CN108285431B - Pirfenidone related substance and preparation method and application thereof - Google Patents
Pirfenidone related substance and preparation method and application thereof Download PDFInfo
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- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract
The invention discloses a pirfenidone related substance, a preparation method and application thereof. The related substances are compounds with the structure shown in the formula I,
Description
Technical Field
The invention relates to a related substance of a medicament, in particular to a pirfenidone related substance and a preparation method and application thereof.
Background
Pirfenidone (PFD) (structure shown in formula II) and chemical name 5-methyl-1-phenylpyridine-2 (1H) -ketone have pharmacological effects, including treating interstitial lung disease (AU5427080) and preventing tissue fibrosis (WO 9426249). In the research of various animal models, the pirfenidone has proved to have a certain inhibiting effect on the fibrosis of tissues and organs such as heart, kidney, lung, liver and blood vessel inner wall. Pirfenidone is also indicated for the treatment of liver injury necrosis and acute lung injury.
The finished product of pirfenidone causes gastrointestinal discomfort and nervous system problems, however, it is not known at present whether these side effects are caused by the structure of pirfenidone itself or by impurities (related substances) contained in the finished product.
The existence of related substances directly relates to the quality and safety of the medicine, and if the related substances in the finished product of the pirfenidone can be separated, identified, subjected to source analysis, safety research, impurity limitation determination and the like, the method has important significance for improving the curative effect and controlling the quality of the pirfenidone.
Therefore, there is a pressing need in the art to provide effective information about related substances in the finished product of pirfenidone.
Disclosure of Invention
The invention aims to provide a pirfenidone related substance, a preparation method and application thereof.
In a first aspect of the invention, there is provided a compound of formula I,
in a second aspect of the present invention, there is provided a process for the preparation of a compound of formula i, said process comprising the steps of:
(1) carrying out bromination reaction on the compound with the structure shown in the formula II to obtain a compound with the structure shown in the formula III; and
(2) mixing a compound with a structure shown in a formula III and 5-methylpyridine-2 (1H) -ketone to obtain a compound with a structure shown in a formula I;
in a third aspect of the invention, the invention provides the use of a compound with a structure shown as formula I for the quality control of pirfenidone or pirfenidone medicaments; and/or as an impurity reference substance during the inspection of the pirfenidone and/or pirfenidone drug related substances.
In a fourth aspect of the invention, a composition is provided, which comprises a compound having the structure shown in formula II and a compound having the structure shown in formula I.
In another preferred embodiment, the content of the compound shown in the formula I is not more than 0.5 wt% based on the total weight of the composition; more preferably, the content of the compound represented by the formula I is not more than 0.1 wt%.
In a fifth aspect of the invention, there is provided the use of a composition as provided by the invention as described above, for the manufacture of a medicament for the prevention of tissue fibrosis, the treatment of necrosis from liver injury, the treatment of interstitial lung disease, or the treatment of acute lung injury.
In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of a composition as provided herein and as described above.
In another preferred embodiment, the dosage form of the pharmaceutical composition is tablets, capsules, injections or pills.
In a seventh aspect of the present invention, there is provided a method for purifying a compound having the structure of formula ii, said method comprising the steps of: and mixing the water solution of the compound with the structure shown in the formula II with activated carbon, and filtering to obtain the purified compound with the structure shown in the formula II.
Accordingly, the present invention provides useful information about related substances in the finished product of pirfenidone.
Drawings
Figure 1 compares the change in body weight of mice in each group (.: p < 0.01;: p < 0.001).
FIG. 2 is a comparison of lung lesions in groups of mice; wherein
A is blank control group, B is low dose group 2.5mg/kg, C is medium dose group 5mg/kg, and D is high dose group 12.5 mg/kg.
FIG. 3 is a pathological section view of the lung; wherein
A is blank control group, B is high dose group 12.5 mg/kg.
FIG. 4 is a liquid chromatogram of crude pirfenidone of example 3.
