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenic acid- Ethanedithiol (I-As.2);

时，为(R)-3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3，4-d]嘧啶-4-砷丁酸-乙二硫醇(I-As-3)。when

When (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenobutanoic acid- Ethanedithiol (I-As-3).

The synthetic route of the preparation method of the covalent targeting arsenic inhibitor of the present invention is shown in Figure 1: firstly, the organic arsenic ligand (arsenoacetic acid/arsenopropionic acid-ethanedithiol/arsenobutyric acid-ethyl dithiol), and then the organic arsenic ligand with the targeting group (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3 , 4-d] pyrimidin-4-amine coupling to obtain a covalent targeting arsenic inhibitor (Fig. 1), which specifically includes the following steps:

(1) Synthesis of organic arsenic ligands

A, synthesis of arsenic acetic acid (As-1-V): 1.2g (6mmol) arsenic trioxide, 1.44g (36mmol) sodium hydroxide and 112mg (0.6mmol) trimethylbenzyl ammonia were dissolved in 12mL water, cooled to Below 20°C, 570 mg (6 mmol) of chloroacetic acid was added to it, and the reaction was carried out at room temperature for 5 h. Reaction finishes, add 2.16g (36mmol) glacial acetic acid and separate out unreacted inorganic arsenic, filter and remove precipitate, insert 2.16g (8.8mmol) barium chloride dihydrate in the filtrate, react at room temperature for 5h, generate white precipitate, filter, use After washing with ice water, the precipitate was ion-exchanged with polystyrene sulfonate hydrogen ion exchange resin (AmberliteR IR-120, H ⁺ ) for 1 h, and the water was spin-off to obtain arsenoacetic acid (0.9 g, 81%) as a white solid. ¹ H NMR (500MHz, D ₂ O) δ 3.62 (s, 2H). ¹³ C NMR (126 MHz, D ₂ O) δ 167.42, 38.29. HRMS (ESI): m/z Calcd for C ₂ H ₅ AsO ₅ [M+H] ⁺ 184.9426, found 184.9440.

B. Synthesis of arsenopropionic acid-ethanedithiol (As-2): 1.4g (7mmol) arsenic trioxide and 1.7g (42mmol) sodium hydroxide were added to 21mL water, stirred and dissolved at 45°C, and 0.73g (7.7mmol) was added dropwise ) 3-chloro-1-propanol, reacted at 55°C for 4h. Adjust the pH to 2 with concentrated hydrochloric acid, concentrate to 5 mL, add 45 mL of ethanol to desalt, spin off the ethanol to obtain a white solid, without further purification, mix the obtained solid with 6.13 g (28.7 mmol) of sodium periodate and 33.6 mg of trichloro Ruthenium chloride was added to a mixed solution of 21 mL of water, 14 mL of acetonitrile and 14 mL of ethyl acetate, and reacted overnight at room temperature. After the reaction was completed, add 25 mL of ethyl acetate to wash 3 times, add 98 mL of water to the lower turbid liquid, acidify with 1M hydrochloric acid to obtain a clear solution, add 245 mL of ethanol for extraction, filter the obtained yellow clear filtrate, concentrate to 70 mL, add 5.26 g (56 mmol) of 1 , 2-ethanedithiol, reacted at room temperature for 2h. The product was purified by reverse-phase chromatographic column to give arsenopropionic acid-ethanedithiol (0.65g, 38.5%) ¹ H NMR (600MHz, CDCl ₃ ) δ3.39-3.31(m, 4H), 2.67(t , J=7.6Hz, 2H), 2.03(t, J=7.6Hz, 2H). ¹³ C NMR (151MHz, CDCl ₃ ) δ179.19, 41.88, 30.36, 30.34. HRMS (ESI) m/z: calcd. for C ₅ H ₉ AsO ₂ S ₂ [M+H]+: 240.9333, found: 240.9335.

