CN115322226A - Covalent targeting arsenic inhibitor and preparation method and application thereof - Google Patents
Covalent targeting arsenic inhibitor and preparation method and application thereof Download PDFInfo
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- CN115322226A CN115322226A CN202210989763.8A CN202210989763A CN115322226A CN 115322226 A CN115322226 A CN 115322226A CN 202210989763 A CN202210989763 A CN 202210989763A CN 115322226 A CN115322226 A CN 115322226A
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- btk
- ethanedithiol
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- GPSQYTDPBDNDGI-MRXNPFEDSA-N 3-(4-phenoxyphenyl)-1-[(3r)-piperidin-3-yl]pyrazolo[3,4-d]pyrimidin-4-amine Chemical group C1=2C(N)=NC=NC=2N([C@H]2CNCCC2)N=C1C(C=C1)=CC=C1OC1=CC=CC=C1 GPSQYTDPBDNDGI-MRXNPFEDSA-N 0.000 claims abstract description 14
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/66—Arsenic compounds
- C07F9/70—Organo-arsenic compounds
- C07F9/80—Heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a covalent targeting arsenic inhibitor, and a preparation method and application thereof.
Background
Arsenic (Arsenic, as) is widely known for its toxicity, but in the early 2000 years, people have begun to use inorganic Arsenic compounds (realgar, arsenic trioxide, etc.) for treating carbuncle and cancerUlcer, cancer, etc. Fowler's solution (1% potassium arsenite) is a common drug for the treatment of syphilis, leukemia, skin cancer and other diseases in the first 19 th to 20 th century. The first chemotherapeutic drug in the modern sense, arsine sodium vandamate, is also an arsenic drug. In particular arsenic (As) 2 O 3 ) Is known for its excellent therapeutic effect on Acute Promyelocytic Leukemia (APL).
The unique biological characteristics of arsenic carcinogenesis and anticancer property reflect, and the arsenic carcinogenesis or anticancer property is caused because the arsenic compound and the metabolite thereof change the conformation and the function of protein through the combination with the sulfhydryl in protein cysteine, influence the physiological activity of arsenic binding protein and cause the arsenic carcinogenesis or the anticancer. As 2 O 3 The specific mechanism for treating APL is that arsenic is directly combined with cysteine residue in zinc finger motif of PML part, the conformation of PML is changed, PML-RAR alpha oligomerization and ubiquitination are induced, and degradation is carried out in proteasome, and finally oncoprotein degradation and APL apoptosis are caused. However, as 2 O 3 Its systemic toxicity and low bioavailability make its therapeutic effect on solid tumors and other cancers insignificant. Organic arsenic is easier than inorganic arsenic in molecular design and chemical modification, and is an important approach for developing arsenic-based drugs. Organic arsenic drugs having different chemical structures, such as Melarsoprol (melarsol), MER1 (S-dimethyl arsenylthiosuccinate), PAzPAO (para-azidophenylarsenic acid), GSAO (4- (N- (S-glutathione acetyl) amino) phenyl arsenious acid) and darinapasin (dimethyl arsenyl glutathione) have been developed for anti-tumor or treatment of other diseases. However, the toxicity of the developing small molecule arsenic drugs to cancer cells has been low, and their IC has been low 50 Still in micromolar level, and the Kd value between arsenic and sulfydryl is in micromolar level, and the affinity is low, so that the prior arsenic medicament can not meet the clinical medication requirement. On the other hand, the existing small molecular arsenic drugs have no specific targeting functional groups, can randomly combine with protein cysteine which is widely existed in cells, and cannot selectively combine with target cancer protein. Therefore, the development of a strategy of covalently targeting arsenic inhibitors with high selectivity and high affinity binding with specific oncoproteins not only provides a new method and thought for developing novel arsenic-based anticancer drugsWays, too, are extremely necessary.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a covalent targeting arsenic inhibitor.
Another object of the present invention is to provide a method for preparing the above-mentioned covalently targeted arsenic inhibitors.
It is a further object of the present invention to provide the use of the above covalently targeted arsenic inhibitors.
