CN111072651A

CN111072651A - Dolasetron oxide and preparation method and application thereof

Info

Publication number: CN111072651A
Application number: CN201911334634.XA
Authority: CN
Inventors: 程训官; 汪东海; 王利; 何志均; 叶飞
Original assignee: Sichuan Haisco Pharmaceutical Co Ltd
Current assignee: Sichuan Haisco Pharmaceutical Co Ltd
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-04-28
Anticipated expiration: 2039-12-24
Also published as: CN111072651B

Abstract

The invention relates to a dolasetron oxide with a structure shown in formula (I) or a salt thereof, a preparation method and application of the compound, wherein the compound has a strong inhibition effect on Lyn and MST1 kinases and can be used as an anti-tumor drug.

Description

Dolasetron oxide and preparation method and application thereof

Technical Field

The invention relates to a compound, a preparation method and application thereof, in particular to a dolasetron oxide, a preparation method and application thereof, belonging to the field of pharmaceutical chemistry.

Background

Nausea and vomiting are the most common adverse reactions in tumor chemotherapy, and more than 75 percent of chemotherapy patients can suffer from nausea and vomiting of different degrees. Serious vomiting not only makes the patient feel uncomfortable, will be unconscious, but also can lead to electrolyte imbalance, nutrition deficiency, seriously weakens the body self resistance, and even some patients often produce fear to chemotherapy because of vomiting reaction, so that interrupt treatment, delay the opportunity of controlling the tumor, finally threaten the life. The antiemetic can not only improve the life quality of patients, but also ensure the successful progress of chemotherapy.

The dolasetron mesylate is an antiemetic, belongs to a novel 5-HT3 receptor antagonist with high selectivity and high affinity, effectively blocks 5-HT3 receptors at the periphery and the center by competing for 5-HT, thereby inhibiting or reducing the occurrence of nausea and vomiting reflex, has the advantages of high efficiency, good tolerance, no extrapyramidal side effect and the like, and has wide market prospect in clinical application. Dolasetron mesylate is the mesylate of dolasetron and has the following structural formula:

however, dolasetron is easily oxidized under light irradiation, and related substances are increased to a certain extent, and reports on separation, structure confirmation and application studies of an oxidized product of dolasetron have not been found so far.

Disclosure of Invention

The invention aims to provide a dolasetron oxide which has a structure shown in a formula (I):

the chemical name is (1R,3R,5S,7R) -7- ((1H-indole-3-carboxyl) oxy) -9-aza [3.3.1] nonane-3-carboxylic acid.

The invention also aims to provide a method for preparing the dolasetron oxide, which comprises the steps of reacting dolasetron or the salt thereof with an oxidant in a reaction solvent, and then adding a reducing agent to quench the reaction to obtain the dolasetron oxide shown in the formula (I).

Further, the reaction solvent is selected from one or more of methanol, ethanol, tetrahydrofuran and dichloromethane; the oxidant is selected from hydrogen peroxide or m-chloroperoxybenzoic acid or a mixture of hydrogen peroxide and m-chloroperoxybenzoic acid; the reducing agent is selected from sodium sulfite or sodium bisulfite or a mixture of sodium sulfite and sodium bisulfite.

Furthermore, the molar feed ratio of the dolasetron to the oxidant is 1: 1.0-20, and the molar feed ratio of the reducing agent to the oxidant is 1: 0.5-2.

Furthermore, the molar feed ratio of the dolasetron to the oxidant is 1: 2-10; the molar feed ratio of the reducing agent to the oxidizing agent is 1:1.

Further, the reaction temperature of the reaction with the oxidant is controlled to be-10 ℃ to 70 ℃, and the reaction temperature of the quenching reaction by adding the reducing agent is controlled to be-10 ℃ to 70 ℃.

Furthermore, the reaction temperature of the reaction with the oxidant is controlled to be 0-50 ℃, and the reaction temperature of the quenching reaction by adding the reducing agent is controlled to be 0-50 ℃.

In one embodiment, the preparative method further comprises the step of performing separation and purification by using high performance preparative liquid chromatography gradient elution under the following chromatographic conditions: adopting a carbon-eighteen bonded silica gel chromatographic column; the mobile phase A is 0.03% ammonia water solution, and the mobile phase B is acetonitrile; the detection wavelength is 210 +/-10 nm.

