CN113637025B - Cefotaxime magnesium compound, preparation method and application thereof - Google Patents

Cefotaxime magnesium compound, preparation method and application thereof Download PDF

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CN113637025B
CN113637025B CN202010393332.6A CN202010393332A CN113637025B CN 113637025 B CN113637025 B CN 113637025B CN 202010393332 A CN202010393332 A CN 202010393332A CN 113637025 B CN113637025 B CN 113637025B
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cefotaxime
magnesium
magnesium compound
compound according
suspension
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CN113637025A (en
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刘翠哲
刘金霞
曹凯
宋成军
金鹏
白红红
杨禄坤
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Chengde Medical University
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    • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
    • C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
    • C07D501/26—Methylene radicals, substituted by oxygen atoms; Lactones thereof with the 2-carboxyl group
    • C07D501/34—Methylene radicals, substituted by oxygen atoms; Lactones thereof with the 2-carboxyl group with the 7-amino radical acylated by carboxylic acids containing hetero rings
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/02—Preparation
    • C07D501/04—Preparation from compounds already containing the ring or condensed ring systems, e.g. by dehydrogenation of the ring, by introduction, elimination or modification of substituents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/02—Preparation
    • C07D501/12—Separation; Purification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a cefotaxime magnesium compound, a preparation method and application thereof. The invention utilizes the reaction of cefotaxime acid and magnesium ions to generate a cefotaxime magnesium compound, improves the stability of the cefotaxime medicament, and provides a choice for patients who are not suitable for sodium salt clinically. The cefotaxime magnesium compound is a magnesium-containing antibiotic, has antibacterial treatment effect and magnesium supplement effect, and has synergistic effect on certain inflammation beneficial parts by combining magnesium element with cefotaxime. The preparation method of cefotaxime magnesium is simple to operate, does not use organic solvent, does not generate toxic and harmful substances, has high product purity, and is convenient for industrial production.

Description

Cefotaxime magnesium compound, preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a cefotaxime magnesium compound, and a preparation method and application thereof.
Background
Cefotaxime, 3-acetoxymethyl-7- [2- (2-amino-4-thiazolyl) -2-methoxyimino]-acetamido-3-cephem-4-carboxylic acid having a molecular formula of C 16 H 17 N 5 O 7 S 2 Molecular weight is 455.46, and the powder is clinically used for injection.
Cefotaxime is the third generation cephalosporin, and is a broad spectrum antibiotic. The action on gram-negative bacilli is obviously better than that of the first and second generation cephalosporins, especially the action on drug-resistant escherichia coli which is stronger than cefotaxime and especially has the strongest action on haemophilus producing penicillinase, and the drug-resistant escherichia coli has stronger action on influenza bacillus, escherichia coli, enterobacter, citrullinia, serratia, klebsiella and beta-lactamase. Cefotaxime treats patients with hematopathy complicated with infection, the clinical response is optimal, the sterilization time is maintained longer, but the sterilization effect on pseudomonas aeruginosa and enterobacter cloacae is poorer.
Cefotaxime is not absorbed in the intestinal tract. The peak blood concentration values after intramuscular injection of 0.5g and 1.0g,0.5h are 12 mug/ml and 25 mug/ml respectively. 1g and 2g are injected into the vein, and the peak value of the blood concentration is 102 mug/ml and 215 mug/ml respectively. 1g of the composition is instilled in an intravenous way within 30min, the blood concentration is 41 mu g/ml after the instillation is finished, and the blood concentration is 1.5 mu g/ml after 4h. The medicine has strong penetrating power in tissue after being absorbed, is widely distributed in body, and can reach effective antibacterial concentration in various tissues, body cavity fluid and body fluid, especially the concentration in bile and urine is higher. Cefotaxime does not easily penetrate normal meninges, but the meninges can increase the penetration amount when the meninges is inflamed. The combination rate of cefotaxime protein is 30-50%. The drug is metabolized in the liver to desacetyl cefotaxime (antibacterial activity is 1/10 of cefotaxime) and other inactive metabolites. The half-life of intramuscular injection and intravenous injection is 0.92-1.35h and 0.84-1.25h respectively. The half-life of the metabolite deacetyl cefotaxime is 1.5h. 80% of cefotaxime can be excreted by kidney, wherein 50% -60% is proto-drug, 10% -20% is deacetyl cefotaxime, and the other 10% -20% is inactive metabolite. The excretion amount of cefotaxime through bile is less, and is about 0.01-0.1% of the administration amount. Hemodialysis can remove 62.3 percent of medicaments from the body, and peritoneal dialysis has no influence on the pharmacokinetics of cefotaxime.