C. Synthesis of arsenic butyric acid-ethanedithiol (As-3): 0.2g (1mmol) arsenic trioxide was dissolved in 3mL 10M sodium hydroxide solution, 1.08g (5mmol) dibromobutane was dissolved in 0.5mL ethanol, at room temperature The dibromobutane solution was added dropwise to the arsenic trioxide solution, and the reaction was refluxed at 80°C for 12h. Regulate pH ≈ 9 with hydrochloric acid, centrifuge to remove unreacted arsenic trioxide, and use the supernatant to obtain a yellowish solid after decompression and rotary evaporation to remove water. Without further purification, the resulting solid is mixed with 0.43g (2.1mmol) sodium periodate and 2.4 mg of ruthenium trichloride was dissolved in 2.1 mL of water, 1.4 mL of acetonitrile and 1.4 mL of ethyl acetate, and reacted overnight at room temperature. After the reaction was completed, add 3 mL of ethyl acetate to wash 3 times, acidify with 1M hydrochloric acid to obtain a clear solution, add 20 mL of ethanol to extract, filter, add 0.38 g (4 mmol) of 1,2-ethanedithiol to the filtrate, and react at room temperature for 2 h. The product was purified by reverse-phase chromatography to give arsenobutyric acid-ethanedithiol (95 mg, 37%) as a white solid product. ¹ H NMR (500 MHz, Chloroform-d) δ3.36-3.30 (m, 4H), 2.45 (t , J=6.9Hz, 2H), 1.90-1.81 (m, 4H). ¹³ C NMR (126MHz, CDCl ₃ ) δ178.88, 41.75, 35.96, 35.01, 21.11. HRMS (ESI): m/z Calcd for C ₆ H ₁₁ AsO ₂ S ₂ [M+H] ⁺ 254.9489, found 254.9504.

(2) The above organic arsenic ligand and (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine -4-amine coupling to obtain the covalently targeted arsenic inhibitor of the present invention:

A, synthesis (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenic acid (I -As-V): 100mg (0.54mmol) As-1-V, 50mg (0.13mmol) (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H -Pyrazolo[3,4-d]pyrimidin-4-amine, 207mg (1.08mmol) EDC and 75mg (0.65mmol) NHS were dissolved in 10mL DMF, and triethylamine was added under ice cooling to adjust the pH=8, and the temperature was naturally raised to Reaction at room temperature for 4h. Centrifuged, after the gained supernatant was desolvated, washed 3 times with 15mL of pure water, dried to obtain the white solid product (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl )-1H-pyrazolo[3,4-d]pyrimidine-4-arsenoacetic acid (43mg, 61%) ¹ H NMR (500MHz, DMSO-d ₆ )δ8.26 (d, J=6.4Hz, 1H) , 7.67(dd, J=8.8, 2.5Hz, 2H), 7.52-7.38(m, 2H), 7.23-7.05(m, 5H), 4.87(m, 0.5H), 4.66(m, 0.5H), 4.56 (br d, J = 12.1Hz, 0.5H), 4.25 (br d, J = 13.2Hz, 0.5H), 4.13 (br d, J = 9.7Hz, 0.5H), 3.97 (br d, J = 13.5Hz , 0.5H), 3.75(m, 0.5H), 3.68(d, J=13.9Hz, 1H), 3.50(d, J=14.2Hz, 1H), 3.23-3.00(m, 1H), 2.87(m, 0.5H), 2.30-2.06(m, 2H), 1.94-1.73(m, 2H). ¹³ C NMR (151MHz, DMSO-d ₆ ) δ163.07, 158.65, 157.60, 156.78, 156.11, 154.38, 143.89, 143.67 , 130.61, 128.36, 124.26, 119.44, 97.82, 55.39, 53.04, 52.49, 50.66, 46.93, _46.08 , 42.07, 30.10, 29.96, 24.86, 23.75. HRMS (ESI): m/z Calcd for C ₂₄ N H ₆ O ₅ [M+H] ⁺ 553.1175, found: 553.1181.

