WO2011085640A1

WO2011085640A1 - Colchiceinamide hydrates, preparation methods thereof, and uses thereof

Info

Publication number: WO2011085640A1
Application number: PCT/CN2011/000057
Authority: WO
Inventors: 刘力
Original assignee: Liu Li
Priority date: 2010-01-13
Filing date: 2011-01-13
Publication date: 2011-07-21
Also published as: CN102126983B; CN102126983A

Abstract

Provided are colchiceinamide hydrates, preparation methods thereof, and uses thereof for the prophylaxis or treatment of diseases of human beings or animals, such as leukaemia, breast cancer, stomach cancer, cervical carcinoma, nasopharyngeal carcinoma, actinic keratoma, basal-cell carcinoma, squamous cell skin carcinoma in situ.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the field of medical technology, and in particular to providing a drug-increase amine hydrate, a preparation method thereof and use thereof. BACKGROUND OF THE INVENTION The published literature only reports colchicine derivatives one by one *J^(C ₂₁ H ₂₄ N ₂ 0 ₅ , molecular weight: 384.43), and so far, there are no published literatures at home and abroad that report the following structures. The cyclic amine crystallization hydrate C ₂₁ H ₂₄ N ₂ 0 ₅ · nH ₂ 0, n=2. 5 - 3. 5 and its preparation method and use. SUMMARY OF THE INVENTION The present invention relates to a colchicine derivative, a preparation method thereof and use thereof, and further relates to an autumn sulphate crystal hydrate [C ₂₁ H ₂₄ N ₂ 0 ₅ · n3⁄40, n=2.5 - 3 5, n may be 2. 5, 2. 75, 3. 0, 3. 5 or a number therebetween] and its preparation method and use.

The crystal cracking amine containing crystal water obtained by the present invention is less toxic than colchicine, and surprisingly, the rhizome amine containing crystal water has much lower wettability than the rhizoma amine containing non-crystal water, and contains crystal water. The autumn crack amine hydrate is more stable than the crystal water-free, easy to store and transport, easy to prepare the preparation, and also shows that the autumn crack amine hydrate of the invention has better storage stability. Further, the deliquescent of the anhydrate causes the air to be prevented from being stuck or the like during the treatment, and the hydrate has a good slidability, thereby improving the operability of the preparation. The inventors based on the above invention have completed the present invention.

Surprisingly, characteristically, the thermogram of the hydrate of the present invention has a corresponding endothermic front under the weightless platform, such as: The thermal analysis map shows the autumn 3 hydrate.

In the embodiment of the embodiment 1, the measurement of the diffraction angle 2 Θ ( 3-60. ) is measured by the powder X-ray diffractometry, and the autumn 3 hydrate of the present invention may include the following

1

Confirmation 2 The position of the threshold has a corresponding feature value.

In still another aspect, the present invention provides a process for the preparation of the autumn crack amine crystal hydrate of the present invention, which comprises the following:

Method 将. The colchicine is dissolved in water or a C1-C6 low molecular alcohol such as methanol, ethanol, isopropanol or C2-C8 lower ether, and ammonia, ammonia, ammonium hydrogencarbonate is added. One or several kinds of ammonium carbonate or a solution thereof, 5 ~ 50 stirred reaction for 2-24 hours, concentrated under reduced pressure, using C1-C6 lower halogenated hydrocarbons, including dichloromethane, chloroform, dichloroethane, One or more of C2-C8 lower esters, such as butyl acetate, ethyl acetate, ethyl formate, etc., are extracted 1-5 times, separated from the water layer, and the organic layer is concentrated under reduced pressure, and water or C1-C6 is low. Molecular alcohols such as methanol, ethanol, isopropanol, C2~C8 lower hydrazine, C1~C6 lower halogenated hydrocarbons, including dichloromethane, chloroform, dichloroethane, etc., C5-C10 linear or branched alkane Or a cycloalkane, including pentane, n-hexane, cyclohexane, petroleum hydrazine, a C6-C10 aromatic hydrocarbon, including one or more of benzene, toluene, etc., C3-C7 low molecular weight, etc. one or more times Crystallize, place, crystallize completely, filter, drain, dry;

Method B. Use colchicine with water or C1-C6 low molecular alcohol such as methanol, ethanol, isopropanol, C2-C8 lower ether, C1-C6 lower halogenated hydrocarbon, including dichloromethane, chloroform, dichloroethane One or more of alkane, etc., as a solvent, to dissolve, add ammonia or ammonia, ammonium bicarbonate, ammonium carbonate or a solution or a solution thereof, stir the reaction for 5 to 50 2- for 2 to 24 hours, and concentrate under reduced pressure. , using CI-C6 lower halogenated hydrocarbons, including dichloromethane, chloroform, dichloroethane, etc., C2-C8 lower fins, such as butyl acetate, ethyl acetate, formate, etc. Extract 1-5 times, separate the water layer, concentrate the organic layer under reduced pressure, use water or C1-C6 low molecular alcohol such as methanol, ethanol, isopropanol, C2 "C8 lower ether, C1-C6 lower halogenated hydrocarbon, Including methylene chloride, chloroform, dichloroethane, etc., C5-C10 linear or branched alkane or cycloalkane, including pentane, n-hexane, cyclohexane, petroleum ether, C6-C10 aromatic hydrocarbons, One or more crystallizations including benzene, toluene, etc., C3-C7 low molecular hydrazine, etc., Home, to complete crystallization, too, drained, and dried to obtain.