The dolasetron oxide can be used as a reference substance for controlling the quality of dolasetron free base or pharmaceutically acceptable salts thereof and preparations thereof and detecting the validity period of the dolasetron free base or pharmaceutically acceptable salts thereof and preparations thereof. It is therefore another object of the present invention to provide the use of dolasetron oxide for the preparation of a reference for dolasetron drugs or formulations thereof.

Still another object of the present invention is to provide a pharmaceutical composition comprising dolasetron or a pharmaceutically acceptable salt thereof and a structural compound represented by formula (i), i.e., a dolasetron oxide of the present invention, in an amount of not more than 0.5% by mass:

the pharmaceutically acceptable salt of dolasetron is selected from any one of maleate, methanesulfonate, sulfonate, sulfate, hydrochloride, hydrobromide, phosphate, nitrate, p-toluenesulfonate, tartrate, fumarate, acetate, formate, benzoate, cinnamate, succinate, malonate and citrate or the combination of the maleate, the methanesulfonate, the sulfonate, the sulfate, the hydrochloride, the hydrobromide and the phosphate.

Further, the content of dolasetron or the pharmaceutically acceptable salt thereof in the pharmaceutical composition is not less than 90%, preferably not less than 95%, and more preferably not less than 98%.

Further, the content of the structural compound represented by the formula (I) is not more than 0.4%, preferably not more than 0.3%, more preferably not more than 0.2%.

Further, the weight ratio of dolasetron or pharmaceutically acceptable salt thereof to the compound of formula (I) is not less than 200:1, preferably not less than 300:1, and more preferably not less than 500: 1.

In one embodiment, the pharmaceutical composition contains dolasetron or pharmaceutically acceptable salts thereof with the content of not less than 98%, dolasetron oxide with the formula (I) with the content of not more than 0.20%, dolasetron related substances A with the content of not more than 1.0% and other related substances.

The preparation method of the pharmaceutical composition comprises the following steps: under the conditions of keeping out of the sun and under the protection of nitrogen or inert gas, reacting endo-hexahydro-8-hydroxy-2.6-methylene-2H-quinolizin-3 (4H) -ketone or salt thereof with indole-3-formic acid to prepare dolasetron, and further reacting dolasetron with acid to prepare pharmaceutically acceptable salt, such as:

the invention also provides a pharmaceutical preparation, which comprises the pharmaceutical composition and an optional pharmaceutically acceptable carrier.

Further, the present invention provides a pharmaceutical formulation wherein the weight ratio between dolasetron or its pharmaceutically acceptable salt and the compound of formula (i) is not less than 200:1, preferably not less than 300:1, more preferably not less than 500: 1.

The pharmaceutical formulations of the present invention may be in a variety of dosage forms well known in the art. The dosage form suitable for the present invention is selected from oral preparations, external preparations or injections, preferably oral preparations or injections, more preferably injections. The injection is selected from injection (injection), transfusion, freeze-dried powder injection or sterile subpackaged preparation, and is preferably injection. The pharmaceutical formulations of the present invention may be prepared using formulation techniques well known in the art.

The pharmaceutically acceptable carrier according to the present invention is a commonly used excipient or adjuvant for preparing the formulation, which is well known in the art. Common excipients or adjuvants for such injections include, but are not limited to: antioxidants, for example phenols such as Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), sulfur-containing compounds such as thiodipropionic acid, sulfites, bisulfites, dithiocarbamates, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate; organic acids/alcohols/esters such as ascorbic acid, citric acid, malic acid, sorbitol, glycerol, propylene glycol, ascorbyl palmitate, esters such as hydroquinone, hydroxycoumarin, vitamin E, amines such as ethanolamine, soybean phospholipids, cephalin, vegetable phospholipids or animal phospholipids, inorganic acids or salts thereof, phosphoric acids or salts thereof, phosphorous acids or salts thereof; osmotic pressure regulators, such as sodium chloride, glucose, potassium chloride, magnesium chloride, calcium chloride, sorbitol, mannitol, and the like, preferably sodium chloride or glucose; bacteriostats such as 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol; a pH adjuster such as any one of hydrochloric acid, tartaric acid, citric acid, potassium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, sodium acetate, lactic acid, citric acid, sodium citrate, sodium hydrogen carbonate, sodium carbonate, or a combination thereof; emulsifiers such as polysorbate-80, sorbitan-kotate, pluronic F-68, lecithin, soy lecithin; solubilizers such as tween-80, bile, glycerol, propylene glycol, lecithin, polyoxyethylene castor oil, and the like; fillers or excipients, for example, lactose, mannitol, sorbitol, dextran, and the like.