Clinically, cefotaxime sodium is frequently used, either singly or together with sulbactam. However, in clinical application, sodium salts are not suitable for patients with high blood sodium.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a cefotaxime magnesium compound with enhanced antibacterial effect and magnesium supplementing effect, and a preparation method and application thereof. The combination of cefotaxime and magnesium can play a role in synergy, cefotaxime can inhibit the synthesis of cell walls of pathogenic bacteria, has strong effect on gram-negative bacteria, and can significantly inhibit the synthesis of the cell walls of the bacteria after being combined with magnesium element, so that the using effect is significantly enhanced. Improves the stability of the cefotaxime medicament, and provides a choice for patients who are not suitable for sodium salt clinically.
The technical scheme adopted by the invention is as follows:
a magnesium cefotaxime compound having the structural formula:
Figure BDA0002486725670000021
the molecular formula is: mg (C) 16 H 17 N 5 O 7 S 2 ) 2 ；
The molecular weight is: 932.9.
the preparation method of the cefotaxime magnesium compound comprises the following steps:
(1) Preparation of cefotaxime acid suspension:
sieving cefotaxime acid, adding into purified water, and mixing to obtain cefotaxime acid suspension;
(2) Preparation of magnesium compound solution or suspension:
pulverizing magnesium compound into fine powder, sieving, adding into purified water, and mixing to obtain magnesium compound solution or suspension;
(3) Reaction:
adding the magnesium compound solution or suspension obtained in the step (2) into the cefotaxime acid suspension obtained in the step (1), fully reacting under the stirring condition, and filtering to obtain a filtrate;
(4) And (3) drying:
and (4) drying and purifying the filtrate obtained in the step (3) to obtain the cefotaxime magnesium compound.
In the step (1), the aperture of the sieved sieve is 60-120 meshes.
The molar ratio of cefotaxime acid in the cefotaxime acid suspension to magnesium element in the magnesium compound solution or suspension is 2.
In the step (2), the magnesium compound is one or a mixture of more of magnesium hydroxide, magnesium bicarbonate and magnesium acetate.
In the step (3), the reaction is carried out at a temperature of 15 to 30 ℃.
In the step (3), a decoloring agent is added while the stirring reaction is carried out.
The decolorizing agent is activated carbon.
In the step (4), the drying is spray drying or freeze drying.
And D, performing spray drying by dispersing the filtrate obtained in the step C into particles by adopting a spray drying device, and enabling the particles to be in contact with hot air to instantaneously remove moisture of the particles to obtain the cefotaxime magnesium compound with the water content of less than 2.0 percent by weight. The spray drying apparatus used in the present invention is a product currently marketed, for example, by dryer plant of the state of the forest, jiangsu, under the trade name QZR-5.
And D, freezing the filtrate obtained in the step C to below 0 ℃ by adopting freeze drying equipment, directly subliming water from a solid state into water vapor by heating under high vacuum, and removing the water to obtain the cefotaxime magnesium compound with the water content of below 2.0 percent by weight. The freeze-drying apparatus used in the present invention is a product currently marketed, for example, by the beijing tetracyclic scientific instruments co.
In the step (4), the purification is performed by recrystallization, an octadecylsilane reverse phase column and preparative liquid chromatography, so that the content of the cefotaxime magnesium compound in the product is more than 95% by weight.
The recrystallization purification was performed in an ethanol-water (1. The octadecylsilane reverse phase column (ODS) is a commonly used reverse phase chromatography column, also called C18 column; because it is a long-chain alkyl bonding phase, has higher carbon content and better hydrophobicity, and has stronger adaptability to various biological macromolecules, the application of the method in biochemical analysis work is the most extensive. The purification using ODS column was carried out using ODS column sold under the trade name GEL C18 AAG12S50 by YMC under the conditions of 5% -50% acetonitrile-methanol-water as eluent.
The apparatus used for purification by preparative liquid chromatography was a preparative liquid sold under the trade name CTO-10A by shimadzu corporation, the preparative column was C18, and the liquid phase conditions were acetonitrile-methanol-water (5.