The number of carbon atoms of the lower ketone or low molecular hydrazine in the present invention is defined as C3-C7 (ie, carbon source) The number of sub-numbers is 3-7, the labeling method for carbon atoms is the same as below), such as acetone, butyl hydrazine, etc.; the number of carbon atoms of lower alcohol or low molecular alcohol is defined as CI-C6 (ie: 1-6 carbon atoms) The number of cesium atoms such as methanol, ethanol, isopropanol, lower hydrazine or low molecular ether is defined as C2-C8, such as diethyl ether, dibutyl ether and the like. The number of carbon atoms of the lower halogenated hydrocarbon is defined as C1-C6; the number of carbon atoms of the lower ester is defined as C2~C8; the number of carbon atoms of the low molecular linear or branched anthracene or cycloalkane is defined as C5-C10, low molecular weight The number of carbon atoms of an aromatic hydrocarbon is defined as C5-C10.

Crystallization or recrystallization solvent of autumn crack amine hydrate, preferably water, methanol, ethanol, isopropanol, n-butanol, acetone, butyl hydrazine, isobutyl, methyl acetate, ethyl acetate, dichloromethane, chloroform, diethyl ether One or more of isopropyl acid, tetrahydrofuran, petroleum ether, and benzene. The autumn crack amine hydrate of the present invention may have different crystal forms, for example, from methanol-water-chloroform, water-chloroform-acetamidine, water-dichloromethane-acetic acid, ethanol-water-dichloromethane-ethyl acetate. The X-ray powder diffraction pattern of the quaternary ammonium amine 2. 5, 3, 3. 5 hydrate prepared by the system of ethanol-water-dichloromethane-acetic acid or the like may be different. The solution of the crystalline hydrate of the present invention or the crystalline hydrate of the examples has a consistency in the retention time of the main peak color under the same HPLC conditions.

The method for obtaining the autumn crack amine anhydrate sample by the special treatment of the autumn crack amine 3 hydrate of the present invention may be at different temperatures (such as 70-105), drying time (8 hours to several ounces), or with other desiccant. (including silica gel, phosphorus pentoxide, anhydrous calcium chloride, anhydrous sodium sulfate, anhydrous carbonic acid pin, anhydrous carbonic acid clock, anhydrous magnesium sulfate, sodium hydroxide, potassium hydroxide, silica gel, etc.) Drying under normal pressure or reduced pressure, or azeotropic boiling water.

The use of the autumn crack amine hydrate of the present invention: the autumn crack amine hydrate of the present invention is used for preparing a lyophilized powder injection preparation for injection, or a large infusion preparation, or a small water injection, a tablet, a capsule, a powder, a granule , as well as ointments, gels, suppositories, etc.

A tablet, capsule, granule for preparing a solid preparation, which may contain a pharmaceutically acceptable filler such as starch, modified starch, lactose, microcrystalline cellulose, cyclodextrin, sorbitol, mannitol, phosphoric acid Calcium, amino acid, etc.; pharmaceutically acceptable disintegrating agents, such as starch, Modified starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low substituted hydroxypropyl cellulose, surfactant; pharmaceutically acceptable wetting agent and chelating agent, such as gelatinized starch , methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinylpyrrolidone, alginic acid and its salts; pharmaceutically acceptable lubricants and glidants, such as stearic acid, stearic acid Magnesium, polyethylene glycol 4000 - 8000, talc, micronized silica gel, magnesium lauryl sulfate, etc.; pharmaceutically acceptable sweeteners and flavors, such as aspartame, cyclamate, sodium saccharin, trichloro Sucrose, edible ^, etc.

The crystal hydrate of the present invention is different from the deliquescent of the anhydrate in that it is isolated from air to prevent blocking or the like during the treatment, and the crystalline hydrate has good slidability, thereby improving the operability of the preparation; and making the prepared solid preparation good. Its dissolution properties make it easy to be absorbed into the Ajfe liquid circulation, improve bioavailability, and facilitate its rapid function.

Preparation method of cartilage or gel of autumn crack amine hydrate: Autumn crack amine hydrate is mixed with 50-95% base, M can be ethanol, glycerin, triethanolamine, glycerin gelatin, polyethylene glycol 200-8000, vaseline , poloxamer, polyvinylpyrrolidone, semi-synthetic fat-soluble monoglyceride, carbomer series (931, 934, 940, 974, AA-1, 1342, etc.), Tween 60-80. The cartilage or gel may contain pharmaceutically acceptable preservatives and stabilizers. The carbomer may be separately dispersed in water during preparation, added with glycerin, polyethylene glycol 200-8000, vaseline, heated in water bath, stirred and mixed, and added. Prescription amount of autumn crack amine hydrate, stirring, adjust the pH = 4. 0 - 8. 5 with pharmaceutically acceptable inorganic organic base, add water to the whole amount, stir until evenly, and dispense.

Preparation method of suppository for autumn 3⁄4 ^ hydrate: 0. 01 - 10% of autumn fU hydrate, the rest consists of suppository matrix, base shield can be ethanol, glycerin, glycerin gelatin, polyethylene glycol 200-8000, poluo Sham, semi-combined ^ fat ^ ϋ, carbomer series (931, 934, 940, 974, ΑΑ-1, 1342, etc.), Tween 60-80 _β preparation method: mixing the main drug with the base shield, water bath heating Stir, melt, stir until it is evenly poured into the suppository that has been coated with the lubricant, until it is slightly spilled, and then flattened after cooling.