The injection is selected from injection, sterile powder for injection and concentrated solution for injection, and can be used for intramuscular injection, intravenous drip, etc. The specification of the injection of the invention is selected from 1mL, 2mL, 5mL, 10mL, 20mL, 50mL, 100mL, 200mL, 250mL or 500 mL.

The pharmaceutical preparation of the present invention may be a unit preparation, and contains 0.01 to 5g of the pharmaceutical composition of the present invention as an essential active ingredient, preferably 0.01 to 3g, more preferably 0.0125g, 0.04g, 0.08g, 0.1g, 0.16g, 0.2g, 0.25g, 0.32g, 0.4g, 0.5g, 0.75g, 1g, 1.25g, 1.5g, 1.75g, 2g, or 2.5g, in terms of dolasetron.

Particularly, the invention also provides a preparation method of dolasetron mesylate injection, which is prepared by mixing the pharmaceutical composition with a pharmaceutically acceptable injection carrier.

Further, the pharmaceutically acceptable injection carrier is selected from any one of an antioxidant, a bacteriostatic agent and a pH regulator or the combination thereof.

Further, the antioxidant is selected from phenols such as Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), sulfur-containing compounds such as thiodipropionic acid, sulfite, bisulfite, dithiocarbamate, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate; organic acids/alcohols/esters such as ascorbic acid, citric acid, malic acid, sorbitol, glycerol, propylene glycol, ascorbyl palmitate, esters such as hydroquinone, hydroxycoumarin, vitamin E, amines such as ethanolamine, soybean phospholipids, cephalin, vegetable phospholipids or animal phospholipids, inorganic acids or salts thereof, phosphoric acids or salts thereof, phosphorous acids or salts thereof.

Further, the pH regulator is selected from any one of hydrochloric acid, tartaric acid, citric acid, potassium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, acetic acid, sodium acetate, lactic acid, citric acid, sodium citrate, sodium bicarbonate and sodium carbonate or the combination thereof.

Further, the bacteriostatic agent is selected from any one of 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol or the combination thereof.

The invention also provides application of the pharmaceutical composition and the pharmaceutical preparation in preparation of antiemetic drugs.

The inventor surprisingly finds that the dolasetron oxide has stronger anti-tumor activity in research. Therefore, another object of the present invention is to provide the use of dolasetron oxide or its composition in preparing anti-tumor drugs, and to provide a novel anti-tumor drug composition containing an effective amount of dolasetron oxide.

The structure of the dolasetron oxide shown in the formula (I) is confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry, and the structure is analyzed as follows (data are derived from the detection result of the dolasetron oxide sample prepared in the example 1 of the invention):

1. mass spectral data are shown in table 1 below:

table 1 mass spectra data for dolasetron oxides of the invention

Mass Spectrometry (MS) shows that the product has [ M + H [ + ] in]⁺The peak proton ratio is 329, which shows that the product has a molecular weight of 328 and a molecular weight (324) higher than that of dolasetron, and shows that the product is a product with a structure rearranged during an oxidation reaction of dolasetron; the deviation between the measured value and the theoretical value of the mass spectrum signal is small, and the molecular formula (C) of the dolasetron oxide₁₈H₂₀N₂O₄) And (6) matching.

2. Nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum

Hydrogen spectra data, as shown in table 2 below:

TABLE 2 NMR Hydrogen spectra data for dolasetron oxides of the invention

Note: s-singlet, d-doublet, m-multiplet, br-broad¹H-NMR data has reasonable attribution, accords with a hydrogen spectrum chemical shift rule, and accords with the structural formula of the dolasetron oxide.