The content of the cefotaxime magnesium compound is determined according to high performance liquid chromatography, the used instrument is Agilent1260, the chromatographic column is ODS C18, the liquid phase condition is 0.1% phosphoric acid water-acetonitrile (70.
One magnesium ion (Mg) 2+ ) Reacts with carboxyl groups on two cefotaxime to generate cefotaxime magnesium with a molecular formula of Mg (C) 16 H 17 N 5 O 7 S 2 ) 2 And the molecular weight is 932.9.
The cefotaxime magnesium compound is applied to the preparation of antibacterial drugs.
Magnesium is an essential element participating in the normal life activities and metabolic processes of organisms. Magnesium affects various biological functions of cells, potassium ion and calcium ion transport, regulates signal transmission, and participates in energy metabolism and protein and nucleic acid synthesis; activation and inhibition of catalytic enzymes and regulation of cell cycle, cell proliferation and cell differentiation; magnesium is also involved in maintaining genomic stability and is also associated with oxidative stress and tumorigenesis in the body. The inventor of the application finds that magnesium and cefotaxime are combined to benefit certain inflammations and play a role in synergistic effect in long-term research.
The beneficial effects of the invention are as follows:
(1) The cefotaxime magnesium compound provided by the invention can play a role in synergy by combining cefotaxime with magnesium, the cefotaxime can inhibit the synthesis of pathogenic bacteria cell walls, the effect on gram-negative bacteria is strong, the synthesis of bacterial cell walls can be obviously inhibited after the cefotaxime magnesium compound is combined with magnesium elements, and the use effect is obviously enhanced. The invention utilizes the reaction of cefotaxime acid and magnesium ions to generate a cefotaxime magnesium compound, improves the stability of cefotaxime medicaments, simplifies the production process of the preparation, and provides a choice for patients who are not suitable for sodium salt clinically. The cefotaxime magnesium compound is a magnesium-containing antibiotic, has antibacterial treatment effects and magnesium supplement effects, is used together with cefotaxime, and has synergistic effects on certain inflammation beneficial parts.
(2) The preparation method of cefotaxime magnesium is simple to operate, does not use organic solvent, does not generate toxic and harmful substances, has high product purity, and is convenient for industrial production.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the embodiments described are merely exemplary of the invention, and not of the invention in its entirety. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
This example provides a method for preparing a cefotaxime magnesium compound having a molecular formula: mg (C) 16 H 17 N 5 O 7 S 2 ) 2 Molecular weight 932.9, structural formula:
Figure BDA0002486725670000051
the preparation method of the cefotaxime magnesium compound comprises the following steps:
(1) Preparation of cefotaxime acid suspension:
sieving 0.2mol of cefotaxime acid by a 60-mesh sieve, adding the powder into 100mL of purified water, and uniformly mixing to obtain cefotaxime acid suspension;
(2) Preparing a magnesium bicarbonate solution:
crushing 0.1mol of magnesium bicarbonate into fine powder, sieving the fine powder by a 60-mesh sieve, adding the fine powder into 20mL of purified water, and uniformly mixing to obtain a magnesium bicarbonate solution;
(3) Reaction:
adding the magnesium bicarbonate solution in the step (2) into the cefotaxime acid suspension in the step (1), stirring at 25 ℃ to fully react, adding 0.1% of active carbon, stirring and decoloring, and filtering to obtain a filtrate;
(4) And (3) drying:
and (3) carrying out freeze drying on the filtrate obtained in the step (3) under the pressure condition of 70 Pa to obtain a primary product with the water content lower than 2%, and then carrying out recrystallization purification by adopting an ethanol-water (1.
The content of the cefotaxime magnesium compound in the product is more than 95 percent by weight through high performance liquid chromatography determination. The high performance liquid chromatography uses Agilent1260 as an instrument, ODS C18 as a chromatographic column, and the liquid phase conditions are 0.1% phosphoric acid water-acetonitrile (70: 30), the flow rate is 1mL/min, and the detection wavelength is 230nm.