Autumn split amine hydrate injection, the preparation method thereof is: The preparation method of the frozen powder injection preparation is as follows: taking the autumn crack amine hydrate, adding a pharmaceutically acceptable auxiliary solvent, a lyophilized support agent or a co-agent, a stabilizer, a water for injection, stirring to dissolve, if necessary, available The pH of the pharmaceutically acceptable acid-base is adjusted to 3. 5 ~ 8. 5, plus activated carbon 0. 005 ~ 0. 5X (W / V) stirring 15 ~ 45fflin, filtration, hydration, sterile filtration, press 2 - 40mg / bottle (based on the main drug), frozen, dried, tampon, finished product.

Autumn split amine hydrate small volume injection and preparation process thereof: autumn crack amine hydrate plus water for injection and pharmaceutically acceptable additive, for example: pharmaceutically acceptable _P H regulator, pharmaceutically acceptable cosolvent The pH is between 3.5 and 8. 5, and the pH is between 3. 5 and 8. 5 .

The pharmaceutically acceptable lyophilized support or adjuvant may contain L sugar, glucose, mannitol, sorbitol, xylitol, dextrose liver, violent acid or a salt thereof (including glycine, taurine, fine One or more of amino acid, sodium dihydrogenate, disodium hydrogen phosphate, sodium deoxycholate, and the like.

The pharmaceutically acceptable pH adjusting agent may be a pharmaceutically acceptable inorganic or organic acid, an inorganic base or an organic base, or a Lewis acid or a base in a broad sense, and may contain one or more kinds, and may be hydrochloric acid. Phosphoric acid, propionic acid, acetic acid and acetate, such as sodium acetate, lactic acid and lactic acid pharmaceutically acceptable salt, citric acid pharmaceutically acceptable salt, sodium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide, phosphoric acid Salt, tartaric acid and its pharmaceutically acceptable salts, borax, boric acid, succinic acid, caproic acid, adipic acid, fumaric acid, maleic acid, trishydroxyaminomethane, diethanolamine, ethanolamine, isopropanolamine , diisopropanolamine, 2-J^-2-(hydroxymethyl) 1, 3-propanediolamine, 1,2-hexanediamine, N-methylglucamine, diisopropylamine and their salts, A hydroxycarboxylic acid and a pharmaceutically acceptable salt, such as one or more of glucose butyric acid, gluconic acid, lactobionic acid, malic acid, threonic acid, glucoheptonic acid, acid, and acid salt.

The pharmaceutically acceptable antioxidants and stabilizers may be arsenic acid, tellurite, bisulfite, pyrosulfite, dithionite, organic compound thiourea, glutathione Peptides, dimercaptopropanol, thioglycolic acid and salts, thiolactic acid and salts, thiodipropionic acid and salts, benzophenone compounds, such as gallic acid and salt, caffeic acid, caffeate, Ferulic acid, ferulic acid, di-tert-butyl-p-phenylene, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoate, salicylic acid or a salt thereof; amino acid and salts thereof; ascorbic acid And ascorbate, isoascorbic acid and isoascorbate, nicotinamide, tartaric acid, nitrate, phosphate, pharmaceutically acceptable salt, citrate, EDTA and EDTA salts, such as disodium EDTA, BDTA tetrasodium, N-di (2 -hydroxyethyl)glycine, α-cyclodextrin, β-cyclodextrin, Υ-cyclodextrin, glucosyl-cyclodextrin (Gt^YD maltosyl- 0-cyclodextrin, hydroxypropyl P- Cyclodextrin, 2-hydroxypropyl cyclodextrin (2-HP-P ~CYD), 3-hydroxypropyl β-cyclodextrin (3-ΗΡ-β-CYD), sulfobutyrate-β-cyclodextrin SBE-β-CD, such as one or more of (SBE ₇ - β -CD ), SBE ₄ - β -CD, and the like.

The pharmaceutically acceptable co-solvent may be glucose, xylitol sorbitol, mannitol, nicotinamide, N-methylglucosamine, ethyl glucosamine, linonic acid, and pharmaceutically acceptable phosphate (which may be phosphoric acid) Sodium dihydrogen, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, etc.), lactic acid, sodium lactate, citric acid, sodium citrate, polyhydroxycarboxylic acid, and pharmaceutically acceptable salts (eg, glucuronic acid, gluconic acid, lactose) Acid, malic acid, threonic acid, glucoheptonic acid, etc. or a pharmaceutically acceptable salt thereof, acid and J acid salt (may be aspartic acid, glutamic acid, etc.), hydrochloric acid, sulfuric acid, tartaric acid, Tween 20-80, poloxamer (including poloxamer 124, 188, 237, 338, 407, etc.), polyethylene glycol 200 - 2000, ethanol, ethylene glycol, 1, 2-propanediol, 1, 3- Propylene glycol, glycerol, glucosyl-cyclodextrin (G-Gy-CYD), maltosyl-β-cyclodextrin, hydroxypropyl hydrazine-cyclodextrin, 2-hydroxypropyl hydrazine-cyclodextrin (2- ΗΡ-β-CYD), 3-hydroxypropyl p-cyclodextrin (3-HP-β-CYD), sulfobutyrate-cyclodextrin (SBE-β ~CD), such as (SBE - β -CD ), SBE ₄ - Etc., and one or more of water.