Carbon spectra data, as shown in table 3 below:

TABLE 3 NMR carbon spectra data for dolasetron oxides of the invention

Note: "+" -a positive peak; "-" -negative peak; "0" -disappearance

¹³C-NMR data have reasonable attribution, conform to the carbon spectrum chemical shift rule and conform to the structural formula of the dolasetron oxide.

Comprehensive analysis of nuclear magnetic resonance spectrum:

(1) according to¹H-NMR is carried out, wherein the nuclear magnetic resonance hydrogen spectrum delta 12.0949-12.1005 of an impurity B test sample is H-19 active hydrogen with drift property, the integral is 1H, the attribution is 1 indole ring NH, the delta 8.7627ppm is H-1, the H-28 active hydrogen has drift property, the integral is 2H, and the attribution is 1 OH and one NH. The hydrogen spectrum shows 10 groups of hydrogen, the integral ratio of the hydrogen from low field to high field is 1:2:2:1:2:1:3:2:4:2, and the total number is 20H, which is consistent with the structural characteristic that the dolasetron oxide has 20 hydrogen.

(2)¹³The C-NMR spectrum has 15 groups of carbon peaks with 18 carbons, which is consistent with the 18-carbon molecular structure of the dolasetron oxide structure.

(3) According to the chemical shift law, C delta 175.7233 is assigned as C-8, C delta 163.2706 is assigned as C-15, and the C-O structure is characterized.

(4) According to the analysis, the characteristic C, H in the sample molecule is attributed, and the attribution is obtained by combining the analysis result of the nuclear magnetic resonance hydrogen spectrum and the COSY spectrum and the analysis result of the carbon spectrum, the DEPT spectrum, the HSQC spectrum and the HMBC spectrum with each C, H in the analyzed sample molecule, so that the molecular structure of the dolasetron oxide is met.

Therefore, according to the comprehensive analysis of mass spectrum, nuclear magnetic resonance H spectrum and C spectrum, the dolasetron oxide is the chemical structure shown in the formula (I).

According to the invention, the dolasetron oxide shown in the formula (I) is found to have strong inhibition effect on Lyn and MST1 through activity research of Eurofins-Cerep SA in France, and the dolasetron oxide has an anti-tumor effect.

Lyn is one of the members of Src family kinases, belongs to the non-receptor tyrosine kinase, and is involved in the binding and dissociation of signal transduction molecules by regulating the tyrosine phosphorylation state. Is closely related to various malignant tumors such as leukemia, lymphoma, prostatic cancer, colon cancer, breast cancer and the like.

MST1 is a core component of the Hippo signaling pathway, and plays an important role in various physiological activities such as cell differentiation, homeostatic control, cell adhesion and migration promotion, autophagy inhibition, apoptosis promotion, and the like through phosphorylation, dimerization, nuclear localization inside and outside, and the like. Is closely related to diseases such as acute leukemia, eczema, thrombocytopenia, immunodeficiency syndrome, autosomal recessive inherited combined immunodeficiency diseases, prostatic cancer, gastric cancer, breast cancer, cervical cancer and the like.

Staurosporine was used as a positive control in this activity study. Staurosporine is a non-selective protein kinase inhibitor and is a common positive control in kinase assays.

Drawings

FIG. 1 is an HPLC chart of a compound of formula (I);

FIG. 2 shows a compound of formula (I)¹H-NMR spectrum;

FIG. 3 shows a compound of formula (I)¹³A C-NMR spectrum;

FIG. 4 is a mass spectrum of a compound having the structure represented by formula (I).

Detailed Description

The present invention is further described below in conjunction with test examples and examples, which enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

Example 1 preparation of dolasetron oxide of the invention

Mixing and stirring dolasetron (10g, 0.0309mol), methanol (200ml) and 30% hydrogen peroxide (50ml, 0.5mol), and heating to 50 ℃ for reaction for 8 hours; a solution of sodium sulfite (63g, 0.5mol) in 500ml of water was added dropwise. Vacuum distilling to remove methanol, filtering the rest water solution to obtain a filter cake as a crude product, and separating the sample by using a preparative liquid phase (a chromatographic column is a carbon-eighteen bonded silica gel column, a mobile phase A is a 0.03% aqueous solution, a B is acetonitrile, gradient elution is carried out, 10-40% B is carried out for 60 minutes, the flow rate is 80ml/min) to obtain 3.65g of a product, and the HPLC purity is 98.7%.