B. Synthesis of (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenoacetic acid-ethyl Dithiol (I-As-1): 185mg (1mmol) As-1-V, 97mg (0.25mmol) (R)-3-(4-phenoxyphenyl)-1-(piperidine-3- Base)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 382mg (2mmol) EDC and 138mg (1.2mmmol) NHS were dissolved in 25mL DMF, and triethylamine was added under ice cooling to adjust the pH=8 , naturally warming to room temperature for 4h. Centrifuge, add 94.12mg (1mmol) 1,2-ethanedithiol to the supernatant, react at room temperature for 2h, and purify the product with a silica gel column, using n-hexane and ethyl acetate as the mobile phase (Rf=0.25, n-hexane:acetic acid Ethyl ester: = 1:1) to give (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d ] Pyrimidine-4-arsenoacetic acid-ethanedithiol (116 mg, 78%). ¹ H NMR (500MHz, Chloroform-d) δ8.26(d, J=20.4Hz, 1H), 7.58(d, J=8.1Hz, 2H), 7.46-7.36(m, 2H), 7.24-7.13(m , 3H), 7.09(d, J=8.0Hz, 2H), 6.33(br d, J=24.4Hz, 2H), 5.01-4.82(m, 1H), 4.74(br d, J=12.7Hz, 0.5H ), 4.55(br d, J=13.3Hz, 0.5H), 4.07(br d, J=17.3Hz, 0.5H), 3.87(br d, J=13.5Hz, 0.5H), 3.72(m, 0.5H ), 3.45-3.28(m, 5H), 3.08-2.92(m, 2H), 2.85(m, 0.5H), 2.42-2.20(m, 2H), 2.12-1.91(m, 1H), 1.82-1.66( m, 1H). ¹³ C NMR (101MHz, CDCl ₃ ) δ169.31, 163.89, 159.76, 155.75, 153.67, 151.56, 145.89, 130.15, 129.78, 124.58, 119.92, 119.26, 97.09, 54.49, 53.45, 45.65, 42.32, 42.26, 41.38, 41.13, 29.97, 25.02, 23.88. HRMS (ESI): m/z Calcd for C ₂₆ H ₂₇ AsN ₆ O ₂ S ₂ [M+H] ⁺ 595.0926, found: 595.0949.

C. Synthesis of (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenic acid- Ethanedithiol (I-As-2): 48mg (0.2mmol) As-2, 76mg (0.2mmol) (R)-3-(4-phenoxyphenyl)-1-(piperidine-3- Base)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 77mg (0.4mmol) EDC and 28mg (0.24mmol) NHS were dissolved in 8mL DMF, and triethylamine was added under ice-cooling to adjust the pH=8 , naturally warming to room temperature for 4h. The product was purified on a silica gel column, using n-hexane and ethyl acetate as mobile phases (Rf=0.3, n-hexane:ethyl acetate:=1:1) to obtain the white solid product (R)-3-(4-phenoxy Phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenopropionic acid-ethanedithiol (72 mg, 59%). ¹ H NMR (500MHz, Chloroform-d) δ8.33 (d, J=18.9Hz, 1H), 7.64 (dd, J=8.3, 5.4Hz, 2H), 7.43-7.36 (m, 2H), 7.22-7.13 ( m, 3H), 7.09(d, J=7.9Hz, 2H), 5.97(br s, 2H), 4.92-4.76(m, 1.5H), 4.51(br d, J=13.4Hz, 0.5H), 4.03 (br d, J=9.1Hz, 0.5H), 3.87 (br d, J=13.3Hz, 0.5H), 3.70(m, 0.5H), 3.39-3.12(m, 5H), 2.88-2.71(m, 2H), 2.65(m, 0.5H), 2.46-2.20(m, 2H), 2.08-1.93(m, 3H), 1.78-1.65(m, 1H). ¹³ C NMR (101MHz, CDCl ₃ ) δ171.39 ，158.75，157.73，156.25，155.17，154.10，144.29，130.03，127.38，124.18，119.63，119.17，98.55，53.38，52.56，49.87，45.95，45.59，42.16，41.61，31.99，30.23，29.85，25.51，25.04，23.89 .HRMS(ESI): m/z Calcd for C ₂₇ H ₂₉ AsN ₆ O ₂ S ₂ [M+H] ⁺ 609.1082, found: 609.1117.

