Hydrophilic berberine-type derivative and application thereof in preparing drug
11725006 · 2023-08-15
Assignee
Inventors
- Hailin Qin (Beijing, CN)
- Lianqiu WU (Beijing, CN)
- Xiang Li (Beijing, CN)
- Haijing ZHANG (Beijing, CN)
- Li Song (Beijing, CN)
- Huachen Song (Beijing, CN)
- Anjun Deng (Beijing, CN)
- Xiaonan Tang (Beijing, CN)
- Zhihui ZHANG (Beijing, CN)
- Zhihong Li (Beijing, CN)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
C07D493/22
CHEMISTRY; METALLURGY
C07D455/03
CHEMISTRY; METALLURGY
International classification
C07D455/03
CHEMISTRY; METALLURGY
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
An 8-dihalomethyl berberine-type quaternary ammonium salt compound represented by general formula (I) or (II) and an application thereof in preparing a drug. The compound shows hydrophilicity and has antimicrobial, anti-inflammatory, anti-ulcerative colitis, and antitumor activities, while having no or low toxicity. ##STR00001##
Claims
1. An 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure shown as general formula I: ##STR00025## wherein: R.sub.2 and R.sub.3 are linked to form alkylene-dioxy; X.sub.1 and X.sub.2 are each selected, independently, from F, Cl, Br, or I; R.sub.9 and R.sub.10 are linked to form alkylene-dioxy and R.sub.11 is H; and X.sub.A is monovalent acid radical ion.
2. The 8-dihalomethyl berberine-type quaternary ammonium salt compounds of quaternized imine-type structure according to any one of claim 1, wherein said monovalent acid radical ion X.sub.A.sup.− is selected from the monovalent inorganic or organic acid radical ion.
3. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to claim 2, wherein said monovalent inorganic acid radical ion is selected from halide anions, hydrogen sulfate ion, hydrogen carbonate ion, dihydrogen phosphate ion, hypohalite ion, halite ion, halate ion, perhalate ion, and nitrate ion; said monovalent organic acid radical ion is selected from formate ion, acetate ion, propionate ion, benzoate ion, p-hydroxybenzoate ion, salicylate ion, protocatechuate ion, ferulate ion, isoferulate ion, homogentisate ion, cinnamate ion, p-hydroxycinnamate ion, caffeate ion, phenylacetate ion, tropate ion, gallate ion, veratrate ion, piperonylate, 3,4,5-trimethoxybenzoate ion, orsellinate ion, shikimate ion, (S)-lactate ion, (R)-lactate ion, (±)-lactate ion, (2R,3R)-(+)-hydrogen tartarate ion, (2S,3S)-(−)-hydrogen tartarate ion, (±)-hydrogen tartarate ion, furoate ion, dihydrogen citrate ion, dihydrogen hydroxycitrate ion, hydrogen maleate ion, hydrogen fumarate ion, L-hydrogen malate ion, D-hydrogen malate ion, (dl)-hydrogen malate ion, hydrogen oxalate ion, hydrogen propanedioate ion, hydrogen succinate ion, hydrogen glutarate ion, hydrogen adipate ion, hydrogen pimelate ion, hydrogen suberate ion, hydrogen azelaate ion, hydrogen sebacate ion, benzenesulfonate ion, gluconate ion, ascorbate ion, sulfamate ion, tosylate ion, methanesulfonate ion, ethanesulfonate ion, 2-naphthalenesulphonate ion, dichloroacetate ion, and difluoroacetate ion.
4. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to claim 2, wherein said monovalent inorganic acid radical ion is selected from halide anions, hydrogen sulfate ion, hydrogen carbonate ion, dihydrogen phosphate ion, hypohalite ion, halite ion, halate ion, perhalate ion, and nitrate ion; said monovalent organic acid radical ion is selected from formate ion, acetate ion, propionate ion, benzoate ion, p-hydroxybenzoate ion, salicylate ion, protocatechuate ion, ferulate ion, isoferulate ion, homogentisate ion, cinnamate ion, p-hydroxycinnamate ion, caffeate ion, phenylacetate ion, tropate ion, gallate ion, veratrate ion, piperonylate, 3,4,5-trimethoxybenzoate ion, orsellinate ion, shikimate ion, (S)-lactate ion , (R)-lactate ion, (±)-lactate ion, (2R,3R)-(+)-hydrogen tartarate ion, (2S,3S)-(−)-hydrogen tartarate ion, (±)-hydrogen tartarate ion, furoate ion, dihydrogen citrate ion, dihydrogen hydroxycitrate ion, hydrogen maleate ion, hydrogen fumarate ion, L-hydrogen malate ion, D-hydrogen malate ion, (dl)-hydrogen malate ion, hydrogen oxalate ion, hydrogen propanedioate ion, hydrogen succinate ion, hydrogen glutarate ion, hydrogen adipate ion, hydrogen pimelate ion, hydrogen suberate ion, hydrogen azelaate ion, hydrogen sebacate ion, benzenesulfonate ion, gluconate ion, and ascorbate ion.
5. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to any one of claim 1, wherein said divalent acid radical ion XA.sup.2- is selected from the divalent inorganic or organic acid radical ion.
6. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to claim 5, wherein said divalent inorganic acid radical ion is selected from sulfate ion, carbonate ion, hydrogen phosphate ion; said divalent organic acid radical ion is selected from (2R,3R)-(+)-tartarate ion, (2S,3S)-(−)-tartarate ion, (±)-tartarate ion, hydrogen citrate ion, hydrogen hydroxycitrate ion, maleate ion, fumarate ion, L-malate ion, D-malate ion, (dl)-malate ion, oxalate ion, propanedioate ion, succinate ion, glutarate ion, adipate ion, pimelate ion, suberate ion, azelaate ion, sebacate ion, benzenesulfonate ion with S.fwdarw.O dipolar bond, benzoate ion with two oxygen anion.
7. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to claim 5, wherein said divalent inorganic acid radical ion is selected from sulfate ion, carbonate ion, hydrogen phosphate ion; said divalent organic acid radical ion is selected from (2R,3R)-(+)-tartarate ion, (2S,3S)-(−)-tartarate ion, (±)-tartarate ion, hydrogen citrate ion, hydrogen hydroxycitrate ion, maleate ion, fumarate ion, L-malate ion, D-malate ion, (dl)-malate ion, oxalate ion, propanedioate ion, succinate ion, glutarate ion, adipate ion, pimelate ion, suberate ion, azelaate ion, and sebacate ion.
8. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to any one of claim 1, wherein said alkylene-dioxy formed by the attachment of R.sub.2 and R.sub.3 and R.sub.9 and R.sub.10 is methylenedioxy.
9. The 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to any one of claim 1, wherein said compound is selected from the group consisting of: TABLE-US-00011
10. The 8-dihalomethyl berberine-type quaternary ammonium salt compounds of quaternized imine-type structure according to any one of claim 1, wherein said compound is selected from the group consisting of: ##STR00039##
11. A method of treating cancers, microbial infections, inflammations, ulcerative colitis in a patient, comprising administration of the 8-dihalomethyl berberine-type quaternary ammonium salt compound of quaternized imine-type structure according to claim 1 to said patients in need thereof, wherein microorganism comprises gram positive and gram negative bacteria, and the cancer is colorectal cancer, liver cancer, lung cancer, breast cancer, osteosarcoma or glioma and the suitable daily dose range of the compound is 0.1-100 mg/kg by body weight.
Description
DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
EMBODIMENTS OF THE INVENTION
(4) The specific embodiments of the invention do not limit the present invention in any way.
(5) The preparation process and structure identification data of the active compounds of the present invention. Wherein, the compound numbers correspond to the specific compound numbers in the invention content.