FIG. 5 is a liquid chromatogram of a compound of formula I after initial purification, i.e., without preparative column purification in example 1.
FIG. 6 is a liquid chromatogram of the purified pirfenidone of example 3.
FIG. 7 is a liquid mass diagram of the mother liquor remaining after the purification of pirfenidone in example 4.
FIG. 8 is a liquid mass diagram of the crude product of formula I in example 4.
FIG. 9 is a liquid mass diagram of the pure product of formula I in example 4.
Detailed Description
The inventor finds that a compound (5-methyl-1- ((6-oxo-1-phenyl-1, 6-dihydropyridin-3-yl) methyl) pyridine-2 (1H) -ketone) with a structure shown in a formula I has an obvious relationship with side reactions of pirfenidone medicaments through extensive and intensive research, and the toxic and side effects tend to be obviously enhanced along with the increase of the impurity concentration. The present invention has been completed based on this finding.
As used herein, "Compound I", "Compound of formula I" and "Compound of formula I" are used interchangeably and refer to the following compounds:
by analogy, other compounds identified using the roman alphabet are also meant.
The main compounds to which the present invention relates are listed below:
as used herein, "related substances" refer to starting materials, intermediates, polymers, side reaction products brought in during production, degradation products during storage, and the like.
As used herein, "pirfenidone," "pirfenidone finished product," or "pirfenidone final product" are used interchangeably and refer to materials prepared according to methods of preparation of compounds having the structure shown in formula ii that are conventional in the art, such as, but not limited to, the methods described in US 39704281, US 8680123. Preferably, the compound of formula II is present in an amount of not less than 95% by weight, based on the total weight of the resulting material.
The formulation of the substance with a pharmaceutically acceptable carrier may also be referred to as a "pirfenidone drug" or "pirfenidone drug".
As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as disintegrants, wetting agents, emulsifiers, pH buffering substances and the like may also be present in these carriers.
As used herein, "HPLC purity" refers to the percentage of a certain peak height or peak area in the total peak height or total peak area as determined by High Performance Liquid Chromatography (High Performance Liquid Chromatography).
Compounds of formula (I) and processes for their preparation
The invention provides a pirfenidone related substance, namely a compound shown in a formula I, and provides a preparation method of the compound, namely, a compound shown in the formula I is obtained by taking a compound shown in the formula II as an initiator through bromination reaction, and then the compound shown in the formula III is reacted with 5-methylpyridine-2 (1H) -ketone.
Specifically, the preparation method of the compound of the formula I comprises the following steps:
firstly, carrying out bromination reaction on a compound shown in a formula II to obtain a compound shown in a formula III;
in the second step, the compound of formula III is mixed with 5-methylpyridin-2 (1H) -one to give the compound of formula I.
Furthermore, the preparation process may have a third step of purifying the compound of formula I.
In one embodiment of the present invention, conventional bromination reagents such as, but not limited to, N-bromosuccinimide, dibromohydantoin, phenyltrimethylammonium tribromide, liquid bromine can be used in the first step.
In one embodiment of the present invention, the reaction temperature of the first step is the reflux temperature of the solvent, and the solvent used includes, but is not limited to, carbon tetrachloride, chloroform, dichloromethane, acetonitrile, DMF, tetrahydrofuran.
In one embodiment of the present invention, an initiator such as, but not limited to, azobisisobutyronitrile, dibenzoyl peroxide, azobisisoheptonitrile may also be used in the first step.
In one embodiment of the present invention, the reaction involved in the second step may use a catalyst, such as, but not limited to, cuprous iodide, cuprous bromide, cuprous chloride, ferrous bromide.
In one embodiment of the present invention, the reaction temperature of the second step is the reflux temperature of the solvent, and the solvent used includes, but is not limited to, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, ethylene glycol, dimethylsulfoxide, acetamide, formamide, N-methylformamide, hexamethylphosphoramide.