D. Synthesis of (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenobutanoic acid- Ethanedithiol (I-As-3): 51mg (0.2mmol) As-3, 76mg (0.2mmol) (R)-3-(4-phenoxyphenyl)-1-(piperidine-3- Base)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 76mg (0.4mmol) EDC and 28mg (0.24mmol) NHS were dissolved in 4mLDMF, and triethylamine was added under ice-cooling to adjust the pH to about 8. Naturally warm up to room temperature and react for 4 hours. The product was purified with a silica gel column, using n-hexane and ethyl acetate as the mobile phase (Rf=0.36, n-hexane:ethyl acetate:=1:1), to obtain the white solid product (R)-3-(4-phenoxybenzene yl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-arsenobutanoic acid-ethanedithiol (70 mg, 54%). ¹ H NMR (500MHz, Chloroform-d) δ8.37 (d, J=22.6Hz, 1H), 7.66 (dd, J=8.4, 6.5Hz, 2H), 7.45-7.38 (m, 2H), 7.22-7.15 (m, 3H), 7.11(d, J=7.9Hz, 2H), 6.25-5.59(br s, 2H), 4.91-4.79(m, 1.5H), 4.58(br d, J=13.3Hz, 0.5H ), 4.05(br d, J=8.8Hz, 0.5H), 3.88(br d, J=13.6Hz, 0.5H), 3.75-3.64(m, 1H), 3.36-3.26(m, 5H), 3.16( m, 0.5H), 2.93-2.71(m, 1.5H), 2.53-2.21(m, 5H), 2.06-1.81(m, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ170.83, 158.71, 157.76 ，156.34，155.36，153.97，144.19，129.95，127.48，124.15，119.61，119.17，98.57，53.48，52.64，49.86，45.73，45.56，41.67，36.88，34.20，31.46，29.71，25.48，24.03，21.60，14.15.HRMS (ESI): m/z Calcd for C ₂₈ H ₃₁ AsN ₆ O ₂ S ₂ [M+H] ⁺ 623.1239, found: 623.1259.

Example 2 Evaluation of the covalent targeting arsenic inhibitor prepared in Example 1 on the inhibition of BTK at the kinase molecular level

Kinase TK试剂盒测量各个化合物对BTK的抑制效率，根据量效曲线(图2)确定各化合物对BTK抑制的IC₅₀值。结果显示，有机砷配体不能抑制BTK的活性，共价靶向砷抑制剂和Ibrutinib都能高效抑制BTK的活性，IC₅₀值在0.9nM-13.9nM(表1)。其中，三价砷药物I-As-1对BTK的IC₅₀为2.3nM，显著强于五价砷药物I-As-V，略强于三价砷药I-As-2和I-As-3。这表明，共价靶向砷抑制剂对BTK有很高的亲和力，是靶向基团和三价砷基团协同作用实现与BTK的高亲和力结合，并且碳链长度的短距离变化对共价靶向砷抑制剂和BTK亲和力的影响很小。BTK molecular level enzymatic analysis adopts

The Kinase TK kit measures the inhibitory efficiency of each compound on BTK, and determines the IC ₅₀ value of each compound on BTK inhibition according to the dose-effect curve (Figure 2). The results showed that organic arsenic ligands could not inhibit the activity of BTK, and both covalently targeted arsenic inhibitors and Ibrutinib could efficiently inhibit the activity of BTK, with IC ₅₀ values ranging from 0.9nM to 13.9nM (Table 1). Among them, the IC ₅₀ of the trivalent arsenic drug I-As-1 on BTK was 2.3nM, significantly stronger than the pentavalent arsenic drug I-As-V, and slightly stronger than the trivalent arsenic drug I-As-2 and I-As- 3. This indicates that the covalently targeted arsenic inhibitor has a high affinity for BTK, and that the targeting group and the trivalent arsenic group act synergistically to achieve high affinity binding to BTK, and the short-distance change of the carbon chain length has a significant effect on the covalent Targeted arsenic inhibitors and BTK affinity had little effect.