Example 2
This example provides a preparation method of the cefotaxime magnesium compound, including the following steps:
(1) Preparation of cefotaxime acid suspension:
sieving 0.2mol of cefotaxime acid by a 120-mesh sieve, adding into 200mL of purified water, and uniformly mixing to obtain cefotaxime acid suspension;
(2) Preparation of a magnesium acetate solution:
crushing 0.1mol of magnesium acetate into fine powder, sieving the fine powder by a 120-mesh sieve, adding the fine powder into 40mL of purified water, and uniformly mixing to obtain a magnesium acetate solution;
(3) Reaction:
adding the magnesium acetate solution in the step (2) into the cefotaxime acid suspension in the step (1), stirring at 15 ℃ to fully react, adding activated carbon, stirring and decoloring, and filtering to obtain filtrate;
(4) And (3) drying:
spray drying the filtrate obtained in the step (3) under the pressure of 10 Pa to obtain a primary product with the water content lower than 2%, and purifying by using an octadecyl silane reverse phase column to obtain the cefotaxime magnesium compound product. The detection proves that the content of the cefotaxime magnesium compound in the product is more than 95 percent by weight.
Example 3
This example provides a preparation method of the cefotaxime magnesium compound, including the following steps:
(1) Preparation of cefotaxime acid suspension:
sieving 0.2mol of cefotaxime acid with a 80-mesh sieve, adding into 150mL of purified water, and uniformly mixing to obtain cefotaxime acid suspension;
(2) Preparation of magnesium hydroxide suspension:
crushing 0.1mol of magnesium hydroxide into fine powder, sieving the fine powder by a 100-mesh sieve, adding 50mL of purified water, and uniformly mixing to obtain magnesium hydroxide suspension;
(3) Reaction:
adding the magnesium hydroxide suspension obtained in the step (2) into the cefotaxime acid suspension obtained in the step (1), stirring at 30 ℃ to fully react, adding activated carbon, stirring and decoloring, and filtering to obtain a filtrate;
(4) And (3) drying:
and (4) spray-drying the filtrate obtained in the step (3) under the pressure of 50 Pa to obtain a primary product with the water content of less than 2%, and purifying by using a preparative liquid chromatography to obtain the cefotaxime magnesium compound product. The detection shows that the content of the cefotaxime magnesium compound in the product is more than 95 percent by weight.
Examples of the experiments
1. The structure of the cefotaxime magnesium compound prepared in example 1 was analyzed by uv absorption spectroscopy (uv absorption spectroscopy analysis equipment Agilent 8453).
The cefotaxime sodium in the prior art and the cefotaxime magnesium prepared by the invention automatically scan ultraviolet absorption spectra by an instrument under 200-400 nm. The ultraviolet absorption spectrum was measured by a comparison method with a control.
2mg of cefotaxime sodium and 2mg of cefotaxime magnesium prepared in example 1 of the present invention were dissolved in 10ml of methanol, respectively, and then the maximum absorption wavelength was measured, and the results thereof are shown in Table 1.
TABLE 1 maximum absorption wavelength of cefotaxime sodium and cefotaxime magnesium
Compound (I) Maximum absorption wavelength
Cefotaxime acid 233nm
Cefotaxime sodium 233nm
Cefotaxime magnesium 233nm
As can be seen from Table 1, the maximum absorption wavelength (. Lamda.) of ultraviolet of the cefotaxime sodium and cefotaxime magnesium compounds max ) Similarly, they all have an absorption maximum at 233 nm; these results of uv absorption spectroscopy indicate that the binding of magnesium ions has little effect on the delocalized resonance structure of cefotaxime.
2. Comparison of solubility of cefotaxime sodium and cefotaxime magnesium compounds in Water
The solubility of cefotaxime sodium and cefotaxime magnesium was determined using conventional solubility determination methods in water at a temperature of 25 c, and the results are shown in table 2.
TABLE 2 solubility of cefotaxime sodium and cefotaxime magnesium compounds in water
Sample (I) Solubility (g/mL)
Cefotaxime sodium 10.34
Cefotaxime magnesium 13.17
As can be seen from table 2, the water solubility of cefotaxime magnesium is better, which is 1.27 times that of cefotaxime sodium.
3. Stability of
Respectively and precisely weighing 10mg of cefotaxime sodium and 10mg of cefotaxime magnesium respectively, placing the cefotaxime sodium and the cefotaxime magnesium in volumetric flasks, adding distilled water to the scales, preparing stock solution with the concentration of 1mg/ml, and placing the stock solution in a refrigerator for later use. Then 0.9 percent of sodium chloride and 5 percent of glucose injection are respectively taken and added with the stock solution to prepare 10ug/ml of sample solution to be detected, each sample is respectively placed in constant-temperature water solution at 35 ℃, the samples are respectively sampled and measured for 1 hour, 3 hours, 5 hours and 8 hours, the drug concentration of each solution to be detected is calculated, and the average value is taken. The results are shown in Table 3.