(6) 1. Examples of the Preparation of Compounds in the Invention
Example 1. Preparation Process and Structure Identification Data of Compound 1
(7) Dissolving coptisine hydrochloride (2.0 g, 5.62 mmol) in 200 ml mixed chloroform-methanol (3:1) solvent in reaction bottle and adding 24 ml concentrated ammonia water, the reaction was carried out under stirring at room temperature for 24 h. Then the chloroform layer was separated, washed using water twice, dried with anhydrous MgSO.sub.4, and filtered. After evaporating the filtrate to dryness, the residue was purified using silica gel column chromatography eluted using dichloromethane, concentrating the eluate to obtain light yellow solid 8-trichloromethyl dihydrocoptisine of 617 mg (yield 25.0%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ: 2.63-2.78 (m, 1H, NCH.sub.2CH.sub.2), 3.35 (m, 1H, NCH.sub.2CH.sub.2), 3.71 (m, 1H, NCH.sub.2CH.sub.2), 3.78-3.90 (m, 1H, NCH.sub.2CH.sub.2), 5.42 (s, 1H, CH—CCl.sub.3), 5.91 (br, 1H, OCH.sub.2O), 5.95 (br, 2H, OCH.sub.2O), 6.02 (br, 2H, OCH.sub.2O, C═CH), 6.61 (s, 1H, ArH), 6.67 (d, J=8.0 Hz, 1H, ArH), 6.86 (d, J=8.0 Hz, 1H, ArH), 7.16 (s, 1H, ArH). Weighing and dissolving 8-trichloromethyl dihydrocoptisine (585 mg, 1.33 mmol) in the mixed solvent of 10 ml t-BuOH and 10 ml DMSO in the reaction bottle, adding in t-BuOK (761 mg, 6.65 mmol), and rising temperature to 80° C. by oil bath on magnetic stirrer, the reaction mixture was reacted for 1.5 h under stirring, with the reaction process being detected by TLC until the reaction was completed. Concentrating the reaction mixture to remove most of the solvents under reduced pressure, ice water was added to the solution until the precipitation was complete. The solution was filtered under reduced pressure to obtain filter cake. The filter cake was washed using water until it was neutral, the filter cake was dried by airing and recrystallized with ethyl acetate to yield 8-dichloromethylene dihydrocoptisine yellow solid 115 mg in yield 21.4%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 3.01 (br, 2H, NCH.sub.2CH.sub.2), 3.60 (br, 2H,NCH.sub.2CH.sub.2), 6.03 (s, 2H, OCH.sub.2O), 6.07 (s, 2H,OCH.sub.2O), 6.66 (s, 1H, ArCH═C), 6.77 (d, J=8.0 Hz, 1H, ArH), 6.82 (s, 1H, ArH), 7.00 (d, J=8.0 Hz, 1H, ArH), 7.43 (s, 1H, ArH); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ: 29.1, 47.1, 98.9, 100.7, 101.1, 103.5, 105.8, 108.38, 108.44, 109.6, 116.1, 122.6, 128.4, 129.7, 134.8, 137.1, 141.3, 145.8, 146.4, 147.3; ESI-MS (m/z): 402.0 [M+H].sup.+. Placing 8-dichloromethylene dihydrocoptisine (100 mg, 0.25 mmol) into the reaction bottle and adding in the methanol solution containing 10% hydrochloric acid 8 ml, the reaction mixture was heated to reflux and the reaction was stopped after 2 hours. The reaction solution was concentrated under reduced pressure to remove most of the solvent, and the residue was obtained. After diluting the residue with a small amount of water, suspending it and precipitating it completely, vacuum filtration was conducted. Washing the filter cake using a small amount of water and drying it via airing, a dark red solid of 8-dichloromethylcoptisine hydrochloride 94 mg was obtained in yield 86.8%. .sup.1H NMR (500 MHz, CD.sub.3OD) δ: 3.33 (br t-like, 2H, NCH.sub.2CH.sub.2), 5.34 (t, J=5.0 Hz, 2H, NCH.sub.2CH.sub.2), 6.13 (s, 2H, OCH.sub.2O), 6.52 (s, 2H, OCH.sub.2O), 7.01 (s, 1H, ArH), 7.72 (s, 1H, ArH), 7.99 (d, J=9.0 Hz, 1H, ArH), 8.01 (d, J=9.0 Hz, 1H, ArH), 8.82 (s, 1H, CHCl.sub.2), 8.97 (s, 1H, ArCH═C); .sup.13C NMR (100 MHz, CD.sub.3OD) δ: 27.6, 54.4, 63.3, 103.9, 105.8, 107.2, 108.9, 113.1, 122.2, 123.4, 125.3, 126.4, 132.9, 136.5, 142.5, 143.2, 148.0, 150.1, 150.7, 152.8; ESI-MS (m/z): 402.0 [M-Cl].sup.+.
Example 2. Preparation Process and Structure Identification Data of Compound 3
(8) Dissolving berberine hydrochloride (2.0 g, 5.38 mmol) in 60 ml chloroform solvent in reaction bottle and adding in 24 ml concentrated ammonia water, the reaction was carried out under stirring at room temperature for 24 h. Then the chloroform layer was separated, washed using water twice, dried with anhydrous MgSO.sub.4, and filtered. After evaporating the filtrate to dryness, the residue was obtained and purified using silica gel column chromatography eluted with dichloromethane. The eluate was concentrated to dryness to obtain light yellow solid 8-trichloromethyl dihydroberberine of 1.775 g (yield 78.9%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ: 2.73 (d, J =15.5 Hz, 1H, NCH.sub.2CH.sub.2), 3.33 (m, 1H, NCH.sub.2CH.sub.2), 3.71 (m, 1H, NCH.sub.2CH.sub.2), 3.87 (ov, 4H, ArOCH.sub.3, NCH.sub.2CH.sub.2), 3.94 (s, 3H, ArOCH.sub.3), 5.65 (s, 1H, CH—CCl.sub.3), 5.94 (s, 2H, OCH.sub.2O), 6.08 (br s, 1H, C═CHAr), 6.61 (s, 1H, ArH), 6.87 (d, J=7.0 Hz, 1H, ArH), 6.97 (d, J=7.0 Hz, 1H, ArH), 7.17 (s, 1H, ArH). Weighing and dissolving 8-trichloromethyl dihydroberberine (814 mg, 1.79 mmol) in the mixed solvent of 15 ml t-BuOH and 15 ml DMSO in the reaction bottle, adding in t-BuOK (1.025 g, 8.95 mmol), and rising temperature to 80° C. by oil bath under stirring, the reaction mixture was reacted for 1.5 h under stirring, with the reaction process being detected by TLC until the reaction was completed. The reaction solution was concentrated under reduced pressure to remove most of the solvents. After adding ice water to the residue for suspension, vacuum filtration was carried out. The filter cake was washed to neutral with water, dried by airing and recrystallized with ethyl acetate to yield 8-dichloromethylene dihydroberberine light yellow solid 218 mg in yield 29.1%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 2.76 (br, 1H, NCH.sub.2CH.sub.2), 3.31 (br, 1H, NCH.sub.2CH.sub.2), 3.46 (br, 1H, NCH.sub.2CH.sub.2), 3.73 (ov, 4H, ArOCH.sub.3, NCH.sub.2CH.sub.2), 3.80 (s, 3H, ArOCH.sub.3,), 6.03 (s, 2H, OCH.sub.2O), 6.56 (s, 1H, ArCH═C), 6.82 (s, 1H,ArH), 6.92 (d, J=8.5 Hz, 1H, ArH), 7.10 (d, J=8.5 Hz, 1H, ArH), 7.43 (s, 1H, ArH); .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ: 29.0, 46.5, 56.2, 60.8, 97.7, 101.0, 103.6, 108.4, 110.4, 114.6, 114.8, 117.6, 122.6, 128.3, 129.5, 135.9, 137.1, 144.3, 146.4, 147.3, 150.0; ESI-MS (m/z): 418.1 [M+H]+. Weighing and placing 8-dichloromethylene dihydrocoptisine (188 mg, 0.45 mmol) in the reaction bottle and adding in the methanol solution containing 10% hydrochloric acid 15 ml, the reaction mixture was heated to reflux and the reaction was carried out under stirring and stopped after 2 hours. The reaction solution was concentrated under reduced pressure to remove most of the solvent, and a small amount of water was added in the residue to dilute until the precipitation was complete, and then filtration was conducted under reduced pressure. Washing the filter cake with a small amount of water and drying it via airing, a dark red solid of 8-dichloromethyl-berberine hydrochloride 115 mg was obtained in yield 56.3%. .sup.1H NMR (500 MHz, CD.sub.3OD) δ: 3.32 (m, 2H, NCH.sub.2CH.sub.2), 4.15 (s, 3H, ArOCH.sub.3), 4.17 (s, 3H, ArOCH.sub.3), 5.37 (m, 2H, NCH.sub.2CH.sub.2), 6.12 (s, 2H, OCH.sub.2O), 7.00 (s, 1H, ArH), 7.73 (s, 1H, ArH), 8.18 (d, J=9.0 Hz, 1H, ArH), 8.23 (d, J=9.0 Hz, 1H, ArH), 8.96 (s, 1H, CHCl.sub.2), 9.40 (s, 1H, ArCH═C); .sup.13C NMR (125 MHz, CD.sub.3OD) δ: 26.6, 53.8, 56.9, 62.6, 62.9, 102.8, 106.3, 107.8, 120.5, 121.1, 124.8, 126.0, 126.6, 132.1, 136.0, 141.8, 143.7, 148.3, 148.9, 151.7, 154.5; ESI-MS (m/z): 418.1 [M-Cl].sup.+.