Example 2 preparation of dolasetron oxide of the invention

Mixing and stirring dolasetron (10g, 0.0309mol), ethanol (200ml) and 30% hydrogen peroxide (10ml, 0.1mol), stirring, and heating to 70 ℃ for reaction for 8 hours; a solution of sodium bisulfite (10.4g, 0.1mol) dissolved in 250ml of water was added dropwise. Distilling under reduced pressure to remove ethanol, and filtering the residual water solution to obtain a filter cake, namely a crude product. The sample is separated by using a preparative liquid phase (the chromatographic column is a carbon-eighteen bonded silica gel column, a mobile phase A is 0.03 percent of aqueous solution, a B is acetonitrile, gradient elution is carried out, the concentration of the acetonitrile is 10 to 40 percent of B, the time is 60 minutes, the flow rate is 80ml/min) to obtain 2.95g of a product, and the HPLC purity is 98.5 percent.

Example 3 preparation of dolasetron oxide of the invention

Mixing and stirring dolasetron (10g, 0.0309mol) and tetrahydrofuran (200ml), adding m-chloroperoxybenzoic acid (8.63g, 0.05mol) in batches, and reacting for 24 hours at 20 ℃; sodium bisulfite (5.20g, 0.05mol) dissolved in 200ml of water was added dropwise, tetrahydrofuran was distilled off under reduced pressure, and the remaining aqueous solution was filtered to give a crude product as a filter cake. The sample is separated by using a preparative liquid phase (a chromatographic column is a carbon-eighteen bonded silica gel column, a mobile phase A is 0.03 percent of aqueous solution, a B is acetonitrile, gradient elution is carried out, 10 to 40 percent of B is carried out, the flow rate is 80ml/min), 2.05g of product is obtained, and the HPLC purity is 98.1 percent.

Example 4 preparation of dolasetron oxide of the invention

Mixing and stirring dolasetron (10g, 0.0309mol) and dichloromethane (300ml), cooling to-10 ℃, adding m-chloroperoxybenzoic acid (17.26g, 0.1mol) in batches, stirring, and reacting for 48 hours at-10 ℃. A solution of sodium bisulfite (10.4g, 0.1mol) dissolved in 150ml of water was added dropwise, dichloromethane was removed by distillation under reduced pressure, and the remaining aqueous solution was filtered to give a crude product as a filter cake. The sample is separated by using a preparative liquid phase (a chromatographic column is a carbon-eighteen bonded silica gel column, a mobile phase A is 0.03 percent of aqueous solution, a B is acetonitrile, gradient elution is carried out, 10 to 40 percent of B is carried out, the flow rate is 80ml/min), 3.17g of product is obtained, and the HPLC purity is 98.4 percent.

Example 5 Activity Studies of dolasetron oxide according to the invention

The experimental method comprises the following steps:

mu.M dolasetron oxide of the invention and positive Staurosporine were preincubated with 0.2mg/mL Lyn kinase in HEPES solution (pH7.4) at 37 ℃ for 15 minutes, and then substrate (0.2mg/mL poly (Glu, Tyr)), 10mMATP and 0.25 mCi. gamma. -32P ] -ATP were added for further incubation for 30 minutes. The reaction was terminated by adding a 3% phosphoric acid solution. Detecting the formation amount of [32P ] poly (Glu: Tyr), and calculating the kinase activity and the compound inhibition rate according to the

formulas

1 and 2.

After pre-incubation of 10. mu.M dolasetron oxide of the invention with positive Staurosporine and 0.25U/mL MST1 kinase, respectively, in MOPS solution (pH7.2) at 37 ℃ for 15 minutes, 50. mu.g/mL Myelin Basic Protein (MBP), 10. mu. MATP and 0.25. mu. Ci. gamma. -32P ] -ATP were added and incubation continued for 30 minutes. The reaction was terminated by adding a 3% phosphoric acid solution. Detecting the formation amount of [32P ] MBP, and calculating the kinase activity and the compound inhibition rate according to the

formulas

1 and 2.

Percent kinase activity ═ to be measured_countPositive for_count) V (vehicle)_countPositive for_count) 100% (formula 1)

Inhibition rate%

Wherein it is to be measured_countIs the isotope content of the substrate of the compound to be tested, positive_countThe content of the substrate isotope of Staurosporine and the solvent_countIs the vehicle control substrate isotope content.