The pharmaceutically acceptable isotonicity adjusting agent may be one or more of glucose, fructose, xylitol, sorbitol, mannitol, invert sugar, maltose, dextran, sodium chloride, potassium chloride, sodium lactate, and the like. .

The de-heating source and the sterilization method of the injection agent of the present invention may be an activated carbon de-heating source with a liquid amount of 0. 005 ~ 3%, a microporous membrane sterilization and hot-press sterilization, or an ultrafiltration sterilization or de-heat source. . In the ultrafiltration method, the ultrafilter can be used in flat, coiled, tubular, Hollow fiber type or round box type, etc., preferably roll type and hollow fiber type ultrafilter, using a filter membrane with a molecular weight of 50,000 to 300,000 to remove most of the heat-generating substances and bacteria, and then using the intercepting relative molecules The ultrafiltration membrane with a mass of 4000 to 30000 removes the remaining heat source, preferably an ultrafiltration membrane with a relative molecular mass of 6000 to 30000.

The autumn crack amine hydrate of the present invention is suitable for preparing human and mammalian leukemia, breast cancer, gastric cancer, cervical cancer, nasopharyngeal carcinoma, actinic keratosis, basal cell carcinoma, squamous cell skin caused by the following application of _β treating or preventing drug and the like in situ

Dosage usage: Under normal circumstances, in adults, intramuscular injection: 2-10mg / time, 1-2 times a day. Intravenous injection: 2- 40 mg/time, diluted with 20 ml of 253⁄44 glucose injection. Intravenous drip: 2- 40 mg, diluted with 53⁄4 glucose injection or 0.9% sodium chloride injection 250 - 500ml, one to two times. Use more than half of the children. Dosage for parenteral administration: 10 ~ 70 kg body weight of human or animal, usually 2-80 mg / day, divided into 1-4 times; children more than half of the use, suppository once a day, one at a time Skin external use depends on the situation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a thermal analysis map of autumn 3 hydrate.

Figure 2 is a thermal analysis map of Qiu 3 hydrate.

Figure 3 shows the DEPT spectrum of the autumn 3 hydrate.

Figure 4 is a powder X-ray diffraction pattern of the autumn Jfe 3 hydrate.

DETAILED DESCRIPTION OF THE INVENTION Powder X-ray diffraction

The D/MX-X-ray diffractometer was used at the Materials Testing Center of Wuhan University of Technology, with a diffraction angle of 2 Θ and a scan range of 3-60. The powder X-ray diffraction pattern of the autumn 3 hydrate was measured. In the embodiment of the embodiment 1, the measurement of the diffraction angle 2 Θ ( 3-60 ° ) is measured by the powder X-ray diffractometry, and the autumn J 3 hydrate of the present invention may include The position of the lower 2 具有 has the corresponding eigenvalue (Fig. 4): about 6.40, 9. 97, 10. 94, 11. 39, 12. 72, 14. 66, 17. 61 , 18. 72, 19 46, 20. 08, 21. 72, 22. 21, 23. 45, 23. 87, 25. 39, 26. 08, 27. 79, 35. 92, 41. 17.

Thermal analysis method

Test conditions: Setaram Setsys 16, NETZSCH STA449C, sample volume about 5mg, heating rate: ΙΟΚ / min, N2 flow rate: 50ml / min, temperature: generally room temperature ~ 400 or so.

Surprisingly, characteristically, the thermal analysis (TG-DTA or TG-DSC) pattern of the hydrate of the present invention has a corresponding endothermic peak under a weightless platform, and the thermal analysis spectrum shows an autumn 3 hydrate.

Wetting test

The autumn crack amine hydrate of the present invention can be stably stored. The autumn crack amine 3 hydrate and the autumn crack amine 3 hydrate of Example 1 were vacuum-dried in the presence of about 95 Torr and phosphorus pentoxide to obtain an autumn § J ^ anhydrate sample (water "2%" separately sealed In the case of a vial, the wettability test is carried out according to the requirements of the Chinese Pharmacopoeia: : An anhydrate and a hydrate of the invention are about 5 g, placed on a dry constant weight surface, accurately weighed, 25 TC;, relative humidity is 75%, respectively The samples were tested at Oh and 48h, and the percentage of wetting gain was calculated. The results showed that the anhydrate was more hygroscopic than the hydrate of the present invention (Table 1);

Table 1. Results of wettigation test

Sampling time (48 hours) Compared with 0 hours, weight gain % autumn 3 hydrate 0. 38

Autumn None 12. 26 Stability Experiment

The crystal hydrate of the rhizoma amine of the present invention can be stably stored stably, and the samples of the anatase of the rhizome and the anatase of the rhizome of Example 1 are respectively obtained under RH 75%, 30-light-proof parts. Accelerated stability test in closed and vials, color components: Column: Kromasi 1 C _Jg (250mm χ 4. 6 legs, 5, mobile phase: acetonitrile - [0 · 02mol / L ^ acid dihydrogen clock solution - triethylamine (phosphoric acid adjusted pH = 5)] (400: 600: 2) detection wavelength 2 ⁴ 8nm, flow rate lml / min, measured was the shield about the rate of increase. the results are shown in Table 2.