Table 1 IC ₅₀ values of arsenic-based compounds on BTK kinase

Example 3 Evaluation of covalent binding of the covalent targeting arsenic inhibitor prepared in Example 1 to BTK in Ramos cells

PCI-33380 probe is an ibrutinib derivative-like BTK covalent binding probe. In cells expressing BTK, the fluorescent bands of PCI-33380 and BTK can be detected by gel electrophoresis and fluorescent gel scanning , and the level of covalent binding between BTK inhibitors and BTK was assessed by competitive labeling. Before Ramos (6×10 ⁶ /2mL) cells were labeled with PCI-33380, they were incubated with different concentrations of covalent targeting arsenic inhibitors for 1h, washed with PBS 3 times to wash off the inhibitors, and then treated with PCI-33380 (2μM) Mark 1h. The cells were washed 3 times with PBS to remove excess probes, the cells were lysed with RIPA (containing phosphatase inhibitors and protease inhibitors) lysate, and then the cell lysates were analyzed by gel electrophoresis and gel fluorescence scanning. As shown in Figure 3, I-As-1 can fully covalently bind to BTK in Ramos cells at 60 nM, while the targeting group and I-As-V cannot covalently bind to BTK in Ramos cells at 35 μM. price combination. These results further prove that the covalently targeted arsenic inhibitor has a high affinity for BTK, and that the covalently targeted arsenic inhibitor has a high affinity for BTK due to the cooperation between the targeting group and the trivalent arsenic group.

Example 4 The effect of the covalently targeted arsenic inhibitor prepared in Example 1 on the BTK-mediated BCR signaling pathway This example studied the effect of the covalently targeted arsenic inhibitor on the Anti- Effects of BTK-mediated BCR signaling pathway activated by IgM stimulation. Ramos (6×10 ⁶ /2 mL) cells were pre-incubated with different concentrations of inhibitors for 1 h, washed 3 times with PBS, then stimulated with Anti-IgM (20 μg/mL) for 10 min, washed 1 time with PBS, and washed with RIPA ( Containing phosphatase inhibitors and protease inhibitors) lysate was lysed, and the cell lysate was analyzed by gel electrophoresis and Western blot. The results are shown in Figure 4, the trivalent arsenic drugs I-As-1, I-As-2 and I-As-3 can effectively target and inhibit the BCR signaling pathway at the nanomolar level. I-As-1 (64nM) significantly blocks autophosphorylation of BTK at Y223, phosphorylation of BTK physiological substrate PLCγ2 (Y1217) and phosphorylation of downstream kinases Erk1/2 (T202/Y204), but does not affect BTK Phosphorylation of the upstream kinase Syk (Y525/526). The pentavalent arsenic inhibitor I-As-V could not significantly affect the phosphorylation of BTK and its downstream kinases at 1 μM. These results suggest that I-As-1 can inhibit BTK activity with high selectivity and high affinity, thereby inhibiting BTK-mediated BCR signaling pathway.

Example 5 Evaluation of the selectivity of the covalent targeting arsenic inhibitor prepared in Example 1 to BTK in Ramos cells

This example compares the in situ response profiles of I-As-1 and Ibrutinib in the proteome of Ramos cells. Ramos (6×10 ⁶ /2mL) cells were incubated with different concentrations of I-As-1 or Ibrutinib (0.001-20μM) for 1h, washed 3 times with PBS to wash off the inhibitors, and then used high-concentration affinity probe PCI -33380 (20μM) labeled for 1h, cells were washed 3 times with PBS to remove excess probes, cells were lysed with RIPA (containing phosphatase inhibitors and protease inhibitors) lysate, detected by gel electrophoresis and fluorescent gel scanning to show off-target Fluorescent strips. In this example, it is observed that I-As-1 can competitively label BTK in Ramos cells with high selectivity in the range of 0.001-20 μM, block the fluorescent labeling of BTK, and do not affect the labeling of other off-target fluorescent bands, indicating that I -As-1 has good selectivity for BTK (Fig. 5).