The experimental results are as follows:

as shown in table 4 below, dolasetron oxide of the present invention was tested for its inhibitory effect on Lyn and MST1 kinase activity at a concentration of 10 μ M.

Table 4: the invention relates to the inhibition rate of dolasetron oxide on the activity of Lyn and MST1 kinase under the concentration of 10 mu M

The above results indicate that IC of the dolasetron oxide on Lyn and MST1 kinase activity inhibition₅₀The value was about 10. mu.M, showing a strong inhibitory activity.

Example 6 preparation of dolasetron of the invention (composition 1) and dolasetron mesylate (composition 2)

Under the protection of light and helium, 10kg of butanone, 0.91kg (5mol) of endo-hexahydro-8-hydroxy-2.6-methylene-2H-quinolizine-3 (4H) -ketone and 0.8kg (5mol) of indole-3-formic acid are added into a reaction kettle; slowly dropwise adding 1.27kg (10mol) of oxalyl chloride into the reaction kettle while stirring; after the addition, heating the reaction solution to reflux reaction for 5 hours, adding 12kg of butanone and 24kg of purified water for extraction after TLC detection shows that no raw material spot exists basically, and separating out a water layer; after the water layer was washed with 12kg of ethyl acetate, solid potassium carbonate was added to adjust the pH to 12-13. Filtering, and vacuum drying the filter cake at 60 ℃ for 5h to obtain 1.23kg of dolasetron (composition 1) with the yield of 75.8%.

Under the protection of light and helium, adding 1kg of acetone and 123g of dolasetron into a reaction kettle, heating, refluxing and dissolving, dropwise adding methane sulfonic acid, and adjusting the pH value of the system to 2-3. The reaction solution was cooled to 20 ℃ and stirred for 2 hours for crystallization. Filtering, and drying the filter cake at 55-65 ℃ under reduced pressure for 5 hours to obtain 135g (composition 2) of dolasetron mesylate with the yield of 84.8%.

EXAMPLE 7 preparation of dolasetron maleate (composition 3) according to the invention

Under the protection of light and nitrogen, 1kg of acetone and 123g of dolasetron prepared in example 5 are added into a reaction kettle, heated, refluxed and dissolved, maleic acid is dropwise added, and the pH value of the system is adjusted to 2-3. The reaction solution was cooled to 20 ℃ and stirred for 3 hours for crystallization. Filtering, and drying the filter cake at 55-65 ℃ under reduced pressure for 5 hours to obtain 133g of dolasetron maleate (composition 3) with the yield of 82.9%.

EXAMPLE 8 preparation of dolasetron phosphate according to the invention (composition 4)

Under the protection of light and nitrogen, 1kg of acetone and 123g of dolasetron prepared in example 5 are added into a reaction kettle, heated, refluxed and dissolved, and added with phosphoric acid dropwise, and the pH value of the system is adjusted to 2-3. The reaction solution was cooled to 20 ℃ and stirred for crystallization for 4 hours. Filtering, and drying the filter cake at 55-65 ℃ under reduced pressure for 5 hours to obtain 132g (composition 4) of dolasetron phosphate with the yield of 86.1%.

EXAMPLE 9 preparation of dolasetron citrate (composition 5) of the invention

Under the protection of light and helium, 1kg of acetone and 123g of dolasetron prepared in example 5 are added into a reaction kettle, heated, refluxed and dissolved, citric acid is dropwise added, and the pH value of the system is adjusted to be 2-3. The reaction solution was cooled to 20 ℃ and stirred for 2 hours for crystallization. Filtering, and drying the filter cake at 55-65 ℃ under reduced pressure for 5 hours to obtain 160g (composition 5) of dolasetron citrate with the yield of 85.3%.