Table 2. Accelerated Stability Test Results

Sampling time (June) 3⁄4 increase in related substances compared with 0. Autumn 3 hydrate 0. 31

Autumn 3⁄4 ^No 1. 21 Example 1 Preparation of autumn split amine 3 hydrate A three-necked flask of 3 g of colchicine and 50 ml of concentrated aqueous ammonia was stirred to dissolve, the temperature was controlled at 25-40 TC, and the reaction was stirred for 12 hours. The organic layer was concentrated under reduced pressure. The organic layer was concentrated under reduced pressure. EtOAc was evaporated from EtOAc EtOAc. After the solids are precipitated, the mixture is filtered with suction, and the precipitate is washed twice with water. The resulting solid is recrystallized from solvent ethanol, chloroform 20 ml, water 2 ml, and petroleum acid 20 ml, and placed under 0 TC. The crystals were dried at about 50*C for 4 hours to obtain yellow crystals of about 1. 9 g. Identification analysis: 2 mg was placed on a white porcelain plate, dissolved in ethanol, and 3 drops of ferric chloride test solution were added dropwise. Immediately dark color, add dilute hydrochloric acid, the color disappears immediately; take 2mg on white porcelain plate, add sodium nitrite crystals and 3 drops of hydrochloric acid, immediately dark brown; melting point: 257-261X (ELECTROTHERMAL MELTING POINT APPARATUS, Uncorrected); room temperature 20- 25TC ratio rotation (10mg/ml chloroform): -182. 2, Karl Fischer method for moisture determination 12.42%, thermal analysis: platform weight loss is about 11.64%, this sample contains 3 crystal water The result (theoretical value 12.33%) is within the error range (see Figure 1); UV: λ makes JiHnnu 248nm, 352nm, ESI-MS: m/z: 383; infrared spectrum: v cm-, 3439 (width ), 3304, 3194, 2994, 2935, 2838, 1665, 1602, 1558, 1508, 1485, 1458, 1415, 1400, 1384, 1349, 1322, 1282, 1143, 1098, 1049, 1009, 856, 750; H- MR (DMSO, D ₂ 0) δ 8. 58 (lH, d, becomes smaller after heavy water exchange, H-8), δ 7. 83 (2Η, Wide single peak, disappeared after heavy water exchange, NH2), δ 7.10 (1H, s, H-12), δ 7.08 (1H, s, H-ll), 56.90 (1Η, d, Η-4), δ 6.74 ( 1Η, s,), δ 4.36 (1Η, m, ) , δ 3.82(3H, s, OCH ₃ ), δ 3.77 (3Η, s, OCH^ , δ 3.47 (3Η, s, 0CH ₃ ) , δ, δ 1.84-2.14 (4H, m, 2 χ C3⁄4), 51.86 (3Η, s, C0CH ₃ ); Nuclear magnetic resonance spectrum ("C-MR" and DEPT spectrum (DEPT 诿 see Figure 3) (Mercury VX-300 NMR Resonance meter): ¹³ C - earn (DMS0): 175.2-C-9; 169.2, C-16; 157.6, C-10; 153.1, C-1; 151. l, C-7a; 150.2, C-3; 141.5, C-2; 138.8, C-12; 135.2, C-12a; 129.7, C-4a; 127.6, C-la; 124.5, C-8; 110.9, C- 11; 108.4, C-4; 61.5, C-2-OCH3; 61.4, C-1-0CH3; 56.6, C-3-OCH3; 52.2, C-7; 37.5, C-6; 30.3, C-5; 22.3, C-17. : C 57.52%, Η6·90%, Ν6.39%; Found: C 57.69%, H6.71%, N6.23%.

Example 2 Preparation of autumn crack amine 3 hydrate 6 g of colchicine and 120 ml of water were placed in a three-necked flask, stirred, 20 g of ammonium hydrogencarbonate, 12 ml of concentrated ammonia water, stirred, controlled temperature 25-50"C, stirring reaction 8-16 hours, concentrated under reduced pressure, extracted three times with 300 ml of chloroform. The organic layer was concentrated under reduced pressure. Under reduced pressure, add 1.5 ml of ethanol, 2 ml of water and then add acetic acid to make micro-turbidity or add a little ethanol to dissolve. Add a small amount of acetic acid. Placed below 0"C, after the solids are precipitated, suction filtration, the precipitate is washed twice, the solid is dissolved in a small amount of ethanol under heating in a water bath, then 3 ml of water, 20 ml of chloroform, and an appropriate amount of diethyl ether are added for recrystallization. Placed below, after precipitation and precipitation is sufficient, suction filtration, the resulting solid is dried at about 60 ° C for about 4 hours to obtain about 3.5 g of yellow crystal; Identification analysis: Take 2 mg on white porcelain plate, add ethanol to dissolve, add three 3 drops of ferric chloride test solution, the solution immediately showed dark color, and then added dilute hydrochloric acid, the color disappeared immediately; take 2mg on white porcelain plate, add sodium nitrite crystals and 3 drops of hydrochloric acid, immediately dark brown; Melting point: 258-2 62TC (ELECTROTHERMAL MELTING POINT APPARATUS, uncorrected), the specific rotation at room temperature (10mg/ml chloroform): -182.6., the moisture content is 12.65% by Cartesian method, thermal analysis (NETZSCH STA449C): platform weight loss is about 12.03%, This is in the error range with the result of the sample containing 3 crystal water (theoretical 12.33%); UV:

248nm, 352nm, Infrared language: ν ^ΚΓ _ϊ αη—, 3439 (width), 3304, 3194, 2994, 2935, 2838, 1665, 1602, 1558, 1508, 1485, 1458, 1415, 1400, 1384, 1349, 1322, 1282, 1143 , 1098, 1049, 1009, 856, 750; ESI-MS: m/z: 383; nuclear magnetic co-pan ( ¹³ C-NMR) and DEPT spectra (Varian Unity-Inova600 NMR, Mercury VX-300 NMR ) : Total carbon number 21, primary carbon 4, secondary carbon 2, tertiary carbon 5, quaternary carbon 10; "C-NMR (DMS0): 175.2, C-9; 169.2, C-16; 157.6, C-10 153.1, C-1; 151. l, C-7a; 150.2, C-3; 141.5, C-2; 138.8, C-12; 135.2, C-12a; 129.7, C-4a; 127.6, C-la 124.5, C-8; 110.9, C-ll; 108.4, C-4; 61.5, C-2-OCH3; 61.4, C-1-0CH3; 56.6, C-3-OCH3; 52.2, C-7; , C-6; 30.3, C-5; 22.3, C-17. Theoretical analysis: C 57.52%, H6.90X, N6.39%; Found: C 57.38%, H7.07%, N6.46 %.

Example 3 Preparation of lyophilized preparation 5 g of autumn crack amine hydrate (Example 1), 15 g of mannitol, 4 g of sodium dihydrogen phosphate, 2 g of citric acid, lg, 1,3-hydroxypropyl cyclodextrin hydrochloride 10g, Tween - 803ml, EDTA di-nano 0.02g, add 20- 50 water for injection about 800ml, stir to dissolve, adjust the _P H to 4.5 ~ 6.8 with citric acid and sodium hydroxide solution of about 1 - 5M, make up water for injection To 1000ml, add activated carbon 0.01 ~ 0.5% (W / V) for 15-30min, filter, filter with 0.22 micron microporous membrane, according to 5, 10mg / bottle, vacuum freeze drying, tampon, freeze-dried Product.

Example 4 Preparation of Autumn Cracked Amine Hydrate Injection Autumn Cracked Amine 3 Hydrate (on a dry basis) 5 g (Example 1), sodium gluconate 4 g, citric acid 5 g, nicotinamide 10 g, glycerol 100 ml, EDTA disodium 0.06g, avoiding strong light, adding water for injection, passing nitrogen, stirring to dissolve, citric acid and sodium citrate solution of about 1-5M to adjust H to 3.5-6.8, add activated carbon 0.01% (W/V) and stir 15~ 45min, filter, hydrate to 2000ml, filter with 0.22 micron microporous membrane or filter with ultrafiltration membrane with molecular weight of 6000 - 20000. The solution is saturated with nitrogen, packed at 5, 10mg, or 20mg / aliquot. Get the finished product.

Example 5 Preparation of Autumn Sodium Sulfate Hydrate Sodium Chloride Infusion: Autumn Split Amine 3 Hydrate lOg (Example 2), sodium chloride 425g, sodium citrate 3g, tartaric acid 5g, Tween-80 5ml, propylene glycol 50ml, nicotinamide 15g, EDTA disodium 0. 6g, added to water for injection, the solution is high purity 5%的活性炭的,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Heat and stir for about 10 to 30 minutes, filter for decarbonization, and then pass through 0.22um membrane fine filtration or filter with ultrafiltration membrane with molecular weight of 6000 ~ 20000. After semi-finished product test, the content, pH value and clarity are qualified. After that, it is filled in a 50ml, 100ml or 250ml bottle or plastic bag under high-purity nitrogen, sterilized, and packaged.

Example 6 Autumn ^ ^ Hydrate tablets or capsules ( 1 Omg / grain)

Prescription: Autumn 3⁄4^3 Hydrate (Example 1) 10g

Microcrystalline cellulose 65g

Starch 20g

Sodium Carboxymethyl Starch 5g

--cyclodextrin 5g

5 % PVP 30 (50% aqueous ethanol solution)

Magnesium stearate 2g

The autumn crack amine hydrate, microcrystalline cellulose, starch, cyclodextrin, sodium carboxymethyl starch over 100 Θ sieve, using 5% PVP 30 of 503⁄4 ethanol aqueous solution to make a soft material for the adhesive, over 18 - The granules are sieved by 24 mesh, dried, and sieved through 14 - 20 mesh sieves, mixed with magnesium stearate, filled or compressed.

Example 7 Autumn ^ hydrate tablet (1 Omg / tablet)

Prescription: Autumn amine 3 hydrate (Example 1) 10g

Microcrystalline cellulose 180g

( ^ hydroxypropyl cellulose 5g

--cyclodextrin 5g

Magnesium stearate 2g

The autumn crack amine hydrate, cyclodextrin, low-substituted hydroxypropyl cellulose through a 100 mesh sieve, The microcrystalline cellulose was passed through 80 mesh, mixed, granulated by a thousand methods, passed through a sieve of 18 - 24 mesh, mixed with magnesium stearate, and compressed.