TABLE 3 stability results for cefotaxime sodium and cefotaxime magnesium
Figure BDA0002486725670000091
As can be seen from Table 3, the cefotaxime magnesium of the invention has stability in different injections, and the content change is smaller than that of cefotaxime sodium, and the stability is better.
4. In vitro bacteriostatic test
An in vitro bacteriostasis experiment is carried out according to pharmacological experimental methodology written by the gentle Shuyun (third edition) and antibacterial drug experimental method in chapter 63.
Preparation of liquid medium: precisely weighing 2g of beef extract, 3g of yeast, 5g of sodium chloride and 700ml of distilled water, mixing, adjusting the pH value of a liquid culture medium to about 7.0, autoclaving in a steam sterilization pot for 30min, adjusting the temperature to 120 ℃, taking out after sterilization, respectively putting into 6 conical flasks, wherein the volume of the liquid culture medium in each conical flask is 100ml, and sealing for later use.
Preparing a liquid medicine: precisely weighing cefotaxime sodium and cefotaxime magnesium according to the number of equimolar beta-lactam rings, adding distilled water to a constant volume of 10ml, diluting to ensure that the concentrations of the cefotaxime sodium and the cefotaxime magnesium are 32 mu g/ml and 31.24 mu g/ml respectively, filtering by adopting a 0.22 mu m filter membrane, and taking the subsequent filtrate for later use.
Preparing a bacterial liquid: respectively picking up an inoculating loop of the second generation bacteria of Escherichia coli and Staphylococcus albus from a nutrient agar slant, inoculating into 2 conical flasks with liquid culture medium, slightly shaking the conical flasks, placing in a gas bath constant temperature oscillator, adjusting the temperature to 37 ℃, culturing for 24-48h, and observing the turbidity of the liquid, namely the bacteria are successfully reactivated.
Turbidimetric experiments: before experiment, the required experimental article is sterilized under high pressure at 120 ℃ for 30 minutes, and the laboratory is disinfected by an ultraviolet lamp for 30 minutes. Under the aseptic operation condition, 32 test tubes are divided into four groups, and each test tube is added with a liquid culture medium to dilute the cefotaxime sodium and cefotaxime magnesium liquid medicine in a multiple ratio. Adding 50 mul of white grape ball bacterial liquid into 16 of the raw materials, adding 50 mul of escherichia coli liquid into the other 16 of the raw materials, putting the raw materials into the constant-temperature incubator again for culturing for 18-24 h, and observing turbidity.
And (3) evaluating the bacteriostatic effect: OD600 values are measured by an ultraviolet spectrophotometer, and the experiments repeated for 3 times all have bacteriostatic effects, so that the bacteriostatic effect is considered to be effective.
The results are shown in tables 4 and 5.
TABLE 4 bacteriostatic Effect of cefotaxime sodium and cefotaxime magnesium on Staphylococcus albus
Figure BDA0002486725670000101
TABLE 5 bacteriostatic effect of cefotaxime sodium and cefotaxime magnesium on E.coli
Figure BDA0002486725670000102
It can be seen from tables 4 and 5 that, according to the measured OD600 values, it is determined that cefotaxime sodium and cefotaxime magnesium all have certain inhibitory action on 2 drug-resistant bacteria, and the antibacterial effect is more obvious with the increase of the drug concentration. By comparing the OD values of cefotaxime sodium and cefotaxime magnesium with the same molar number of beta-lactam rings, the antibacterial effect of the cefotaxime magnesium is respectively stronger than that of the cefotaxime sodium with the same molar number of beta-lactam rings corresponding to the cefotaxime sodium at the concentrations of 31.24, 7.81, 3.9, 1.95 and 0.49 mu g/ml on the inhibition effect of staphylococcus albus; on the inhibition effect on escherichia coli, when the cefotaxime magnesium is at the concentrations of 3.9 and 1.95 mu g/ml, the antibacterial effect is respectively stronger than that of cefotaxime sodium at the concentrations of 4 and 2 mu g/ml corresponding to the number of beta-lactam rings of the same mole, and comprehensively considered, the cefotaxime magnesium salt has stronger inhibition effect on staphylococcus albus and is stronger than the cefotaxime sodium.