Example 3. Preparation Process and Structure Identification Data of Compound 5
(9) 8-Dichloromethylene dihydrocoptisine (100 mg, 0.249 mmol) was placed into the reaction bottle, then 1.5 ml 95% ethanol was added in and the mixture was well-mixed. The 98% concentrated sulfuric acid (41 μl , 0.745 mmol) was added into 0.4 ml water to dilute to dilute sulfuric acid solution. This dilute sulfuric acid solution was dropped into the aforementioned reaction bottle containing 8-dichloromethylene dihydrocoptisine and 95% ethanol solution under stirring condition. The reaction was carried out for 1 h under 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using a small amount of 95% ethanol. The filter cake was dried by airing to yield 8-dichloromethyl-coptisine hydrogen sulfate dark red solid 114 mg in yield 91.7% (Combined with the structural identification of 8-dichloromethyl-coptisine hydrogen maleate and 8-dichloromethyl-berberine hydrogen maleate, compound 5 was confirmed to be 8-dichloromethyl-coptisine hydrogen sulfate). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.20 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, ArH), 6.20 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.29 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2).
Example 4. Preparation Process and Structure Identification Data of Compound 7
(10) 8-Dichloromethylene dihydroberberine (5.5 g, 13.149 mmol) was weighed and placed into the reaction bottle, and 130 ml tetrahydrofuran was added in the reaction bottle and the mixture was well-mixed. 98% concentrated sulfuric acid (2.1 ml, 39.448 mmol) was added into 12 ml purified water to dilute to dilute sulfuric acid solution. This dilute sulfuric acid solution was added dropwise into the aforementioned reaction solution containing 8-dichloromethylene dihydroberberine and tetrahydrfuran solution. Then, the reaction mixture was reacted for 1.5 h under stiring and refluxing on 70° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine hydrogen sulfate red solid 6.20 g in yield 91.3% (Combined with the structural identification of 8-dichloromethyl-coptisine hydrogen maleate and 8-dichloromethyl-berberine hydrogen maleate, compound 7 was confirmed to be 8-dichloromethyl-berberine hydrogen sulfate). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ:9.22 (s, 1H, ArCH═C), 9.20 (s, 1H, CHCl.sub.2), 8.33 (d, J=9.1 Hz, 1H, ArH), 8.15 (d, J=9.1 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.06 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ: 153.38, 150.34, 147.66, 147.03, 142.61, 140.74, 134.86, 131.59, 126.67, 125.35, 124.50, 120.59, 119.84, 107.81, 106.02, 102.17, 62.83, 62.37, 57.24, 52.96, 25.79.
Example 5. Preparation Process and Structure Identification Data of Compound 9
(11) 8-Dichloromethylene dihydrocoptisine (100 mg, 0.249 mmol) was weighed and placed into the reaction bottle, and 8 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. 85% phosphoric acid (230 3.735 mmol) was added in dropwise. Then, the reaction mixture was reacted for 2 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran and a small amount of 95% ethanol solution. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine dihydrogen phosphate dark red solid 116 mg in yield 93.3%. .sup.1H NMR (500 MHz, CD3OD) δ: 8.97 (s, 1H, ArCH═C), 8.82 (s, 1H, CHCl.sub.2), 7.99 (s, 2H, ArH), 7.71 (s, 1H, ArH), 7.01 (s, 1H, ArH), 6.52 (s, 2H, OCH.sub.2O), 6.14 (s, 2H, OCH.sub.2O), 5.34 (br t-like, 2H, NCH.sub.2CH.sub.2), ˜3.30 (ov, 2H, NCH.sub.2CH.sub.2).
Example 6. Preparation Process and Structure Identification Data of Compound 11
(12) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 20 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. 85% phosphoric acid (440 μl , 7.172 mmol) was added in dropwise. Then, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine dihydrogen phosphate red solid 130 mg in yield 52.7%. .sup.1-H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.33 (d, J=9.0 Hz, 1H, ArH), 8.14 (d, J=9.0 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t-like, 2H, NCH.sub.2CH.sub.2), ˜4.88 (br, OH).
Example 7. Preparation Process and Structure Identification Data of Compound 13
(13) 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was weighed and placed into the reaction bottle, and 8 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, hydrobromic acid (172 μl, 1.492 mmol) was added in dropwise. The reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine hydrobromate red solid 216 mg in yield 90.0%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.22 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 8.00 (d, J=8.8 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t-like, 2H, NCH.sub.2CH.sub.2), 3.29 (t-like, 2H, NCH.sub.2CH.sub.2); .sup.13C NMR (100 MHz, DMSO-d.sub.6) 8: 150.25, 148.44, 147.68, 145.59, 141.29, 140.32, 134.23, 131.33, 125.10, 123.60, 122.08, 120.73, 111.16, 107.84, 105.94, 104.13, 102.16, 61.79, 52.52, 25.67.
Example 8. Preparation Process and Structure Identification Data of Compound 15
(14) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 6 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, hydrobromic acid (166 μl, 1.434 mmol) was added in dropwise. The reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine hydrobromate red solid 185 mg in yield 77.5%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 2H, ArCH═C, CHCl2), 8.33 (d, J=9.1 Hz, 1H, ArH), 8.15 (d, J=9.1 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2); .sup.13C NMR (100 MHz, DMSO-d.sub.6) 153.37, 150.34, 147.66, 147.03, 142.62, 140.74, 134.86, 131.59, 126.67, 125.32, 124.49, 120.60, 119.85, 107.81, 106.01, 102.17, 62.83, 62.37, 57.24, 52.97, 25.80.
Example 9. Preparation Process and Structure Identification Data of Compound 17
(15) 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was weighed and placed into the reaction bottle, and 4 ml 95% ethanol solution was added in the reaction bottle and the mixture was well-mixed. The concentrated nitric acid (102 μl 1.492 mmol) was added into 1 ml water to dilute to dilute nitric acid solution and this dilute nitric acid solution was dropped into the reaction bottle. The reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine nitrate red crystal 187 mg in yield 80.8%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.20 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.30 (t, 2H, J =6.0 Hz, NCH.sub.2CH.sub.2).
Example 10. Preparation Process and Structure Identification Data of Compound 19
(16) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 4 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. The concentrated nitric acid (98 μl 1.434 mmol) was added into 1 ml water to dilute to dilute nitric acid solution and this dilute nitric acid solution was dropped into the reaction bottle. The reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine nitrate red solid 184 mg in yield 80.0%. .sup.11H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.32 (d, J=9.2 Hz, 1H, ArH), 8.14 (d, J=9.2 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=5.6 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.33 (ov, 2H, NCH.sub.2CH.sub.2).
Example 11. Preparation Process and Structure Identification Data of Compound 21
(17) Oxalic acid dihydrate (190 mg, 1.492 mmol) was weighed and placed into the reaction bottle, and 10 ml methanol was added in the reaction bottle and the mixture was well-mixed. 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was added to the above solution batchwise. The reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. Most solvent was removed via concentration under reduced pressure. 4 ml water was added in. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-copyisine hydrogen oxalate dark red solid 237 mg in yield 96.8%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 7.99 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.30 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2).
Example 12. Preparation Process and Structure Identification Data of Compound 23
(18) Oxalic acid dihydrate (190 mg, 1.434 mmol) was weighed and placed into the reaction bottle, and 10 ml methanol was added in the reaction bottle and the mixture was well-mixed via stirring. Then, 8-dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating until the reaction was completed. The reaction mixture was cooled to room temperature. The solvent was concentrated under reduced pressure to dryness. 10 ml of water was added into the residue in the reaction bottle, and then the mixture was well-shaken and filtered under reduced pressure. The filter cake was washed using ether to yield 8-dichloromethyl-berberine hydrogen oxalate red solid 98 mg in yield 40.3%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.33 (d, J =8.8 Hz, 1H, ArH), 8.14 (d, J=8.8 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (br t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (br t-like, 2H, NCH.sub.2CH.sub.2).
Example 13. Preparation Process and Structure Identification Data of Compound 25
(19) Maleic acid (190 mg, 1.492 mmol) was weighed and placed into the reaction bottle, and 1 ml water and 10 ml 95% ethanol was added in the reaction bottle and the mixture was mixed up. Then, 8-dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 2 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. Most of the solvent was removed via concentrating under reduced pressure. 4 ml of tetrahydrofuran was added in the reaction mixture. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine hydrogen maleate red solid 208 mg in yield 80.7%. .sup.11H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J =8.8 Hz, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 6.01 (s, 2H, HOOCCH═CHCOO.sup.−), 5.22 (t, 2H, J=6.4 Hz, NCH.sub.2CH.sub.2), 3.30 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2).