The technical scheme of the invention is as follows:
a covalent targeting arsenic inhibitor has a structural formula
Wherein R is
In a preferred embodiment of the invention, R is
The preparation method of the covalent targeting arsenic inhibitor comprises the following steps:
(1) Dissolving organic arsenic ligand, (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, EDC and NHS in DMF, adding triethylamine under ice bath to adjust the pH to 7.5-8.4, naturally heating to room temperature, and reacting for 3-5H; the organic arsenic ligand is arsenic acetic acid, arsenic propionic acid-ethanedithiol or arsenic butyric acid-ethanedithiol;
(2) And (2) purifying the material obtained in the step (1) by using a silica gel column to obtain the covalent targeting arsenic inhibitor.
In a preferred embodiment of the invention, the organic arsenic ligand is arsenic acetic acid, (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine, EDC and NHS in a molar ratio of 1: 0.25: 2: 1.2.
In a preferred embodiment of the invention, the organic arsenic ligand is arsenopropionic acid-ethanedithiol, the molar ratio of arsenopropionic acid-ethanedithiol, (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine, EDC and NHS is 1: 2: 1.2.
In a preferred embodiment of the invention, the organic arsenic ligand is arsenobutyric acid-ethanedithiol, (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine, EDC and NHS in a molar ratio of 1: 2: 1.2.
In a preferred embodiment of the present invention, n-hexane and ethyl acetate are mobile phases in the silica gel column purification.
The covalent targeting arsenic inhibitor is applied to the preparation of antitumor drugs.
An antitumor drug comprises the covalent targeting arsenic inhibitor as an active ingredient.
In a preferred embodiment of the invention, the active ingredient is the above-mentioned covalently targeted arsenic inhibitors.
The beneficial effects of the invention are: the covalent targeting arsenic inhibitor has a targeting group and trivalent arsenic (As) III ) The covalent targeting arsenic inhibitor can highly specifically target BTK, is covalently bound with BTK cysteine residue (Cys 481) with high affinity, effectively inhibits a BTK-mediated B Cell Receptor (BCR) signal pathway, leads to Ramos cell death, and has better in-vivo anti-tumor proliferation activity.
Drawings
FIG. 1 is a block diagram of an organic arsenic ligand and a covalently targeted arsenic inhibitor of example 1 of the present invention.
FIG. 2 is a graph showing the results of experiments in example 2 of the present invention, which shows dose-response curves of arsenic-based compounds and Ibrutinib for the inhibition of BTK kinase activity.
FIG. 3 is a graph showing the results of experiments in example 3 of the present invention in which the covalent binding ability of a covalently targeted arsenic inhibitor to BTK in Ramos cells was evaluated by competition for placeholder fluorescent labels. After the Ramos cells are marked by PCI-33380, gel electrophoresis and gel fluorescence scanning are used for detecting an electrophoresis result chart of the marked probes (about 78kDa, molecular weight of BTK), and a band is proved to be BTK by Western blot detection.
FIG. 4 is a graph showing the results of inhibition of BTK-mediated BCR signaling pathway induced by Anti-IgM stimulation in Ramos cells by I-As-1, I-As-2, I-As-3 and I-As-V in example 4 of the present invention, labeled with corresponding antibodies in Western blot experiment.
FIG. 5 is a graph showing the results of detection of 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 arrows indicate the major bands of probe labeling (about 78kDa, expected molecular weight of BTK), which was confirmed to be BTK by Western blot detection.
FIG. 6 is a graph showing the results of experiments in example 6 of the present invention, in which (A) the dose-response curves of covalently targeted arsenic inhibitors and Ibrutinib on the antiproliferative activity of Ramos cells and (B) the uptake results of Ramos cells at different concentrations of I-As-1, I-As-2, I-As-3, I-As-V and Ibrutinib.
FIG. 7 is a graph showing the results of inhibition of mouse graft tumors by I-As-1 in example 7 of the present invention, wherein (A) I-As-1 was more effective in preventing tumor growth in a mouse graft tumor model experiment, and (B) the body weight of mice was varied with time during the study and the body weight of mice in each group was slightly increased at the end of the study.