5. In vivo antibacterial experiments
(1) Detection method
The standard strain of Streptococcus pneumoniae was dissolved in a liquid medium and added to a nutrient broth containing 10% calf serum, and the mixture was incubated at 37 ℃ for 24 hours with 7.5% carbon dioxide. The recovered bacteria were subcultured on blood agar plate medium with 7.5% carbon dioxide at 37 ℃ for 24h. Before use, the bacteria were picked up on a sterile super clean bench using a pick-up ring, diluted to an appropriate concentration with sterile physiological saline and detected to have an absorbance at 600nm (A600 nm) value of 2.489.
After one week of acclimatization, rats were monitored for body temperature in the morning and evening, and continuously for 3 days. Rats with body temperature fluctuation of less than 0.5 ℃ are selected to be included in the experiment and are randomly divided into 6 groups, namely a blank group, a model group, a cefotaxime sodium low-dose group, a cefotaxime sodium high-dose group, a cefotaxime magnesium low-dose group and a cefotaxime magnesium high-dose group, wherein 10 rats are selected in each group. After anesthetizing the rat, the oral cavity of the rat is opened, the throat part is fully exposed, the trachea is slowly inserted into the oral cavity by using a tracheal catheter connected with an injector, 0.6ml of bacteria solution is pushed in, and the blank group is pushed in normal saline with the same volume. Immediately after inoculation, the rats were allowed to stand upright for about 20 seconds to ensure that the inoculum solution gravitated into the lungs.
The low-dose group of cefotaxime sodium comprises 22.5 mg/kg of water, the high-dose group of cefotaxime sodium comprises 45 mg/kg of water, the low-dose group of cefotaxime magnesium comprises 21.2 mg/kg of water, and the high-dose group of cefotaxime magnesium comprises 42.4 mg/kg of water. The tail vein injection was administered twice daily for three consecutive days, and equal volumes of saline were administered to the blank group and the model group.
(2) Observation content and detection index
Observing whether the physiological and pathological conditions of the rat actively ingest food and absorb water; whether or not it is dull, hairy or somnolence; nasal cavity and orbit with or without secretion; whether or not there is shortness of breath, whether or not there is wheezing sound; urine color, volume and feces changes.
After 12h of the last administration, 4% chloral hydrate 10mg/kg is injected into the abdominal cavity for anesthesia, 1.5mL of abdominal aorta blood is taken in an anticoagulant vacuum blood collection tube added with EDTA, the mixture is uniformly mixed, and a blood analyzer is used for measuring the WBC count and classification of whole blood.
Fixing the two lungs with 10% formaldehyde solution, embedding in paraffin, slicing, staining with hematoxylin-eosin (HE), and observing the histopathological changes of the liver under a light microscope.
The experimental data were processed using SPSS 19.0 statistical software and the metric data are expressed in x. + -.s. The mean between groups was statistically significant using one-way analysis of variance with P < 0.05.
(3) The result of the detection
(3.1) general State
On day 1 after animal inoculation, the dosing and model groups began to develop tachypnea, lethargy, reduced feeding, frizzy, and poor response to the surrounding environment. On day 2, the animals had significantly decreased food intake and activity, hair was straight and vertical, curly around the corners of the cages, especially in the model group. The low and high dose groups of cefotaxime sodium are lower than those of cefotaxime magnesium, the high dose groups of cefotaxime sodium are rapid to breathe, and the food intake is obviously less than that of the cefotaxime magnesium. On day 3, the appetite and activity of animals in the administration group are increased, rats in the cefotaxime sodium group are still quickly absorbed compared with those in the cefotaxime magnesium group, rats in the model group can hear and obviously breathe, asthma and creme, the physical quality is reduced, and obvious bone feeling is caused. The appetite, activity, body mass and body temperature of the rats in the blank group have no obvious change compared with those before inoculation.