Example 6 Evaluation of the Antiproliferative Activity of the Covalently Targeted Arsenic Inhibitor Prepared in Example 1 on Ramos Cells

In order to evaluate the antitumor activity of the covalently targeted arsenic inhibitor, this example evaluates its antiproliferative activity on Ramos cells. RBmos cells were seeded in 96-well plates (1×10 ⁴ cells/well/100 μL) and cultured for 12 hours, then treated with different concentrations of inhibitors (DMSO, 0.5%) for 72 hours, and the cell viability was measured with CCK-8 kit. The _IC50 values of the inhibitors on Ramos cells were determined according to the dose-response curve (Fig. 6A). The results are very exciting. Compared with Ibrutinib, trivalent arsenic compounds I-As-1, I-As-2 and I-As-3 all showed stronger anti-proliferation ability of Ramos cells. In particular, the IC ₅₀ value of I-As-1 for inhibiting the proliferation of Ramos was 0.5 μM, which was 24 times higher than that of Ibrutinib and more than 200 times higher than that of pentavalent arsenic I-As-V (Table 2). This example analyzes the possible biological basis of these results through cell uptake experiments. Ramos (5×10 ⁶ cells/2 mL) cells were cultured with different concentrations of I-As-1, I-As-2, I-As-3, I-As-V or Ibrutinib for 1 h, then washed 3 times with PBS, The cell extract was detected by HPLC-ICP-MS to detect the uptake of arsenic compounds by Ramos cells, and the uptake of Ibrutinib by Ramos cells was detected by HPLC-ESI-MS. As shown in Figure 6B, in the range of 2 μM-20 μM, the uptake of I-As-1 was 6.3-13.8 times and 19.0-54.8 times that of Ibrutinib and I-As-V, respectively. The mechanism should be that I-As-1 is more lipophilic than I-As-V and Ibrutinib, leading to easier uptake of I-As-1 by Ramos cells, which explains the higher lipophilicity of I-As-1 than Ibrutinib and I-As -V has stronger cytotoxicity. On the other hand, this example notes that I-As-1 is more cytotoxic than I-As-2 and I-As-3, but the cellular uptake of I-As-1 is slightly lower than that of I-As-2 and I-As-3. The main reason is that the affinity between I-As-1 and BTK is higher, which is more conducive to the covalent reaction with intracellular BTK, and inhibits the BCR signaling pathway to kill Ramos cells. Overall, covalently targeted arsenic inhibitors can selectively bind BTK with high affinity, inhibit the BCR signaling pathway, and potently kill Ramos cells.

Table 2 IC ₅₀ values of arsenic-based inhibitors on Ramos cells

Example 7 In vivo anti-tumor activity evaluation of the covalent targeting arsenic inhibitor prepared in Example 1

HS 15)、I-As-1(10mg/kg、20mg/kg).和Ibrutinib(10mg/kg)治疗14天，给药体积为0.1mL/d/只。如图7A所示，由于Ramos细胞形成的小鼠异种移植瘤松散且不致密，当可以确认肿瘤形成时，肿瘤的初始大小较大，达到约300mm³。当治疗结束后，与未治疗组相比，Ibrutinib治疗组(10mg/kg)的肿瘤体积只减小了15％(P＝0.513)，相同剂量I-As-1治疗组的肿瘤体积减小了17％(P＝0.192)。令人惊喜的是，I-As-1(20mg/kg)治疗组的肿瘤体积减小了33％(P＝0.02)。另一方面，Ibrutinib给药15mg/kg后，小鼠出现死亡，因此未评估Ibrutinib(20mg/kg)剂量组。治疗14天后，各组小鼠体重略有增加，治疗期间体重无明显波动，表明I-As-1毒性低，具有进一步开发应用的前景(图7B)。In order to further evaluate the antitumor activity of the covalently targeted arsenic inhibitor targeting BTK, in this example, SCID mice were inoculated with Ramos cells (5×10 ⁶ /0.1mL/only) to construct a mouse xenograft tumor model, and I- As-1 and Ibrutinib were compared to evaluate the antitumor activity of the drug on xenografted tumors in mice. When the tumor volume was measurable, the mice were randomly divided into four groups (5 mice in each group), and Vehicle (75% normal saline, 5% DMSO and 20%