FIG. 8 is a graph showing the results of biodistribution of I-As-1 in the heart, liver, spleen, lung, kidney, tumor and blood of mice in example 7 of the present invention.
Detailed Description
The technical solution of the present invention is further illustrated and described by the following detailed description in conjunction with the accompanying drawings.
The chemical structural formula of the covalent targeting arsenic inhibitor is shown in the specification
Wherein:
when in use
When it is (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenic acetic acid (I-As-V);
when in use
When is (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenoacetic acid-ethanedithiol (I-As-1);
when in use
When is (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenopropionic acid-ethanedithiol (I-As.2);
when the temperature is higher than the set temperature
When is (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenobutyric acid-ethanedithiol (I-As-3).
The synthetic route of the preparation method of the covalent targeting arsenic inhibitor is shown in figure 1: firstly, preparing carboxyl modified organic arsenic ligand (arsenic acetic acid/arsenic propionic acid-ethanedithiol/arsenic butyric acid-ethanedithiol), then coupling the organic arsenic ligand with a targeting group (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine to obtain a covalent targeting arsenic inhibitor (figure 1), and specifically comprising the following steps:
(1) Synthesis of organic arsenic ligands
A. Synthesis of arsenic acetic acid (As-1-V): 1.2g (6 mmol) of arsenic trioxide, 1.44g (36 mmol) of sodium hydroxide and 112mg (0.6 mmol) of trimethylbenzylamine are dissolved in 12mL of water, cooled to below 20 ℃ in an ice bath, 570mg (6 mmol) of chloroacetic acid are added thereto, and reacted at room temperature for 5 hours. After the reaction was completed, 2.16g (36 mmol) of glacial acetic acid was added to precipitate unreacted inorganic arsenic, the precipitate was removed by filtration, 2.16g (8.8 mmol) of barium chloride dihydrate was added to the filtrate, and the reaction was carried out at room temperature for 5 hours to produce a white precipitate, which was filtered, washed with ice water, and the precipitate was washed with polystyrene sulfonic acid hydrogen ion exchange resin (Amberlite R IR-120, H + ) Ion exchange was carried out for 1h and water was removed to give the product as arsenic acetic acid (0.9g, 81%) as a white solid. 1 H NMR(500MHz，D 2 O)δ3.62(s，2H). 13 C NMR(126MHz，D 2 O)δ167.42，38.29.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 (7 mmol) of arsenic trioxide and 1.7g (42 mmol) of sodium hydroxide are added to 21mL of water, dissolved by stirring at 45 ℃, 0.73g (7.7 mmol) of 3-chloro-1-propanol is added dropwise, and the reaction is carried out for 4h at 55 ℃. The resulting solid was added with 6.13g (28.7 mmol) of sodium periodate and 33.6mg of ruthenium trichloride to a mixed solution of 21mL of water, 14mL of acetonitrile and 14mL of ethyl acetate without further purification, and reacted overnight at room temperature. After the reaction is finished, 25mL ethyl acetate is added for washing for 3 times, 98mL water is added into lower turbid liquid, 1M hydrochloric acid is used for acidification to obtain clear solution, 245mL ethanol is added for extraction, yellow clear filtrate obtained through filtration is concentrated to 70mL, 5.26g (56 mmol) of 1, 2-dithioglycol is added, and the reaction is carried out for 2 hours at room temperature. The product was purified by reverse phase chromatography to give a white solid product, 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 Arsenobutyric acid-ethanedithiol (As-3): 0.2g (1 mmol) of arsenic trioxide was dissolved in 3mL of 10M sodium hydroxide solution, 1.08g (5 mmol) of dibromobutane was dissolved in 0.5mL of ethanol, the dibromobutane solution was dropwise added to the arsenic trioxide solution at room temperature, and the reaction was refluxed at 80 ℃ for 12 hours. Adjusting pH to be approximately 9 by hydrochloric acid, centrifuging to remove unreacted arsenic trioxide, carrying out reduced pressure rotary evaporation on supernate to remove water to obtain a yellowing solid, dissolving the yellowing solid with 0.43g (2.1 mmol) of sodium periodate and 2.4mg of ruthenium trichloride with 2.1mL of water, 1.4mL of acetonitrile and 1.4mL of ethyl acetate without further purification, and reacting at room temperature overnight. After the reaction is finished, 3mL ethyl acetate is added for washing for 3 times, 1M hydrochloric acid is used for acidification to obtain clear solution, 20mL ethanol is added for extraction, filtration is carried out, and the filtrate is added with0.38g (4 mmol) of 1, 2-ethanedithiol are reacted at room temperature for 2h. The product was purified by reverse phase chromatography to give a white solid product, arsenobutyric acid-ethanedithiol (95mg, 37%) 1 H NMR(500MHz，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) Coupling the organic arsenic ligand with (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine to obtain the covalent targeted arsenic inhibitor of the invention:
A. synthesis of (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenic acetic acid (I-As-V): 100mg (0.54 mmol) As-1-V,50mg (0.13 mmol) (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine, 207mg (1.08 mmol) EDC and 75mg (0.65 mmol) NHS were dissolved in 10mL DMF, adjusted pH =8 with triethylamine in ice bath, and allowed to warm to room temperature for 4h. Centrifuging, washing the obtained supernatant with 15mL pure water for 3 times, and drying to obtain 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 H 25 AsN 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-ethanedithiol (I-As-1): 185mg (1 mmol) As-1-V, 97mg (0.25 mmol) (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine, 382mg (2 mmol) of EDC and 138mg (1.2 mmol) of NHS were dissolved in 25mL of DMF, adjusted to pH =8 with triethylamine in ice bath, and allowed to warm to room temperature naturally for 4h. Centrifuging, adding 94.12mg (1 mmol) of 1, 2-ethanedithiol to the supernatant, reacting at room temperature for 2H, purifying the product with silica gel column, and using n-hexane and ethyl acetate as mobile phase (Rf =0.25, n-hexane: ethyl acetate: =1: 1) to obtain (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3, 4-d) as white solid]Pyrimidine-4-arsenoacetic acid-ethanedithiol (116mg, 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 )δ169.31，163.89，159.76，155.75，153.67，151.56，145.89，130.15，129.78，124.58，119.92，119.26，97.09，54.49，53.45，50.75，46.72，45.65，42.32，42.26，41.38，41.13，29.97，25.02，23.88.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-arsenopropionic acid-ethanedithiol (I-As-2): 48mg (0.2 mmol) As-2, 76mg (0.2 mmol) (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine, 77mg (0.4 mmol) EDC and 28mg (0.24 mmol) NHS were dissolved in 8mL DMF, pH =8 was adjusted with triethylamine under ice bath, and the reaction was allowed to warm to room temperature for 4h. Purifying the product with silica gel column and n-hexaneAnd ethyl acetate as a mobile phase (Rf =0.3, n-hexane: ethyl acetate: = 1), to give the product (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidine-4-arsenopropionic acid-ethanedithiol (72mg, 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-arsenobutyric acid-ethanedithiol (I-As-3): 51mg (0.2 mmol) As-3, 76mg (0.2 mmol) (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d]Pyrimidin-4-amine, 76mg (0.4 mmol) EDC and 28mg (0.24 mmol) NHS were dissolved in 4mL DMF, adjusted to pH 8 with triethylamine in ice bath, and allowed to warm to room temperature naturally for 4h. The product was purified by a silica gel column using n-hexane and ethyl acetate as mobile phases (Rf =0.36, n-hexane: ethyl acetate: =1: 1) to give the product (R) -3- (4-phenoxyphenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3, 4-d) as a white solid]Pyrimidine-4-arsenobutyric acid-ethanedithiol (70mg, 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 inhibition of BTK at the kinase molecular level by the covalently targeted arsenic inhibitor prepared in example 1
BTK molecular level enzymatic analysis
The Kinase TK kit measures the BTK inhibition efficiency of each compound, and the IC of each compound for BTK inhibition is determined according to a dose-response curve (figure 2) 50 The value is obtained. The results show that the organic arsenic ligand can not inhibit the activity of BTK, and the covalent targeting arsenic inhibitor and Ibrutinib can both effectively inhibit the activity of BTK, IC 50 Values ranged from 0.9nM to 13.9nM (Table 1). Wherein the trivalent arsenic drug I-As-1 is IC for BTK 50 Is 2.3nM, significantly stronger than pentavalent arsenic drugs I-As-V, slightly stronger than trivalent arsenic drugs I-As-2 and I-As-3. This indicates that the covalent targeted arsenic inhibitor has high affinity for BTK, and is high affinity binding with BTK achieved by synergistic effect of the targeting group and the trivalent arsenic group, and the short-distance change in carbon chain length has little effect on the affinity of the covalent targeted arsenic inhibitor and BTK.