Example 8 Autumn crack amine hydrate particles including autumn crack amine 3 hydrate particles (2.5 mg/bag)

Prescription: Autumn 3⁄4 ^ Hydrate 2. 5g

Mannitol 47g

Microcrystalline cellulose 150g

Solid consumption lg

5 % hydroxypropyl methylcellulose

The autumn crack amine hydrate, mannitol, microcrystalline cellulose, food flavor through a 100 mesh sieve, using 5% hydroxypropyl methylcellulose in 503⁄4 ethanol aqueous solution to make a soft material for the adhesive, after 18 - 24 The mesh is granulated, dried under 60TC, and sieved after 14-20 mesh.

Example 9 an autumn crack amine 3 hydrate gel

Prescription: Autumn 3 hydrate (Example 2)

Polyethylene glycol 6000

Polyethylene glycol 400

Glycerin

Carbomer 934

Carbomer 1342

Water

Carbomer 1342 and Carbomer 934 will be dispersed with water, glycerin, polyethylene glycol 6000, polyethylene glycol 400, stirred and mixed, added with rhizome amine 3 hydrate, heated in water bath, stirred until homogeneous, with phosphoric acid The appropriate amount of sodium disodium hydrogenate and sodium dihydrogen phosphate solution is adjusted to pH = 4. 0 ~ 7. 5, and it is available in separate parts.

Example 10 Suppository of autumn $3 hydrate (12 mg/granule) Prescription: Autumn^3 hydrate (Example 1) 1. 2g (100 pellets) Polyethylene glycol 4000 180g Glycerin 5ml

Polosham 50g

Tween 80 2ml

Mix the autumn amine 3 hydrate, glycerin, polyethylene glycol 4000, poloxamer, Tween 80, heat in a water bath, stir, melt, stir until thoroughly, and pour into the suppository with lubricant. * Medium, to slightly overflow the plug mold, to be flattened after cooling, and the mold is obtained.

Example 11, Pharmacological Test

1. Inhibition of gastric cancer

The human gastric adenocarcinoma SGC-7901 cell line in logarithmic growth phase was digested and prepared into a single cell suspension of about 2 x ^{10 7} /ml in PBS, in BALB/c mice (body weight).

18-22g, female half) 0.2ml subcutaneously injected into the right lower back. After 2 days, the rats were randomly divided into blank control group, administration group pyrimidine, 5-FU group, and administration group (autumn 3 hydrate group). Group 10 only. The blank control group was intraperitoneally injected with 0.2 ml of normal saline once a day, 5-FU group was injected with 5-FU (physiological salt release, volume 0.2 ml), 20 mg/kg, once every other day; autumn split amine 3 hydrate group was injected with 1 mg. /kg, 5 times a week, continuous injection for 2 weeks. After 24 hours of the last administration, the animals were sacrificed, the animals were sacrificed, the tumors were removed, the tumor weight was weighed, and the tumor inhibition rate was calculated (the average tumor weight of the control group - the average tumor weight of the administration group) / the average tumor weight of the control group was 100%. The results are shown in Table 3 (P value is the result of the administration group compared with the blank control group, the same below).

Table 3. Inhibition of gastric cancer

Group n mean tumor weight (g) tumor inhibition rate (%) P value

Blank control group 10 1.88 ±0.69

5-FU group 10 0.96 ± 0.24 46.07 < 0.05 Autumn 3 hydrate group 10 1.24 ± 0.43 34.04 < 0.05

(Embodiment 1)

2. Inhibition of cervical cancer

Hela cell line of human uterine head cancer in logarithmic growth phase, prepared after digestion with PBS Single cell suspension at a concentration of approximately 2 χΐ θ ⁷ / ml, female mice in Kunming (body weight)

18-22 g) 0.2 ml subcutaneously, and 3 days after planting, the mice were randomly divided into a blank control group, a drug-administered group (cyclophosphamide group), and a drug-administered group (autumn 3 hydrate group, taken from Example 2). ), 10 in each group. Oral administration, the blank control group was given 0.2 ml of normal saline once a day, and the cyclophosphamide group was injected with cyclophosphamide (physiological salt release, volume 0.2 ml), 25 mg/kg, once a day; autumn amine 3 hydrate Group 1.5mg/kg (salt saline-prepared suspension, volume

0.2ml), once a day for 2 weeks. After 24 hours of the last administration, the animals were sacrificed, the tumor was excised, the tumor weight was weighed, and the tumor rate was calculated - (the average tumor weight of the control group - the average tumor weight of the administration group) / the average tumor weight of the control group was xl003⁄4, and the results were shown in Table 4.

Table 4. Inhibition of cervical cancer

Group n mean tumor weight (g) tumor inhibition rate (%) P value

Blank control group 10 1.67 ±0.49

Ringworm amide group 10 0.75 ±0.37 55.09 < 0.05 autumn crack amine 3 hydrate group 10 0.93 ± 0.42 44.31 < 0.05

3. Inhibition of breast cancer

The human breast cancer MA-737 cell line was subcutaneously injected into the right back of the BALB/c mouse (body weight 18-22 g, female) by 0.2 ml. On the third day after the implantation, the mice were randomly divided into a blank control group and administered. Group (^^ pyrimidine, 5-FU group), administration group (autumnamine 3 hydrate group, Example 1), 10 per group. The blank control group was intraperitoneally injected with 0.2 ml of normal saline once a day. The fluorouracil group was injected with pyrimidine (physiological salt release, volume 0.2 ml), 50 mg/kg, once every other day; the autumn crack amine 3 hydrate group was injected with 1.2 mg/kg. (saline-modulated suspension, volume

0.2 ml), once a day, for 2 weeks. After 24 hours of the last administration, the animals were sacrificed, the tumor was removed, the tumor weight was weighed, and the tumor inhibition rate was calculated (the average tumor weight of the control group - the average tumor weight of the administration group) / the average tumor weight of the control group was 1003⁄44, and the results were shown in Table 5. .