Example 10 stability Studies of pharmaceutical compositions of the invention

Taking a proper amount of the dolasetron prepared in example 6 (composition 1), dolasetron mesylate (composition 2) and commercial dolasetron mesylate and dolasetron mesylate samples, placing the samples into a weighing bottle, wherein the thickness of the samples is about 5mm, the samples are respectively placed in an opening under the environment of high temperature (60 ℃) and high humidity (25 ℃, relative humidity (90 +/-5)%), and illumination (intensity is (4500 +/-500) lx) for 10d, the samples are respectively taken at the 5d and the 10d, the appearance and the color of the samples are observed to be unchanged (white powder), substances related to HPLC (high performance liquid chromatography) are determined, and compared with the samples before (0d) under each environment, the results are shown in the following table 5:

table 5 stability test results of the pharmaceutical composition of the present invention and the existing product (%)

Chromatographic conditions are as follows: a chromatographic column: ZORBAX SB C8 reverse phase column (250X 4.6 mm); mobile phase A: 0.01mol/L sodium dihydrogen phosphate buffer solution (pH6.8-7.0) -acetonitrile (1000:53), mobile phase B: methanol, gradient elution; detection wavelength: 210 nm.

Table 5 above shows that the

compositions

1 and 2 of the present invention have lower levels of dolasetron oxide than the commercial products, and the related substance a and total impurities thereof increase much less under high temperature, high humidity and light conditions, which means that controlling the levels of dolasetron oxide of the present invention is advantageous for controlling the levels of related substance a and total impurities in the compositions. Research shows that when the content of the dolasetron oxide exceeds 0.5%, the total impurities increase more quickly in stability investigation.

Example 11 preparation of dolasetron mesylate injection according to the invention

Prescription:

the preparation process comprises the following steps:

weighing glycerol with the prescription amount, completely dissolving the glycerol with 70% of injection water with the prescription amount which is cooled to room temperature, adding dolasetron mesylate (composition 2) with the prescription amount, stirring and dissolving (concentrated preparation), adding medicinal activated carbon with the concentration volume of 0.05% (W/V), stirring uniformly, standing and adsorbing for 10min, and filtering to remove carbon; adding water for injection to 95% of the total amount, stirring uniformly, adjusting pH to 3.0 with 0.1mol/L hydrochloric acid solution, adding water for injection to the total amount, and stirring uniformly; sampling and measuring the properties, pH value, content and bacterial endotoxin, fine filtering, filling nitrogen, sealing by fusion after the materials are qualified, and performing moist heat sterilization at 121 ℃ for 12-15 minutes to obtain the product. Through high performance liquid chromatography detection, the mass ratio of the dolasetron mesylate to the compound of the formula (I) in the dolasetron mesylate injection is 580: 1.

Example 12 preparation of dolasetron mesylate injection of the invention

Prescription:

the preparation process comprises the following steps:

weighing mannitol with a prescription amount, completely dissolving the mannitol with 70% injection water with the prescription amount which is cooled to room temperature, adding dolasetron mesylate (composition 2) with the prescription amount, stirring and dissolving (concentrated preparation), adding medicinal activated carbon with the concentration volume of 0.05% (W/V), stirring uniformly, standing and adsorbing for 10min, and filtering to remove carbon; adding water for injection to 95% of the total amount, stirring uniformly, adjusting pH to 3.8 with 0.1mol/L hydrochloric acid solution, adding water for injection to the total amount, and stirring uniformly; sampling and measuring the properties, pH value, content and bacterial endotoxin, fine filtering, filling nitrogen, sealing by fusion after the materials are qualified, and performing moist heat sterilization at 121 ℃ for 12-15 minutes to obtain the product. Through high performance liquid chromatography detection, the mass ratio of the dolasetron mesylate to the compound of the formula (I) in the dolasetron mesylate injection is 1070: 1.

Claims

1. A dolasetron oxide is characterized by having a structure shown as a formula (I):

2. a process for preparing dolasetron oxide according to claim 1, which is obtained by reacting dolasetron or a salt thereof with an oxidizing agent in a reaction solvent and then quenching the reaction with a reducing agent.

3. The preparation method according to claim 2, wherein the reaction solvent is selected from one or more of methanol, ethanol, tetrahydrofuran and dichloromethane; the oxidant is selected from hydrogen peroxide or m-chloroperoxybenzoic acid or a mixture of hydrogen peroxide and m-chloroperoxybenzoic acid; the reducing agent is selected from sodium sulfite or sodium bisulfite or a mixture of sodium sulfite and sodium bisulfite.