HS 15), I-As-1 (10mg/kg, 20mg/kg). And Ibrutinib (10mg/kg) were treated for 14 days, and the administration volume was 0.1mL/d/monkey. As shown in FIG. 7A , since the mouse xenograft tumor formed by Ramos cells was loose and not dense, when tumor formation could be confirmed, the initial size of the tumor was large, reaching about 300 mm ³ . When the treatment ended, compared with the untreated group, the tumor volume of the Ibrutinib treatment group (10mg/kg) was only reduced by 15% (P=0.513), and the tumor volume of the same dose I-As-1 treatment group was reduced 17% (P=0.192). Surprisingly, the tumor volume of the I-As-1 (20 mg/kg) treatment group was reduced by 33% (P=0.02). On the other hand, mice died after administration of 15 mg/kg Ibrutinib, so the Ibrutinib (20 mg/kg) dose group was not evaluated. After 14 days of treatment, the body weight of mice in each group increased slightly, and there was no significant fluctuation in body weight during the treatment, indicating that I-As-1 has low toxicity and has the prospect of further development and application (Fig. 7B).

On the other hand, arsenic is an elemental mass label that can be used to conveniently detect the biodistribution of arsenic drugs in mice by ICP-MS. After the tumor treatment experiment, this example detects the arsenic content in the mouse heart, liver, spleen, lung, kidney, tumor and blood by ICP-MS, and observes the arsenic accumulation of I-As-1 in the main organs or tissues . The results are shown in Figure 8, I-As-1 is the most abundant in tumor tissue and tends to target tumors.

In summary, the present invention designs and synthesizes a covalently targeted arsenic inhibitor with high selectivity and high affinity covalently binding to the oncoprotein BTK, inhibits the BCR signaling pathway mediated by BTK, efficiently inhibits the activity of Ramos cells, and can Inhibits the growth of xenografted tumors in mice. The results show that our strategy provides a clear and reasonable design idea and scheme for the development of new arsenic-based antitumor drugs.

The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention In the range.

Claims (10)

1. A covalent targeting arsenic inhibitor, characterized in that: its structural formula is

Among them, R is

2. A kind of covalent target arsenic inhibitor as claimed in claim 1, is characterized in that: described R is

3. The preparation method of a covalent targeting arsenic inhibitor according to claim 1 or 2, characterized in that: comprising the steps of: (1) Organic arsenic ligand, (R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine- 4-amine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in DMF, and three Ethylamine adjusts the pH to 7.5-8.4, then naturally warms up to room temperature and reacts for 3-5 hours; the organic arsenic ligand is arsenoacetic acid, arsenopropionic acid-ethanedithiol or arsenobutyric acid-ethanedithiol; (2) The material obtained in step (1) is purified with a silica gel column or directly spin-dried and washed with water to obtain the covalently targeted arsenic inhibitor. 4. preparation method as claimed in claim 3 is characterized in that: described organic arsenic ligand is arsenic acetic acid, arsenic acetic acid, (R)-3-(4-phenoxyphenyl)-1-(piperidine The molar ratio of -3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:0.25:2:1.2. 5. The preparation method according to claim 3, characterized in that: the organic arsenic ligand is arsenopropionic acid-ethanedithiol, arsenopropionic acid-ethanedithiol, (R)-3-(4- The molar ratio of phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:1:2:1.2 . 6. The preparation method according to claim 3, characterized in that: the organic arsenic ligand is arsenobutyric acid-ethanedithiol, arsenobutyric acid-ethanedithiol, (R)-3-(4- The molar ratio of phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, EDC and NHS is 1:1:2:1.2 . 7. The preparation method according to any one of claims 3 to 6, characterized in that: in the silica gel column purification, n-hexane and ethyl acetate are mobile phases. 8. The application of the covalent targeted arsenic inhibitor according to claim 1 or 2 in antitumor drugs. 9. An antineoplastic drug, characterized in that its active ingredient comprises the covalent targeting arsenic inhibitor according to claim 1 or 2. 10. An antineoplastic drug as claimed in claim 9, characterized in that its active ingredient is the covalent targeting arsenic inhibitor as claimed in claim 1 or 2.

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