In one embodiment of the present invention, the third step is performed by chromatographic purification, such as, but not limited to, high performance liquid chromatographic separation and purification.
Use of compounds of formula I
The inventor firstly discovers that the compound in the formula I can cause toxic reactions such as obvious reduction of mouse activity, obvious reduction of body weight, pulmonary congestion, ataxia and the like, and is related to dosage, so that if the compound is identified and limited by impurities in the production process of the pirfenidone, the quality control of a pirfenidone final product can be effectively realized.
For example, but not limited to, the compound of formula I prepared is used as an impurity control to confirm the proportion of the compound of formula I in the finished product or medicament of pirfenidone.
Purification of pirfenidone
The present invention provides a method for the purification of pirfenidone, particularly for the removal of compounds of formula I from pirfenidone.
In one embodiment of the invention, the purification step comprises:
dissolving pirfenidone in water to obtain a pirfenidone aqueous solution;
secondly, mixing the pirfenidone aqueous solution with active carbon, and filtering to remove the active carbon;
and thirdly, centrifuging the filtrate obtained in the second step to obtain the compound with the structure shown in the formula II.
In the first step, an aqueous solution of pirfenidone is obtained at 90-95 ℃.
In the second step, active carbon is added into pirfenidone water solution at the temperature of 90-95 ℃, the mixture is stirred at a constant temperature, the content of the compound of the formula I or the compound of the formula II is determined to determine whether the mixture is qualified or not, the mixture is filtered when the mixture is hot after the mixture is qualified, and the active carbon is filtered.
In the third step, the filtrate is cooled to 10-15 ℃ and then kept warm for a period of time (0.5-2 hours), and white crystals are obtained by centrifugation.
The invention provides a purified pirfenidone containing, based on total weight, no more than 0.5 wt% of a compound of formula I; preferably not more than 0.1 wt%.
The invention provides a purified pirfenidone, which contains a compound shown in a formula II in an amount of not less than 99.5 wt% based on the total weight of the pirfenidone; preferably not less than 99.9 wt%.
The invention also uses the purified pirfenidone for preventing tissue fibrosis, treating liver injury necrosis, treating interstitial lung disease, or treating acute lung injury, and can effectively reduce the toxic and side effects caused by the low content of the compound in the formula I, thereby effectively improving the medication safety and improving the compliance of patients.
The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and purified pirfenidone provided by the invention. In one embodiment of the present invention, the pharmaceutical composition is in the form of a tablet, capsule, injection, or pill.
The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
The main advantages of the invention are:
1. the compound of formula I in pirfenidone is found to cause toxic side effects for the first time and has dose dependency, thereby bringing the quality control of pirfenidone into play.
2. Provides a simple method for preparing the compound shown in the formula I, thereby obtaining a standard product of the compound shown in the formula I and enabling the quality control of pirfenidone.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified. The weight volume percentage units in the present invention are well known to those skilled in the art and refer to, for example, the weight of solute in a 100 ml solution. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
SUMMARY
1H-NMR spectroscopy was carried out using a Bruker-400 NMR spectrometer. The abbreviations used are as follows: s, single multiplet; d, doublet; t, triplet; q, quartet; qu, quintet; m, multiplet; br, spectrum. Mass spectrometry was performed using an agilent mass spectrometer.
A liquid phase assay method for pirfenidone and a compound of formula i:
chromatographic column C18 column 4.6 x 250mm, 5 μm
Mobile phase A phase 10mmol/L ammonium acetate B phase methanol
A:B＝50:50
Flow rate 1.0mL/min
Detection wavelength of 225nm
Example 1
Preparation of Compounds of formula I
Synthesis of
5-bromomethyl-1-phenylpyridin-2 (1H) -one: 5-methyl-1-phenylpyridin-2 (1H) -one (18.5 g, 0.10mol), N-bromosuccinimide (17.8 g, 0.10mol), azobisisobutyronitrile (3.2 g, 0.02mol) in carbon tetrachloride (750mL) was stirred under gentle reflux for 4 hours under irradiation with a 450W medium pressure mercury lamp. Cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, followed by column chromatography (10% petroleum ether: 90% ethyl acetate) to give 5-bromomethyl-1-phenylpyridin-2 (1H) -one (16.3 g, yield 61.7%) as a pale yellow solid.