4. The preparation method according to claim 3, wherein the molar charge ratio of the dolasetron to the oxidant is 1: 1.0-20, and the molar charge ratio of the reducing agent to the oxidant is 1: 0.5-2.

5. The preparation method according to claim 4, wherein the molar charge ratio of the dolasetron to the oxidant is 1: 2-10; the molar feed ratio of the reducing agent to the oxidizing agent is 1:1.

6. The preparation method according to any one of claims 2 to 5, wherein the reaction temperature for the reaction with the oxidizing agent is controlled to be-10 ℃ to 70 ℃, and the reaction temperature for the quenching reaction by adding the reducing agent is controlled to be-10 ℃ to 70 ℃.

7. The preparation method according to claim 6, wherein the reaction temperature for the reaction with the oxidizing agent is controlled to be 0 ℃ to 50 ℃, and the reaction temperature for the quenching reaction by adding the reducing agent is controlled to be 0 ℃ to 50 ℃.

8. The method according to any one of claims 2 to 7, further comprising a step of separation and purification using HPLC gradient elution under the following chromatographic conditions: adopting a carbon-eighteen bonded silica gel chromatographic column; the mobile phase A is 0.03% ammonia water solution, and the mobile phase B is acetonitrile; the detection wavelength is 210 +/-10 nm.

9. Use of dolasetron oxide according to claim 1 as a standard or control.

10. A pharmaceutical composition, which comprises dolasetron or pharmaceutically acceptable salt thereof and a structural compound shown as a formula I with the mass percentage not higher than 0.5 percent:

the pharmaceutically acceptable salt is selected from any one of maleate, methanesulfonate, sulfonate, sulfate, hydrochloride, hydrobromide, phosphate, nitrate, p-toluenesulfonate, tartrate, fumarate, acetate, formate, benzoate, cinnamate, succinate, malonate, citrate or a combination thereof.

11. The pharmaceutical composition according to claim 10, wherein the dolasetron or the pharmaceutically acceptable salt thereof is contained in an amount of not less than 90%, preferably not less than 95%, more preferably not less than 98%.

12. Pharmaceutical composition according to claim 10, characterized in that the content of compound of formula i is not higher than 0.4%, preferably not higher than 0.3%, more preferably not higher than 0.2%.

13. The pharmaceutical composition according to claim 10, wherein the weight ratio between the dolasetron or the pharmaceutically acceptable salt thereof and the compound of the formula I is not less than 200:1, preferably not less than 300:1, more preferably not less than 500: 1.

14. The pharmaceutical composition according to any one of claims 10 to 13, which comprises dolasetron or a pharmaceutically acceptable salt thereof in an amount of not less than 98%, dolasetron oxide represented by formula i in an amount of not more than 0.20%, dolasetron-related substance a in an amount of not more than 1.0%, and the balance other related substances.

15. A process for preparing a pharmaceutical composition according to any one of claims 10 to 14, wherein endo-hexahydro-8-hydroxy-2.6-methylene-2H-quinolizin-3 (4H) -one or a salt thereof is reacted with indole-3-carboxylic acid in the absence of light and under nitrogen or inert gas to prepare dolasetron, which is further reacted with an acid to prepare a pharmaceutically acceptable salt.

16. A pharmaceutical formulation comprising a pharmaceutical composition according to any one of claims 10 to 14, and optionally a pharmaceutically acceptable carrier.

17. A pharmaceutical formulation according to claim 16, wherein the weight ratio between dolasetron or its pharmaceutically acceptable salt and the compound of formula I is not less than 200:1, preferably not less than 300:1, more preferably not less than 500:1,

18. a method for preparing dolasetron mesylate injection, which is characterized in that the dolasetron mesylate injection is prepared by mixing the raw material medicament as defined in any one of claims 10 to 14 with a pharmaceutically acceptable injection carrier.

19. Use of a pharmaceutical composition according to any one of claims 10 to 14 or a pharmaceutical formulation according to claim 16 or 17 for the manufacture of an anti-emetic medicament.

20. An antitumor pharmaceutical composition comprising an effective amount of dolasetron oxide according to claim 1.

21. Use of the dolasetron oxide according to claim 1 or the antineoplastic pharmaceutical composition according to claim 20 in the preparation of antineoplastic drugs.