Example 14. Preparation Process and Structure Identification Data of Compound 27
(20) Maleic acid (166 mg, 1.434 mmol) was weighed and placed into the reaction bottle, and 10 ml methanol was added in the reaction bottle and the mixture was mixed up via stirring. Then, 8-dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating until the reaction was completed. The reaction mixture was cooled to room temperature. The solvent was evaporated under reduced pressure to dryness. 10 ml of water was added into the residue in the reaction bottle, and then well-shaken and filtered under reduced pressure. The filter cake was washed using ether to yield 8-dichloromethyl-berberine hydrogen maleate red solid 120 mg in yield 47.0%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.32 (d, J=9.0 Hz, 1H, ArH), 8.14 (d, J =9.0 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 6.03 (s, 2H, HOOCCH═CHCOO.sup.−), 5.26 (br t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.06 (s, 3H, ArOCH.sub.3), 3.32 (ov, 2H, NCH.sub.2CH.sub.2).
Example 15. Preparation Process and Structure Identification Data of Compound 29
(21) P-toluenesulfonic acid (p-TsOH, 128 mg, 0.746 mmol) was weighed and placed into the reaction bottle, and 8 ml methanol was added in the reaction bottle and the mixture was well-mixed. Then, 8-dichloromethylene dihydrocoptisine (100 mg, 0.249 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. Most solvent was removed via concentration under reduced pressure. 2 ml water was added in the reaction mixture. After full precipitation of product, filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-copyisine tosylate red solid 138 mg in yield 96.6%. .sup.11H NMR (400 MHz, DMSO-d.sub.6) δ: 9.20 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.16 (d, J=8.8 Hz, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.47 (d, J=8.0 Hz, 2H, ArH), 7.15 (s, 1H, ArH), 7.10 (br d, J=8.0 Hz, 2H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.20 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=6.4 Hz, NCH.sub.2CH.sub.2), 3.29 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2), 2.28 (s, 3H, ArCH.sub.3).
Example 16. Preparation Process and Structure Identification Data of Compound 31
(22) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 8 ml tetrahydrofuran was added in the reaction bottle and the mixture was well-mixed. Then, p-toluenesulfonic acid (p-TsOH, 247 mg, 1.434 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 70° C. oil bath under heating. The reaction mixture was cooled to room temperature. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine tosylate red solid 186 mg in yield 65.9%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.32 (d, J=9.0 Hz, 1H, ArH), 8.14 (d, J=9.0 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.47 (d, J=7.5 Hz, 2H, ArH), 7.16 (s, 1H, ArH), 7.11 (d, J=7.5 Hz, 2H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (br t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (br t-like, 2H, NCH.sub.2CH.sub.2), 2.29 (s, 3H, CH.sub.3ArSO.sub.3).
Example 17. Preparation Process and Structure Identification Data of Compound 33
(23) 8-Dichloromethylene dihydrocoptisine (100 mg, 0.249 mmol) was weighed and placed into the reaction bottle, and 6 ml tetrahydrofuran was added in the reaction bottle and the mixture was well-mixed. Sulfamic acid (36 mg, 0.373 mmol) was weighed and dissolved with 1 ml DMSO. This sulfamic acid solution was added into the reaction solution dropwise and, after finishing, the reaction mixture was reacted for 2 h under stirring and refluxing on 70° C. oil bath under heating. The reaction mixture was cooled to room temperature. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine sulfamate red solid 110 mg in yield 88.6%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.22 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.6 Hz, 1H, ArH), 8.00 (d, J=8.6 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (br s, 3H, ArH, NH.sub.2SO.sub.3.sup.−), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (br t-like, 2H, NCH.sub.2CH.sub.2), 3.29 (br t-like, 2H, NCH.sub.2CH.sub.2); HRMS(ESI) (m/z): C.sub.20H.sub.14O.sub.4NCl.sub.2 [M-NH.sub.2SO.sub.3.sup.−].sup.+ calculated: 402.02944; discovered: 402.02994.
Example 18. Preparation Process and Structure Identification Data of Compound 35
(24) 8-Dichloromethylene dihydroberberine (100 mg, 0.239 mmol) was weighed and placed into the reaction bottle, and 5 ml absolute methanol was added in the reaction bottle and the mixture was well-mixed. Then, sulfamic acid (70 mg, 0.717 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. The solvent was evaporated under reduced pressure to dryness. 9 ml tetrahydrofuran was added in the reaction mixture to precipitate the product. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine sulfamate red solid 114 mg in yield 92.5%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.20 (s, 1H, CHCl.sub.2), 8.33 (d, J=9.2 Hz, 1H, ArH), 8.14 (d, J=9.2 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.19 (br s, 2H, NH.sub.2SO.sub.3.sup.−), 7.17 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (br t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ: 153.37, 150.34, 147.66, 147.03, 142.61, 140.75, 134.86, 131.59, 126.67, 125.34, 124.49, 120.60, 119.85, 107.81, 106.01, 102.17, 62.83, 62.37, 57.23, 52.96, 25.79; HRMS(ESI) (m/z): C.sub.21H.sub.18O.sub.4NCl.sub.2[M-NH.sub.2SO.sub.3].sup.+ calculated: 418.06074; discovered: 418.06021.
Example 19. Preparation Process and Structure Identification Data of Compound 37
(25) 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, methanesulfonic acid (97 μl, 1.492 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature and the product was precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran and diethyl ether anhydrous until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine methanesulfonate red solid 224 mg in yield 90.4%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 7.99 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.29 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2), 2.29 (s, 3H, CH.sub.3SO.sub.3.sup.−).
Example 20. Preparation Process and Structure Identification Data of Compound 39
(26) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, methanesulfonic acid (93 μl, 1.434 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. 10 ml diethyl ether anhydrous was added in the reaction mixture to make the product precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using diethyl ether anhydrous and tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine methanesulfonate red solid 213 mg in yield 86.6%. NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.20 (s, 1H, CHCl.sub.2), 8.33 (d, J =9.0 Hz, 1H, ArH), 8.15 (d, J=9.0 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (br t-like, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.06 (s, 3H, ArOCH.sub.3), 3.30 (br t-like, 2H, NCH.sub.2CH.sub.2), 2.31 (s, 3H, CH.sub.3SO.sub.3); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ: 153.37, 150.33, 147.66, 147.02, 142.61, 140.74, 134.86, 131.59, 126.67, 125.35, 124.50, 120.60, 119.84, 107.81, 106.02, 102.17, 62.83, 62.37, 57.24, 52.96, 25.79, 18.45.
Example 21. Preparation Process and Structure Identification Data of Compound 41
(27) 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, ethanesulfonic acid (122 μl, 1.492 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran and diethyl ether anhydrous and until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine ethanesulfonate red solid 171 mg in yield 67.1%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.16 (d, J=8.8 Hz, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.29 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2), 2.35 (q, 2H, J=7.2 Hz, CH.sub.3CH.sub.2SO.sub.3.sup.−), 1.03 (t, J=7.2 Hz, 3H, CH.sub.3CH.sub.2SO.sub.3).
Example 22. Preparation Process and Structure Identification Data of Compound 43
(28) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, ethanesulfonic acid (117 μl, 1.434 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. 10 ml diethyl ether anhydrous was added in the reaction mixture to make the product precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using diethyl ether anhydrous and tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine ethanesulfonate red solid 193 mg in yield 76.4%. NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.33 (d, J =9.2 Hz, 1H, ArH), 8.14 (d, J=9.2 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 2.37 (q, J=7.6 Hz, 2H, CH.sub.3CH.sub.2SO.sub.3), 1.05 (t, J=7.6 Hz, 3H, CH.sub.3CH.sub.2SO.sub.3); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ: 153.37, 150.34, 147.66, 147.03, 142.62, 140.74, 134.86, 131.59, 126.67, 125.34, 124.50, 120.60, 119.85, 107.81, 106.01, 102.17, 62.83, 62.37, 57.24, 52.96, 45.05, 25.79, 9.79.
Example 23. Preparation Process and Structure Identification Data of Compound 45
(29) 8-Dichloromethylene dihydrocoptisine (500 mg, 1.243 mmol) was weighed and placed into the reaction bottle, and 7 ml 95% ethanol was added in the reaction bottle and mixed up. Then, benzenesulfonic acid (588 mg, 3.728 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1.5 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature and the product was precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using a small amount of 95% ethanol solution. The filter cake was dried under vacuum to yield bis(8-dichloromethyl-coptisine) monobenzenesulfonate red solid 574 mg in yield 96.0%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.20 (s, 1H, ArCH═C), 8.68 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.7 Hz, 1H, ArH), 7.99 (d, J=8.7 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.58 (m, 1H, ArH), 7.30 (m, 1.5 H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 3.29 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2).