(3.2) blood routine
(3.2.1) Effect on leukocyte count
Compared with a blank group, the white blood cell number of the model group rats is obviously increased (P is less than 0.01); compared with the model group, the number of the white blood cells of the rats in each administration group is obviously reduced (P is less than 0.01), which indicates that the cefotaxime sodium and the cefotaxime magnesium have treatment effect on pneumonia of the rats, and the number of the white blood cells of the low-dose group of the cefotaxime magnesium is obviously reduced (P is less than 0.05) compared with the low-dose group of the cefotaxime sodium. Compared with a blank group, the difference of the cefotaxime sodium high-dose group is obvious (P is less than 0.01), and the difference of the cefotaxime magnesium high-dose group is not obvious.
(3.2.2) Effect on the percentage of neutrophils
Compared with a control group, the percentage of the neutrophils in the model group rats is obviously increased (P is less than 0.01); compared with the model group, the number of the neutral granulocytes of the rats in each administration group is obviously reduced (P is less than 0.01, P is less than 0.05), which shows that the cefotaxime sodium and the cefotaxime magnesium have treatment effect on pneumonia of the rats, and the percentage of the neutral granulocytes in the cefotaxime magnesium low-dose group is more obviously reduced (P is less than 0.05) compared with the cefotaxime sodium low-dose group. Compared with a blank group, the difference of the cefotaxime sodium high-dose group is obvious (P is less than 0.01), and the difference of the cefotaxime magnesium high-dose group is not obvious.
(3.2.3) Effect on lymphocyte percentage
The percentage of the model rat lymphocytes is obviously reduced compared with the control group (P is less than 0.01); compared with the model group, the number of lymphocytes of the rats in each administration group is obviously increased (P < 0.01, P < 0.05). The results show that both cefotaxime sodium and cefotaxime magnesium have therapeutic effect on rat pneumonia, and the percentage increase of lymphocytes in the low-dose group of cefotaxime magnesium is more obvious (P is less than 0.05) compared with the low-dose group of cefotaxime sodium.
The results are shown in Table 6.
TABLE 6 in vivo efficacy test results for cefotaxime magnesium
Figure BDA0002486725670000131
Figure BDA0002486725670000141
Note: in comparison with the set of models, * p＜0.05， ** p is less than 0.01; in comparison to the blank set, the results, # p＜0.05， ## p is less than 0.01; compared with the low-dose group of cefotaxime sodium, Δ P＜0.05， ΔΔ P＜0.01
the above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (10)

1. A magnesium cefotaxime compound characterized by the structural formula:
Figure FDA0003865834070000011
the molecular formula is: mg (C) 16 H 16 N 5 O 7 S 2 ) 2 ；
The molecular weight is: 932.9.
2. a process for preparing a magnesium cefotaxime compound according to claim 1, comprising the steps of:
(1) Preparation of cefotaxime acid suspension:
sieving cefotaxime acid, adding into purified water, and mixing to obtain cefotaxime acid suspension;
(2) Preparation of magnesium compound solution or suspension:
pulverizing magnesium compound into fine powder, sieving, adding into purified water, and mixing to obtain magnesium compound solution or suspension;
(3) Reaction:
adding the magnesium compound solution or suspension obtained in the step (2) into the cefotaxime acid suspension obtained in the step (1), fully reacting under the stirring condition, and filtering to obtain a filtrate;
(4) And (3) drying:
and (4) drying and purifying the filtrate obtained in the step (3) to obtain the cefotaxime magnesium compound.
3. A process for preparing a cefotaxime magnesium compound according to claim 2, wherein in step (1), the sieved mesh size is 60-120 mesh.
4. A process for the preparation of a cefotaxime magnesium compound according to claim 2, wherein the molar ratio of cefotaxime acid in the cefotaxime acid suspension to magnesium element in the magnesium compound solution or suspension is 2.
5. The method for preparing a cefotaxime magnesium compound according to claim 2, wherein in the step (2), the magnesium compound is one or a mixture of magnesium hydroxide, magnesium bicarbonate and magnesium acetate.
6. A process for preparing a magnesium cefotaxime compound according to claim 2, wherein the reaction is carried out at a temperature of 15-30 ℃ in step (3).
7. The process for producing a cefotaxime magnesium compound according to claim 2, wherein in step (3), a decoloring agent is further added after the stirring reaction.
8. The process for preparing a magnesium cefotaxime compound according to claim 7, wherein the decolorizing agent is activated carbon.
9. The method for preparing a magnesium cefotaxime compound according to claim 2, wherein in step (4), the drying is spray drying or freeze drying.
10. Use of a magnesium cefotaxime compound according to claim 1 for the manufacture of an antibacterial medicament.
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