TABLE 1 IC of arsenic-based Compounds on BTK kinase 50 Value of
Example 3 evaluation of covalent binding of covalently targeted arsenic inhibitors prepared in example 1 to BTK in Ramos cells
The PCI-33380 probe is an ibutinib derivative BTK covalent binding probe and expresses BTIn K cells, the fluorescent band of PCI-33380 bound to BTK can be detected by gel electrophoresis and fluorescent gel scanning, and the level of covalent binding between BTK inhibitor and BTK can be assessed by competitive labeling. Ramos (6X 10) 6 2 mL) cells were incubated with various concentrations of covalently targeted arsenic inhibitor for 1h, washed 3 times with PBS to wash out the inhibitor, and then labeled with PCI-33380 (2. Mu.M) for 1h before labeling with PCI-33380. Cells were washed 3 times with PBS to remove excess probe, lysed with RIPA (phospho-and protease inhibitor) -lysis solution, and then analyzed by gel electrophoresis and gel fluorescence scanning. As shown in FIG. 3, I-As-1 was able to bind covalently to BTK in Ramos cells at 60nM, while the targeting group and I-As-V were still unable to bind covalently to BTK in Ramos cells at 35. Mu.M. These results further demonstrate that the covalently targeted arsenic inhibitors have a high affinity for BTK, and that the targeting group and trivalent arsenic group act synergistically to provide a high affinity for BTK for the covalently targeted arsenic inhibitors.
Example 4 effect of covalently targeted arsenic inhibitors prepared in example 1 on BTK-mediated BCR signaling pathway the effect of covalently targeted arsenic inhibitors on Anti-IgM stimulated activated BTK-mediated BCR signaling pathway in Ramos cells was investigated by immunoblotting (Western blot). Ramos (6X 10) 6 /2 mL) cells were preincubated for 1h with different concentrations of inhibitor, washed 3 times with PBS, then stimulated with Anti-IgM (20. Mu.g/mL) for 10min, cells washed 1 time with PBS and lysed with RIPA (phospho-and proteinase inhibitor) -lysate, and cell lysates were analyzed by gel electrophoresis and Western blot. As shown in FIG. 4, the trivalent arsenic drugs I-As-1, I-As-2 and I-As-3 can effectively target and inhibit BCR signaling pathway at nanomolar level. I-As-1 (64 nM) significantly blocked the autophosphorylation of BTK at Y223, phosphorylation of the BTK physiological substrate PLC γ 2 (Y1217) and phosphorylation of the downstream kinase Erk1/2 (T202/Y204), but did not affect phosphorylation of the BTK upstream kinase Syk (Y525/526). The pentavalent arsenic inhibitor I-As-V still does not significantly affect the phosphorylation of BTK and its downstream kinases at 1. Mu.M. These results indicate that I-As-1 can inhibit BTK activity with high selectivity and high affinity, thereby inhibiting the BTK-mediated BCR signaling pathway.