Example 24. Preparation Process and Structure Identification Data of Compound 47
(30) 8-Dichloromethylene dihydroberberine (500 mg, 1.195 mmol) was weighed and placed into the reaction bottle, and 3.5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, benzenesulfonic acid (567 mg, 3.585 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1.5 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to 0° C. gradually to precipitate the crystal. Filtration was conducted under reduced pressure. The filter cake was washed using a small amount of 95% ethanol solution. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine benzenesulfonate red solid 366 mg in yield 53.1%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.32 (d, J =9.2 Hz, 1H, ArH), 8.15 (d, J=9.2 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.59 (m, 2H, ArH), 7.31 (m, 3H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2).
Example 25. Preparation Process and Structure Identification Data of Compound 49
(31) 8-Dichloromethylene dihydrocoptisine (200 mg, 0.497 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, naphthalenesulfonic acid (311 mg, 1.492 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature and the product was precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using tetrahydrofuran until the filtrate was colourless. The filter cake was dried under vacuum to yield 8-dichloromethyl-coptisine 2-naphthalenesulphonate red crystalline 250 mg in yield 82.4%. .sup.11H NMR (400 MHz, DMSO-d.sub.6) δ: 9.20 (s, 1H, ArCH═C), 8.67 (s, 1H, CHCl.sub.2), 8.17 (d, J=8.8 Hz, 1H, ArH), 8.13 (br s, 1H, ArH), 7.98 (d, J=8.8 Hz, 1H, ArH), 7.97 (ov, 1H, ArH), 7.89 (m, 1H, ArH), 7.85 (d, J=8.8 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.70 (dd, J=8.4, 1.2 Hz, 1H, ArH), 7.52 (m, 2H, ArH), 7.16 (s, 1H, ArH), 6.57 (s, 2H, OCH.sub.2O), 6.21 (s, 2H, OCH.sub.2O), 5.22 (t, 2H, J=5.6 Hz, NCH.sub.2CH.sub.2), 3.29 (t, 2H, J=6.0 Hz, NCH.sub.2CH.sub.2).
Example 26. Preparation Process and Structure Identification Data of Compound 51
(32) 8-Dichloromethylene dihydroberberine (200 mg, 0.478 mmol) was weighed and placed into the reaction bottle, and 5 ml 95% ethanol was added in the reaction bottle and the mixture was well-mixed. Then, 2-naphthalenesulfonic acid (299 mg, 1.434 mmol) was added in batchwise and, after finishing, the reaction mixture was reacted for 1 h under stirring and refluxing on 80° C. oil bath under heating. The reaction mixture was cooled to room temperature. 10 ml diethyl ether anhydrous was added in the reaction mixture to make the product precipitated. Filtration was conducted under reduced pressure. The filter cake was washed using diethyl ether anhydrous. The filter cake was dried under vacuum to yield 8-dichloromethyl-berberine 2-naphthalenesulphonate red solid 62 mg in yield 20.7%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.21 (s, 1H, ArCH═C), 9.19 (s, 1H, CHCl.sub.2), 8.32 (d, J=9.2 Hz, 1H, ArH), 8.14 (d, J=9.2 Hz, 1H, ArH), 8.13 (s, 1H, ArH), 7.96 (m, 1H, ArH), 7.89 (m, 1H, ArH), 7.85 (m, 2H, ArH), 7.70 (dd, J=8.4, 1.6 Hz, 1H, ArH), 7.52 (m, 2H, ArH), 7.16 (s, 1H, ArH), 6.21 (s, 2H, OCH.sub.2O), 5.26 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 4.12 (s, 3H, ArOCH.sub.3), 4.05 (s, 3H, ArOCH.sub.3), 3.30 (t, J=6.0 Hz, 2H, NCH.sub.2CH.sub.2); NMR (100 MHz, DMSO-d.sub.6) δ: 153.36, 150.34, 147.66, 147.02, 145.57, 142.60, 140.74, 134.85, 132.56, 132.02, 131.57, 128.31, 127.31, 127.14, 126.65, 126.26, 126.14, 125.33, 124.49, 123.88 (2×C), 120.59, 119.83, 107.81, 106.00, 102.17, 62.82, 62.36, 57.22, 52.96, 25.79.
(33) 2. Test Examples of Solubility Examination on the Compounds of the Invention
Example 1. Test Example of Hydrosolubility Examination on the Compounds of the Invention
(34) The compounds of the invention were weighed and placed into a certain amount of pure water solvent under 25° C.±2° C., respectively. The water solution containing the compounds of the invention was shaken forcefully every 5 minutes for 30 seconds and the dissolution status within 30 minutes was observed. If there were no visible solute particle, it would be regarded as complete dissolution.
(35) Experimental results: The solubility was determined at 25° C.±2° C. of temperature and the amount of the compounds of the invention dissolved in one milliliter of purified water are 8-dichloromethyl coptisine hydrochloride (1) 5 mg, 8-dichloromethyl berberine hydrochloride (3) 35 mg, 8-dichloromethyl coptisine hydrogen oxalate (21) 5.25 mg, 8-dichloromethyl berberine hydrogen oxalate (23) 5.1 mg, 8-dichloromethyl coptisine hydrogen maleate (25) 3.71 mg, 8-dichloromethyl berberine hydrogen maleate (27) 3.83 mg, 8-dichloromethyl coptisine tosylate (29) 1.5 mg, 8-dichloromethyl berberine tosylate (31) 5.0 mg, 8-dichloromethyl coptisine benzenesulfonate (45) 8.9 mg, and 8-dichloromethyl berberine benzenesulfonate (47) 6.45 mg, respectively. In parallel determination, the amount of quaternary coptisine chloride and quaternary berberine chloride as quaternary ammonium salt substrates of berberine-type alkaloids that can be dissolved in each milliliter of water is less than 1 mg and 2 mg, respectively.
Example 2. Test Example of Alcohol-Solubility Examination on the Compounds of the Invention
(36) The compounds of the invention were weighed and placed into different amount of 95% ethanol solvent under refluxing, respectively. The dissolution status within 30 minutes was observed. If there were no visible solute particle, it would be regarded as complete dissolution.
(37) Experimental results: When examining under refluxing condition, the amount of 95% ethanol solvent required for dissolving each gram of the compounds of the invention are 8-dichloromethyl coptisine hydrochloride (1) 23 ml, 8-dichloromethyl coptisine hydrogen maleate (25) 23 ml, 8-dichloromethyl berberine hydrogen maleate (27) 5.8 ml, 8-dichloromethyl coptisine tosylate (29) 24 ml, 8-dichloromethyl berberine tosylate (31) 7 ml, and 8-dichloromethyl berberine benzenesulfonate (47) 7 ml, respectively.
(38) 3. Experimental Examples of the Pharmacological Efficacy and Toxicology Evaluation on the Compounds of the Invention
Example 1: Evaluation on the Anti-Microbial Activity of the Compounds of the Invention
(39) 1. Materials and Methods
(40) (1) Strains: Staphylococcus aureus, Escherichia colt, Candida albicans MCC (F) 98001.
(41) (2) Experimental method: the anti-bacterial activity of the compounds of the invention was evaluated by determining the minimum inhibitory concentration (MIC) using the twofold microdilution broth method. The compounds of the invention and the reference substances (quaternary ammonium salt substrates of berberine-type alkaloids and levofloxacin) were prepared into a 20000 μg/ml stock solution with DMSO for use. S. aureus and E. coli were diluted using MHB (Mueller Hinton Broth) medium, and C. albicans using Sabouraud medium, all prepared into 10.sup.6 CFU (colony forming unit) concentration as bacterial suspensions for use. The determination steps of MIC are as follows. First, 180 μl of MHB medium (for S. aureus and E. coli) or Sabouraud Medium (for C. albicans) was transferred to the first well in each row of the sterile 96 well plate, and 100 μl to the second to the 11th wells of each row, and 200 μl to the 12th well as negative blank control well. 20 μl sample stock solution was taken and added to the first well. After mixed up, 100 μl was taken from the first well and transferred to the second well. The same as mentioned above, the second well was mixed up, 100 μl of mixed content was taken from the second well and added to the third well, as described above. As such, the same operation was continued till the 10th well, and finally 100 μl of content was drawn from the 10th well and discarded. The bacterial suspension of 100 μl was added to each of the aforementioned from first to eleven wells and mixed up. Three parallel groups for each sample were set up. The 96 well plates were put into the incubator to culture for 24 h. The temperature to determine the MIC of the compounds of the invention against S. aureus and E. was set at 37° C. and that of C. albicans at 25° C. The MIC values of each sample to be tested as well as those of the references were observed and recorded with the naked eyes.