Table 5. Inhibition of breast cancer

Group η Average tumor weight (g) Tumor inhibition rate (%) P value Blank control group 10 1. 89士0. 66

Fluorouracil 10 0. 92 ± 0. 37 51. 32 < 0. 05

Autumn 3⁄4Jfe3 hydrate group 10 1. 17 ± 0. 51 38. 10 < 0. 05 4, inhibition of leukemia

The long-term L615 leukemia cells were prepared in PBS with a concentration of about 2×10′/ml of single-cell sputum, and subcutaneously injected into 615 inbred mice (body weight 18-22 g, male and female). 2 ml, seed ^ day 3, the mice were randomly divided into a blank control group, a drug-administered group (fluorouracil, 5-FU group), a drug-administered group (autumnamine 3 hydrate group, Example 1), each group 10 only. All are administered by gavage. The blank control group was given 0.2 ml of normal saline once a day; the 5-FU group was administered by 30 mg/kg intragastrically once every other day; the autumn split amine 3 hydrate group was 1 mg/kg (physiological saline-prepared suspension) , volume 0. 2ml), once a day, until the animal died naturally, calculate the survival extension rate = (average survival time of the administration group - average survival time of the control group) / average survival time of the control group X 100%, the results are shown in Table 6.

Table 6. Effect on survival rate of leukemia

Group n survival time (days, d) survival extension rate (%) P value blank control group 10 7. 3 ± 0. 9

5-FU group 10 10. 8 ± 1. 2 47. 9 < 0. 05 Autumn ^ Hydrate group 10 9. 6 ± 1. 0 31. 5 < 0. 05 Understandable, from the professional point of view, many details Variations are possible, and thus do not limit the scope and spirit of the invention, and the invention is not limited to the embodiments described above.

Claims

Claim

1. The number of the autumn crack amine hydrate, its molecule? C ₂₁ H _M Na0s · nH ₂ 0, n=2.5.

The autumn crack amine hydrate according to claim 1, which is an autumn crack amine 3 hydrate, wherein n = 3 _e

3. The autumn hydrate according to claim 2, characterized in that the powder is utilized

X-ray diffraction method, measured in the diffraction angle 2Θ (3>60.), the hydrate has a corresponding characteristic value at a position including the following 2 Θ value: about 6.40, 9. 97, 10. 94, 11 39, 12. 72 , 14. 66 , 17. 61, 18. 72 , 19. 46 , 20. 08 , 21. 72, 22, 21, 21. 45, 23. 87, 25. 39, 26. 08 , 21, 19, 35, 52, 41. 17.

A method of producing the autumn crack amine hydrate according to any one of claims 1 to 3, which comprises:

Method A. One or more of colchicine water or C1-C6 low molecular alcohol, C2-C8 lower hydrazine is used as a solvent to dissolve, add ammonia, ammonia, ammonium bicarbonate, ammonium carbonate or Several or the solution, 5 ~ 501C stirred reaction for 2-24 hours, concentrated under reduced pressure, extracted with one or more of C1-C6 lower halogenated hydrocarbon, C2-C8 lower ester, etc., divided into water Layer, organic layer concentrated under reduced pressure, water or C1-C6 low molecular alcohol, C2-C8 lower acid, C1-C6 lower halogenated hydrocarbon, C5-C10 linear or branched alkane or cycloalkane, petroleum ether, C6- One or more of C10 aromatic hydrocarbons, C3-C7 low molecular weight cesium are crystallized one or more times, placed, crystallized completely, filtered, drained, dried; or

Method B. Using colchicine with water or one or more of C1-C6 low molecular alcohol, C2-C8 lower ether, CI-C6 lower halogenated hydrocarbon as solvent, dissolve, add ammonia gas, ammonia water, carbon ^ ^ One or several of ammonium or ammonium carbonate or a solution thereof, stirring reaction for 5-24 hours for 2-24 hours, concentration under reduced pressure, using one or several of C1-C6 lower halogenated hydrocarbons, C2-C8 lower esters Extract 1-5 times, separate the water layer, concentrate the organic layer under reduced pressure, use water or C1-C6 low molecular alcohol C2~C8 lower acid, C1-C6 lower 3⁄4 generation, C5-C10 linear or branched Terpene or naphthenic, stone One or more of the oil ether, the C6~C10 aromatic hydrocarbon, and the C3-C7 low molecular ketone are crystallized one or more times, placed to complete the crystallization, filtered, drained, and dried.

A pharmaceutical composition comprising the autumn crack amine hydrate hydrate according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is prepared as a lyophilized powder injection preparation for injection, a large infusion preparation, a small water injection, a solid preparation, an ointment preparation, a gel preparation, a suppository, The solids thereof include tablets, capsules, and granules.

The use of the autumn crack amine hydrate according to any one of claims 1 to 3 for the preparation of a medicament, which is characterized in that it is suitable for the preparation of the following human and mammalian leukemia, breast cancer, gastric cancer, cervix The use of drugs for the treatment or prevention of cancer, nasopharyngeal carcinoma, actinic keratosis, basal cell carcinoma, squamous cell skin carcinoma in situ.