Example 5 evaluation of the selectivity of covalently targeted arsenic inhibitors prepared in example 1 for BTK in Ramos cells
This example compares the in situ reaction profiles of proteomes for I-As-1 and Ibrutinib in Ramos cells. Ramos (6X 10) 6 /2 mL) cells were incubated with different concentrations of I-As-1 or Ibrutinib (0.001-20. Mu.M) for 1h, washed 3 times with PBS to wash away the inhibitor, then labeled with an excess concentration of affinity probe PCI-33380 (20. Mu.M) for 1h, washed 3 times with PBS to remove excess probe, lysed with RIPA (phosphatase inhibitor and protease inhibitor) lysate, gel electrophoresis and fluorescence gel scan to reveal off-target fluorescent bands. In the example, I-As-1 is observed to be capable of competitively labeling BTK in Ramos cells with high selectivity in the range of 0.001-20 mu M, blocking the fluorescent labeling of BTK, and not affecting the labeling of other off-target fluorescent bands, which shows that I-As-1 has good selectivity to BTK (figure 5).
Example 6 evaluation of antiproliferative Activity of covalently targeted arsenic inhibitors prepared in example 1 on Ramos cells
To evaluate the antitumor activity of the covalently targeted arsenic inhibitors, the antiproliferative activity against Ramos cells was evaluated in this example. RBmos cells were seeded in 96-well plates (1X 10) 4 cells/well/100 μ L) for 12h, followed by 72h treatment with different concentrations of inhibitor (DMSO, 0.5%), and cell viability was determined using the CCK-8 kit. Determination of IC of inhibitors on Ramos cells from dose-response curves (FIG. 6A) 50 The value is obtained. The result is very exciting, and the trivalent arsenic compounds I-As-1, I-As-2 and I-As-3 have stronger anti-Ramos cell proliferation capacity compared with Ibrutinib. In particular, I-As-1 inhibits Ramos proliferation 50 The value is 0.5 mu M, which is improved by 24 times compared with Ibrutinib and is improved by more than 200 times compared with pentavalent arsenic I-As-V (Table 2). This example analyzes the possible biological basis of these results by cell uptake experiments. Culturing Ramos (5X 10) with different concentrations of I-As-1, I-As-2, I-As-3, I-As-V or Ibrutinib 6 cells/2 mL) for 1h, then washing with PBS for 3 times, detecting the uptake of arsenic-containing compounds by Ramos cells by using HPLC-ICP-MS of cell extract, and detecting the uptake of Ibrutinib by Ramos cells by HPLC-ESI-MS of cell extract. As shown in FIG. 6B, the uptake of I-As-1 was in the range of 2. Mu.M to 20. Mu.MThe amount is 6.3-13.8 times and 19.0-54.8 times of 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, resulting in I-As-1 being more readily taken up by Ramos cells, which explains I-As-1 is more cytotoxic than Ibrutinib and I-As-V. On the other hand, this example notes that I-As-1 is more cytotoxic than I-As-2 and I-As-3, but that I-As-1 is taken up by cells in slightly lower amounts than 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 favorable for generating covalent reaction with BTK in cells and inhibiting BCR signal channel to kill Ramos cells. In general, the covalent targeting arsenic inhibitor can selectively and high-affinity target-bind BTK, inhibit a BCR signal channel and kill Ramos cells strongly.
TABLE 2 IC of arsenyl inhibitors on Ramos cells 50 Value of
Example 7 evaluation of in vivo antitumor Activity of covalently targeted arsenic inhibitors prepared in example 1
To further evaluate the antitumor activity of BTK-targeted covalently targeted arsenic inhibitors, SCID mice were inoculated with Ramos cells (5 × 10) 6 0.1 mL/mouse) to construct a mouse graft tumor model, and the antitumor activity of the drug on the mouse graft tumor is evaluated by the relative ratio of I-As-1 and Ibrutinib. When tumor volume was measurable, mice were randomly divided into four groups (5 groups), and Vehicle (75% physiological saline, 5% dmso and 20% w/w) was administered to the tail vein separately
HS 15), I-As-1 (10 mg/kg, 20 mg/kg), and Ibrutinib (10 mg/kg) for 14 days at a dose volume of 0.1 mL/d/tube. As shown in FIG. 7A, since mouse xenograft tumors formed by Ramos cells were loose and not dense, when tumor formation could be confirmed, the initial size of the tumor was large, reaching about 300mm 3 . When the treatment was over, the tumor volume was only reduced by 15% in the Ibrutinib-treated group (10 mg/kg) compared to the untreated group (P = 0.513), and the same dose of I-As-1 treatment resulted in a tumorTumor volume decreased by 17% (P = 0.192). Surprisingly, the tumor volume in the I-As-1 (20 mg/kg) treated group was reduced by 33% (P = 0.02). On the other hand, mice died after administration of 15mg/kg of Ibrutinib, and therefore the Ibrutinib (20 mg/kg) dose group was not evaluated. After 14 days of treatment, the body weight of each group of mice is slightly increased, and the body weight does not fluctuate obviously during the treatment period, so that the I-As-1 has low toxicity and has the prospect of further development and application (figure 7B).