(42) 2. Experimental Results
(43) See table 1
(44) TABLE-US-00003 TABLE 1 MIC values determination result of the compounds of the invention in anti-microbial experiment MIC (ug/mL) Compounds S. aureus E. coli C. albicans berberine hydrochloride 125 250 250 coptisine hydrochloride >250 >250 250 1 15.6 62.5 <1.95 (0.78).sup.a 3 31.2 >250 3.9 Levofloxacin <1.95 <1.95 >250 Note: “.sup.-” indicates no determination being conducted; “.sup.a” indicates that when the concentration of the stock solution of compounds to be tested was reduced to 2000 μg/ml to further determine the MIC value, the test result was 0.78 μg/ml.
(45) Although the action intensity of the compounds of the invention against S. aureus and E. coli is lower than that of positive control levofloxacin in MIC evaluation, because the compounds of the invention are nontoxic and hypotoxic bioactive compounds, they have a broader application prospect even than levofloxacin. And the intensity of pharmacological action of the compounds of the invention against C. albicans is significantly higher in MIC evaluation than that of the positive control levofloxacin (the MIC of levofloxacin is more than 250 μg/ml).
Example 2: Anti-Inflammatory Pharmacodynamics Evaluation of the Compounds of the Invention with the Acute Ear Swelling Model of Mice Caused by Croton Oil
(46) 1. Materials and Methods
(47) (1) Animals: Balb/c mice, male (20-22 g); eight mice in each group.
(48) (2): Grouping: The experiment was divided into model group, positive drug indomethacin (Sigma company) group, the invention compound 1 administration group, and the invention compound 3 administration group.
(49) (3) Dosage and times of administration: 5 mg/kg for positive drug; on test of compounds 1 and 3 of the invention, the dosage was both 100 mg/kg, once a day for three days.
(50) (4) Experimental methods: the mice in the model group were administrated 0.9% normal saline (dosage volume: 10 ml/kg) by the intragastrical administration. In the positive drug group, the positive drug was prepared by 5 mg/kg of dosage using 0.9% normal saline as the solvent and was administrated by gavage (dosage volume: 10 ml/kg). In the compounds of the invention administration groups, the compounds were prepared by 100 mg/kg of dosage using 0.9% normal saline as the solvent and were administrated by gavage (dosage volume: 10 ml/kg). After the last administration, all mice were induced to inflammation by daubing 20 μl of croton oil acetone solution on both sides of the right ear, the left ear not daubed as normal control ear. After 4 hours of modeling, the mice were killed, the ears were cut off along the auricle baseline, and the ear pieces of each subject were taken off at the counterpart place of each ear with a perforator and weighed. The difference between the weight of the right ear piece and that of the left ear piece indicated the degree of inflammatory swelling. The inhibition rate of drugs on ear swelling (%) was calculated by comparing the ear swelling degree values of each administration group and positive drug group with the data of model group.
Inhibition rate (%)=[(swelling degree of model group−swelling degree of administration group)/swelling degree of model group]×100 (%)
(51) (5) Statistical analysis: The experimental results were expressed as ‘mean±sd’. The statistical differences between the two groups were calculated and analyzed by t-test method. .sup.#, indicates P<0.05 (compared with the model group); and .sup.##, P<0.01 (compared with the model group).
(52) 2. Experimental Results
(53) See Table 2.
(54) TABLE-US-00004 TABLE 2 The swelling inhibition test results of compounds 1 and 3 of the invention in the acute swelling model of mouse ear caused by croton oil Number Degree of Inhibition of cases swelling rate Groups (pieces: start/end) (mg) (%) Model(croton oil) 8/8 94.95 ± 18.42 0 Indomethacin (5 mg/kg) 8/8 70.26 ± 20.97.sup.# 26.01.sup.# 1 (100 mg/kg) 8/8 41.24 ± 16.01.sup.## 56.59.sup.## 3 (100 mg/kg) 8/8 85.19 ± 39.24 10.33 Note: .sup.#P <0.05, .sup.##P <0.01, compared with the model group.
3. Results Analysis
(55) (1) Compared with the model group, indomethacin, a positive drug, had a strong inhibition effect on ear swelling (.sup.#P<0.05), suggesting that the experimental system was reasonable, accurate, and reliable.
(56) (2) Compared with the model group, compound 1 group showed significant anti-inflammatory activity, statistically significant difference being obtained, .sup.##P<0.01. Compared with the model group, compound 3 showed anti-inflammatory activity, but the anti-inflammatory activity was weaker than compound 1 and positive control.
(57) 4. Conclusion: In the case of 100 mg/kg dosage for intragastric administration, compound 1 has strong anti-swelling activity for mouse ear caused by croton oil, while compound 3 has anti-inflammatory activity, but it is relatively weak.
(58) Although the administration dosage of the compounds of the invention in the experiment was greater than 5 mg/kg of the positive control drug indomethacin, also in view of the fact that the compounds of the invention have the prominent features of being nontoxic or hypotoxic bioactive compounds, and the compounds of the invention are not homologous compound of indomethacin, and, considering the effectiveness and safety of drugs comprehensively (drug research cannot only consider the intensity of pharmacological action, the safety of drugs is even more important), the fact that in the case of large dosage of the compounds of the invention, the anti-inflammatory effect was stronger than that of the positive control drug and the experimental animal can endure high doses of the compounds of the invention just demonstrate that the compounds of the invention have more application value than the positive control drug in the preparation of drugs for preventing, alleviating and/or treating inflammation.
Example 3: Biological Research and Implementation Example of the Compounds of the Invention Against Ulcerative Colitis
(59) 1. Materials and Methods
(60) (1) Animals: C57b1/6j mice, male (20-22 g); seven mice in each group.
(61) (2) Grouping: The experiment was divided into normal control group, dextran sodium sulfate (DSS) model group, the first positive drug (SASP) group, the second positive drug (dihydroberberine) group, and the invention compound 1 and 3 administration groups.
(62) (3) Dosage and times of administration: The positive drug was 500 mg/kg, both dihydrocoptisine and the compounds of the invention were 100 mg/kg, once a day for eight days.
(63) (4) Experimental methods: The method of this experiment was a literature method: see Zhi-Hui Zhang, Hai-Jing Zhang, An-Jun Deng, Bo Wang, Zhi-Hong Li,
(64) Yang Liu, Lian-Qiu Wu, Wen-Jie Wang, and Hai-Lin Qin. Synthesis and Structure-activity Relationships of Quaternary Coptisine Derivatives as Potential Anti-ulcerative Colitis Agents. Journal of Medicinal Chemistry. 2015, 58, 7557-7571.
(65) 2. Results and discussion: The compounds of the invention had significant therapeutic effect on acute C57b1/6j mice ulcerative colitis induced by dextran sodium sulfate (DSS) in vivo.
(66) (1) The compounds of the invention could effectively reduce the body weight loss of C57b1/6j mice model of ulcerative colitis induced by DSS (see Table 3).
(67) It can be seen from Table 3 that, after the ending of the experiment, compared with the initial value of body weight of animals in each group, the body weight of animals in the normal control group increased by 0.09%. In the model group, the body weight of animals decreased by 27.61% (**, P<0.01, compared with the normal control group). The body weight of animals in positive drug SASP group decreased by 27.70% at the administration dosage of 500 mg/kg, and the body weight of the animals in the positive drug dihydrocoptisine administration group decreased by 18.77%.sup.# (.sup.#P<0.05, compared with the model group) at the administration dose of 100 mg/kg. While, the body weight of the animals in the compounds of the invention 1 and 3 administration groups decreased by 8.19%.sup.## (.sup.##P<0.01, compared with the model group) and 25.54%, respectively, at the administration dose of 100 mg/kg. Therefore, the compounds of the invention can slow down or significantly slow down the body weight loss of model animals, statistically significant difference from the model group being shown. The experimental results showed that, at the dose of 100 mg/kg, the compounds of the invention can effectively alleviate the body weight loss of C57b1/6j mice with experimental ulcerative colitis to a certain extent. In addition, compound 1 is apparently superior to dihydrocoptisine in improving the body weight loss of ulcerative colitis model animals (in fact, there is a huge difference between the two structures), and compound 3 is also superior to the positive drugs.
(68) TABLE-US-00005 TABLE 3 Influence of the compounds of the invention on the body weight of C57b1/6j mice with ulcerative colitis Number of cases Body weight (g) X ± SD Body weight Groups (pieces: start/end) Start End change rate (%) Normal control group 7/7 22.84 ± 0.42 22.86 ± 0.90 +0.09 Model group (DSS induction) 7/7 22.94 ± 1.32 16.65 ± 1.95 −27.61** SASP group (500 mg/kg).sup.a 7/7 22.10 ± 1.42 15.86 ± 1.58 −27.70 Dihydrocoptisine (100 mg/kg).sup.a 7/7 22.69 ± 0.77 18.43 ± 2.01 −18.77.sup.# 1 (100 mg/kg) 7/7 21.34 ± 0.50 19.60 ± 0.93 −8.19.sup.## 3 (100 mg/kg) 7/7 21.86 ± 0.59 16.28 ± 0.69 −25.54 Notes: .sup.aPositive control group; **P <0.01, compared with the normal control group; .sup.#P <0.05 and .sup.##P <0.01, compared with model group.
(69) (2) The improving effect of the compounds of the invention on the colon contracture of C57b1/6j mice model of ulcerative colitis induced by DSS (see Table 4).
(70) Table 4 shows the colon length of each group and the percentage of colon contracture compared with the normal control group in the end of the experiment. The results showed that, compared with the normal control group which had the colon length of 8.19 cm, the colon length of the model group mice was significantly shorter, which was 5.21 cm (**, P<0.01, compared with the normal control group). At the administration dose of 100 mg/kg used in the experiment, compared with the model group mice, the colon length of the mice in each administration group of the compounds of the invention was significantly longer. The colon length of mice of compound 1 group was 6.85 cm.sup.## (.sup.##, P<0.01, compared with the model group), and compound 3 was 5.83 cm (.sup.#, P<0.05, compared with the model group). The colon length of mice in positive drug SASP group was 5.90 cm.sup.# (.sup.#, P<0.05, compared with the model group) at the administration dose of 500 mg/kg. The colon length of mice in positive drug dihydrocoptisine administration group was 6.23 cm (.sup.##, P<0.01, compared with the model group) at the administration dose of 100 mg/kg. Compared with the normal control group, compound 1 of the invention showed obvious improvement effect on the colon contracture of C57b1/6j mice model of ulcerative colitis induced by DSS, superior to the positive drug and the active compound dihydrocoptisine. Compound 3 also showed obvious activity.
(71) TABLE-US-00006 TABLE 4 The effect of the compounds of the invention on the colon contracture in C57b1/6j mice ulcerative colitis model animals induced by DSS Number of cases Colon Percentage of colon Groups (pieces: start/end) length (cm) contracture (%) Normal control group 7/7 8.19 ± 0.39 0 Model group (DSS induction) 7/7 5.21 ± 0.48** 36.34** SASP group (500 mg/kg).sup.a 7/7 5.90 ± 0.30.sup.# 28.43.sup.# Dihydrocoptisine (100 mg/kg).sup.a 7/7 6.23 ± 0.14.sup.## 23.93.sup.## 1 (100 mg/kg) 7/7 6.85 ± 0.56.sup.## 16.37.sup.## 3 (100 mg/kg) 7/7 5.83 ± 0.27.sup.# 28.83.sup.# Notes: .sup.aPositive control group; **P <0.01, compared with the normal control group; .sup.#P <0.05 and .sup.##P <0.01, compared with model group.
(72) (3) The effect of the compounds of the invention on disease activity index (DAI) and DAI inhibition rate of C57b1/6j mice model of ulcerative colitis induced by DSS (Table 5).
(73) DAI score was used to evaluate therapeutic effect of active compounds via body weight loss percentage of animals, fecal characteristics, and fecal blood, and the like, which are closely related to clinical symptoms of ulcerative colitis. The lower DAI score and the higher DAI inhibition rate indicated that the model animal was more close to the normal physiological status after treatment. The effect of the compounds of the invention on the disease activity index (DAI) and the inhibition rate of DAI in the C57b1/6j mice model of ulcerative colitis induced by DSS was investigated. The results showed that the compounds of the invention have significant anti-ulcerative colitis activity. At the administration dose of 100 mg/kg used in the experiment, the anti-ulcerative colitis activity of the compounds of the invention were significantly higher than that of positive drug SASP at the administration dose of 500 mg/kg, and also higher than that of positive drug dihydrocoptisine at the administration dose of 100 mg/kg. In Table 5, **, P<0.01, compared with the normal control group; .sup.#, P<0.05 and .sup.##, P<0.01, compared with the model group.
(74) TABLE-US-00007 TABLE 5 The effect of the compounds of the invention on disease activity index (DAI) and DAI inhibition rate of C57b1/6j mice model of acute ulcerative colitis induced by DSS Number of DAI cases (pieces: inhibition Groups start/end) DAI rate (%) Normal control group 7/7 0.17 ± 0.18 − Model group (DSS induction) 7/7 4.00 ± 0.00** 0 SASP group (500 mg/kg).sup.a 7/7 3.07 ± 1.01.sup.# 23.33.sup.# Dihydrocoptisine (100 mg/kg).sup.a 7/7 2.24 ± 0.97.sup.## 44.03.sup.## 1 (100 mg/kg) 7/7 1.39 ± 0.61.sup.## 65.17.sup.## 3 (100 mg/kg) 7/7 1.96 ± 0.87.sup.## 51.00.sup.## Notes: .sup.aPositive control group; **P <0.01, compared with the normal control group; .sup.#P <0.05 and .sup.##P <0.01, compared with model group.
Example 4: The Regulation Experiment and Results of Compounds 1 and 3 of the Invention on Tumorigenesis-Related Target Molecules in Six Different Tumor Cells
(75) 1. Materials and Methods
(76) (1) Cells: Rat glioma cell C6, human large cell lung cancer cell NCIH460, human breast cancer cell MDA-MB-231, human colon cancer cell HCT116, human liver cancer cell 7721, and human osteosarcoma cell MG63.
(77) (2) Experimental method: Rat glioma cell C6, human large cell lung cancer cell NCIH460, human breast cancer cell MDA-MB-231, human colon cancer cell HCT116, human liver cancer cell 7721, and human osteosarcoma cell MG63 were cultured in vitro conventionally. When the above six cells grew to 80% confluent state, they were divided into six well plates. Pre-protection was carried out for three hours with the compounds 1 and 3 of the invention with a concentration of 1 μM. After that, LPS was added to stimulate for 24 hours, and the samples were collected and frozen.
(78) The prepared cell samples were broken by ultrasound in RIPA lysate. After 4-degree cracking for 30 min and centrifuging at 13000 rpm for 10 min, the supernatant was taken. The protein concentration was determined by Brandford method. According to the protein concentration, the same amount of protein was taken and detected by Western blot (WB).
(79) The operation of WB experimental detection is as follows: 4% concentrated glue and 8% separating glue were prepared according to standard SDS-PAGE method. The cytolysis supernatant containing the same concentration of protein was mixed with 5×SDS sample-adding buffer, which was boiled for 5 min and sample-added after cooling. After electrophoresis, it was transferred to PVDF membrane using wet transfer. TBST (0.1% Tween-20; 10 mmol/L Tris-Cl, pH7.5; 3% BSA; 150 mmol/L NaCl) was used to block the non-specific binding site at 4° C. overnight. Membrane washing was done with TBST solution, 10 min/time×3 times. The membrane was incubated with diluted primary antibody (1:500) at room temperature for 3 h. Membrane washing was done with TBST solution, 10 min/time×3 times. The membrane was transferred into the second antibody (1:1000 dilution) and reacted at room temperature for 2 h. Membrane washing was done with TBST solution, 10 min/time×3 times. The membrane was made flat, the luminescent liquid was added dropwise, and imaging was done with chemiluminescence. The results showed that compounds 1 and 3 of the invention have regulatory effects on the signal molecules of STAT3, NF-κB, E-Cadherin, and PCNA closely related to the pathogenesis of glioma, lung cancer, colon cancer, liver cancer, breast cancer, and osteosarcoma at the protein level, exhibiting obvious anticancer activity, as shown in
(80) 2. Experimental Results
(81) Under this experimental system, 1×10.sup.−8 mol/L (i. e. 10 nM) of the series compounds of the invention either had significant inhibitory effects on STAT3, NF-κB, and PCNA, which are signal target molecules closely related to tumorigenesis in tumor cells of colorectal cancer, liver cancer, lung cancer, breast cancer, osteosarcoma, and glioma cells, or promoted the expression of E-cadherin to a certain extent. Thus, they have anticancer activity in the prevention, alleviation and/or treatment of diseases related to colorectal cancer, liver cancer, lung cancer, breast cancer, osteosarcoma, and brain tumor.