On the other hand, arsenic is an element mass label, and the biodistribution condition of the arsenic drug in the mouse body can be conveniently detected through ICP-MS. After the tumor treatment experiment is finished, the content of arsenic in heart, liver, spleen, lung, kidney, tumor and blood of the mouse is detected by ICP-MS, and the accumulation condition of arsenic in main organs or tissues of I-As-1 is observed. As shown in FIG. 8, I-As-1 was found to be present in the tumor tissue at the highest concentration, and thus, it tended to target tumors.
In conclusion, the invention designs and synthesizes the covalent targeting arsenic inhibitor which has high selectivity and high affinity and is covalently combined with oncoprotein BTK, inhibits a BCR signal channel mediated by BTK, efficiently inhibits the activity of Ramos cells, and can inhibit the growth of mouse transplanted tumors in vivo. The results show that the strategy provides clear and reasonable design ideas and schemes for developing novel arsenic-based antitumor drugs.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A covalently targeted arsenic inhibitor characterized by: the structural formula is
Wherein R is
2. As claimed in claimThe covalently targeted arsenic inhibitor of claim 1, wherein: r is
3. The method of preparing a covalently targeted arsenic inhibitor of claim 1 or 2, wherein: the method comprises the following steps:
(1) Dissolving an organic arsenic ligand, (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in DMF, adding triethylamine under ice bath to adjust the pH to 7.5-8.4, naturally heating to room temperature, and reacting for 3-5H; the organic arsenic ligand is arsenic acetic acid, arsenic propionic acid-ethanedithiol or arsenic butyric acid-ethanedithiol;
(2) Purifying the material obtained in the step (1) by using a silica gel column or directly spin-drying and washing to obtain the covalent targeting arsenic inhibitor.
4. The method of claim 3, wherein: the organic arsenic ligand is arsenic acetic acid, (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-group) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, EDC and NHS, the molar ratio is 1: 0.25: 2: 1.2.
5. The method of claim 3, wherein: the organic arsenic ligand is arsenic propionic acid-ethanedithiol, the molar ratio of the arsenic propionic acid-ethanedithiol, (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, EDC and NHS is 1: 2: 1.2.
6. The method of claim 3, wherein: the organic arsenic ligand is arsenic butyric acid-ethanedithiol, (R) -3- (4-phenoxyphenyl) -1- (piperidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine, EDC and NHS, and the molar ratio is 1: 2: 1.2.
7. The production 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 used as mobile phases.
8. Use of the covalently targeted arsenic inhibitor of claim 1 or 2 in antitumor drugs.
9. An antitumor agent characterized by: the active ingredient comprises the covalent targeting arsenic inhibitor as claimed in claim 1 or 2.
10. An antitumor drug as claimed in claim 9, characterized in that: the active ingredient of which is the covalently targeted arsenic inhibitor of claim 1 or 2.
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| CN115819486A (en) * | 2022-05-12 | 2023-03-21 | 厦门大学 | Arsenic-sulfydryl molecular beacon compound and application thereof |
| CN115819486B (en) * | 2022-05-12 | 2024-08-02 | 厦门大学 | Arsenic-sulfhydryl molecular beacon compound and application thereof |
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