(82) 3. Conclusions
(83) The series compounds of the invention have significant regulatory effect on signal molecules related to tumorigenesis, shown as either having inhibitory effect to a certain extent on target molecules of STAT3, NF-κB, and PCNA related to tumorigenesis, or having promoting effect on the expression of E-cadherin protein.
(84)
(85)
(86)
(87)
(88)
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(90)
Example 5: Toxicity Test Results of Compounds 1 and 3 of the Invention on 293T Normal Cell Line Cells Cultured in Vitro
(91) (1) Experimental method:: 293T normal cell line cells cultured in vitro grown to 90% confluent state were digested with 0.1% trypsin/0.1% EDTA and inoculated into 96 well cell culture plate, 3×10.sup.3 cells per well. On the next day of culture, the original culture medium was removed, 1×10.sup.−5 mol/L (i.e. 10 μM) of working solution of the compounds of the invention were added into each well to continue culture. The same volume of complete cell culture medium was added into the blank control well. The cytotoxicity of the compounds of the invention on the 293T cell was detected by CCK8 method after the 293T cell was co-cultured with the compounds of the invention for 48 hours (n=6). The toxic effect of the compounds of the invention on 293T normal cell line cells was calculated according to the following formula, and expressed in inhibition rate:
Inhibition rate (%)=[(control well absorbance−drug well absorbance)/control well absorbance]×100 (%)
(92) (2) Results: Within the time range of experiment to test, 1×10.sup.−5 mol/L (i.e. 10 μM) of the series compounds of the invention had no obvious cytotoxicity on 293T normal cell line cells, the inhibition rate was—3.18%-8.54, and there was no significant difference statistically. See Table 6 for cell growth inhibition rate.
(93) TABLE-US-00008 TABLE 6 Toxicity test results (inhibition rate) of the compounds of the invention on 293T cells Sample The compounds of the concentration inhibition rate invention (μM) (%) 1 10 8.54 3 10 -3.18
(94) (3) Conclusion: The series compounds of the invention have no obvious toxicity on the growth of 293T normal cell line cells, and are suitable for downstream activity screening experiment.
Example 6: Acute Toxicity Test Results of the Compounds of the Invention
(95) Kunming mice (18-22 g) were divided into groups, ten mice in each group, half male and half female. Eight dose groups were set up. According to bliss method, the dose of each administration dose group was set up by the design from the highest dose of 5 g/kg in equal order of 1:0.8. Mice were administrated by gavage. The night before the drug administration, animals were not allowed to eat any food but were allowed to drink water. The mice were given normal diet four hours after administration. After a single administration, the indices of mental state, body weight, diet, behavior, secretion, excreta, death, and toxic reaction, and the like, of the animals were observed for 14 consecutive days, and LD.sub.50 value was calculated. The acute toxicity test results of compounds 1 and 3 of the invention indicated that their LD50 values are as follows: Compound 1 was more than 5.0 g/kg (i.e. LD50 value was not detected), and compound 3 was 600 mg/kg. Compound 1 is a nontoxic specific antibacterial, anti-inflammatory, anti-ulcerative colitis, and anti-tumor compound. Compound 3 is a kind of special antibacterial, anti-inflammatory, anti-ulcerative colitis, and anti-tumor compound with hypotoxicity.
Example 7: The Inhibitory Effect of Compound 1 of the Invention on Tumorigenesis and Growth of Colorectal Cancer Model Mice Induced by (Azoxymethane, AOM)/DSS
(96) 1. Experimental Animals
(97) Male C57b1/6j mice with a body weight range of 18-20 g (purchased from Beijing Huafukang Biotechnology Co., Ltd., License No.: SCXK(Jing)2014-0004).
(98) 2. Experimental Groupings
(99) (1) Normal control group; (2) Model group (AOM/DSS group); (3) Positive drug group (Capecitabine group); (4) Compound 1 group of the invention.
(100) 3. Administration Dosage
(101) (1) Positive drug Capecitabine: 250 mg/kg;
(102) (2) Compound 1 of the invention: 50 mg/kg.
(103) 4. Experimental Method
(104) C57b1/6j mice were randomly grouped according to the above experimental grouping scheme, five mice in the normal control group, and ten other groups.
(105) (1) Normal Control Group:
(106) After one week's normal pre-feeding, normal feeding was done for another two weeks. From the 3rd week on, 0.5% carboxymethylcellulose sodium water solution was administered by gavage in the amount of 10 ml/kg. Administration mode was such that the drug was administered once a day for 12 weeks until the end of the experiment.
(107) (2) Model Group
(108) After one week of normal pre-feeding, AOM was given by intraperitoneal injection at a dose of 10 mg/kg (Prepared by a concentration of 10 mg AOM in 10 ml of normal saline). Then the animals were fed normally for one week, which was recorded as the first week of the experiment. At the beginning of the second week, mice were allowed to drink the drinking aqueous solution containing 2% DSS freely for one week. The mice exhibited obvious symptoms of bloody stool and loose stool, etc. From the 3rd week on, 0.5% carboxymethylcellulose sodium water solution was administrated by gavage at the amount of 10 ml/kg. The administration mode was such that the drug was administered once a day for 12 weeks until the end of the experiment. (3) Positive Drug Capecitabine Group
(109) After one week of normal pre-feeding, AOM was given by intraperitoneal injection at a dose of 10 mg/kg (Prepared by a concentration of 10 mg AOM in 10 ml of normal saline). Then the animals were fed normally for one week, which was recorded as the first week of the experiment. At the beginning of the second week, mice were allowed to drink the drinking water solution containing 2% DSS freely for one week. The mice exhibited obvious symptoms of bloody stool and loose stool, etc. From the 3rd week on, Capecitabine (prepare by a concentration of 250 mg capecitabine in 10 ml of 0.5% sodium carboxymethylcellulose aqueous solution and store at 4° C.) was administered by gavage at a dose of 250 mg/kg (Capecitabine was administered once a day for two consecutive doses. The drug was stopped for one week for each consecutive two week administration), up to the end of the experiment. The administration lasted for 12 weeks (including the weeks of drug withdrawal).
(110) (4) Compound 1 Group of the Invention
(111) After one week of normal pre-feeding, AOM was given by intraperitoneal injection at a dose of 10 mg/kg (prepared by a concentration of 10 mg AOM in 10 ml of normal saline). Then the animals were fed normally for one week, which was recorded as the first week of the experiment. At the beginning of the second week, mice were allowed to drink the drinking water solution containing 2% DSS freely for one week. The mice exhibited obvious symptoms of bloody stool and loose stool, etc. From the 3rd week on, the compound 1 of the invention (prepare by a concentration of 50 mg of compound 1 of the invention in 10 ml of 0.5% sodium carboxymethylcellulose aqueous solution and store at 4° C.) was administered consecutively by gavage at a dose of 50 mg/kg. The administration mode was such that the drug was administered once a day, up to the end of the experiment. The administration lasted for 12 weeks.
(112) At the end of the experiment, the mice were decapitated and killed, changes of spleen weight and colonic tumor burden of the animals after treatment were observed. The experimental results showed that the compounds of the invention exhibited significant antitumor activity in animal in vivo experiment to treat colorectal cancer, and the curative effect was significantly superior to the positive control drug. Specific data is shown in Table 7,
(113) 5. Experimental Results
(114)
(115)
(116) TABLE-US-00009 TABLE 7 Effect of compound 1 of the invention on spleen weight of colorectal cancer model mice Number of Groups cases (mice) Spleen (Dosage) start/end weight (g) Normal control group 5/5 0.088 ± 0.009 Model group (AOM/DSS induction) 10/10 0.183 ± 0.026* Capecitabine (250 mg/kg) 8/10 0.228 ± 0.029 Compound 1 (50 mg/kg) 8/10 0.113 ± 0.013.sup.# Note: *P <0.05, compared with normal control group; .sup.#P <0.05, compared with model group.
(117)
(118)
(119) The calculation method of calculated tumor burden value is as follows:
Tumor burden=(Mean tumor diameter/numbers of mice).sup.2×(Tumor number/numbers of mice)
(120) TABLE-US-00010 TABLE 8 Effect of compound 1 of the invention on colorectal tumor burden of colorectal cancer model mice Number of Tumor Groups cases (mice) burden (Dosage) start/end (piece) Normal control group 5/5 0 Model group (AOM/DSS induction) 10/10 32.76 ± 8.17** Capecitabine (250 mg/kg) 8/10 42.69 ± 10.26 Compound 1 (50 mg/kg) 8/10 1.13 ± 1.00.sup.## Note: **P <0.01, compared with normal control group; .sup.##P <0.01, compared with model group.