5-methyl-1- ((6-oxo-1-phenyl-1, 6-dihydropyridin-3-yl) methyl) pyridin-2 (1H) -one: a solution of 5-bromomethyl-1-phenylpyridin-2 (1H) -one (15.1 g, 0.057mol), 5-methylpyridin-2 (1H) -one (6.2 g, 0.057mol), potassium carbonate (11.8 g, 0.086mol), cuprous iodide (1.1 g, 0.0057mol) in N, N-dimethylformamide (200mL) was stirred under gentle reflux for 7 hours. Cooled to room temperature, filtered, and the filtrate concentrated under reduced pressure, and subjected to column chromatography (50% petroleum ether: 50% ethyl acetate) to give 5-methyl-1- ((6-oxo-1-phenyl-1, 6-dihydropyridin-3-yl) methyl) pyridin-2 (1H) -one (7.4 g, yield 44.3%) as an off-white solid.
1H NMR(400MHz,MeOD)δ7.77(d,J＝2.1Hz,1H),7.69(dd,J＝9.4,2.5Hz,1H),7.60(s,1H),7.57–7.46(m,3H),7.45–7.36(m,3H),6.60(d,J＝9.4Hz,1H),6.52(d,J＝9.2Hz,1H),4.96(s,2H),2.12(s,3H)
13CNMR(101MHz,MeOD)δ164.03,163.93,145.00,143.22,142.02,140.17,136.89,130.55,130.04,127.80,121.79,120.57,118.92,117.64,50.31,16.94
m/e＝293(M+H)
Purification of
The instrument model is as follows: wates 2767/Qda
Column type: waters Xbridge Prep C1810 μm OBO 19 × 250mm
Column flow rate: 30ml/min
Wavelength: 214nm/254nm
A trigger: 254nm
Mobile phase A: h2O(0.1％NH3·H2O)
Mobile phase B: ACN (acetonitrile)
Gradient method:
| Time | A | B％ | |
| 0 | 70 | 30 | |
| 11 | 50 | 50 | |
| 11.2 | 5 | 95 | |
| 13 | 5 | 95 | |
| 13.2 | 95 | 5 | |
| 15 | 95 | 5 |
the compound of formula I was obtained in 98% HPLC purity by purification and used in the toxicity study examples described below.
Example 2
Toxicity Studies of Compounds of formula I
First, experimental material
1. The test substance:
the compound of the formula I obtained in example 1 (5-methyl-1- ((6-oxo-1-phenyl-1, 6-dihydropyridin-3-yl) methyl) pyridin-2 (1H) -one) has an HPLC purity of 98% and a molecular weight of 292.12.
2. Solvent:
0.5% CMC-Na from Shanghai Rui star Gen tech Ltd
3. Reagent:
sodium carboxymethylcellulose was purchased from Sigma, Lot No: SLBJ9393V
PBS was purchased from seimer feishale biochemicals (beijing) ltd,
4. the instrument equipment comprises:
mouse laminar flow rack purchased from Suzhou von Willebrand Experimental animals Equipment Co., Ltd
5. Animals:
a total of 32 male BALB/c mice, 18-20g, were purchased from Shanghai Sphere-Bikai laboratory animals Co. The week of age of the animals when they arrive at the animal room of this company is 5-6 weeks. Upon arrival, animal house personnel transfer the animals from shipping packages to squirrel cages and examine each animal. The examination range includes appearance, limbs, cavities and the like, and whether the animal has abnormal performance in posture or movement.
Laboratory mice were placed in mouse cages (260mm x 160mm x 120mm) of an animal house, 4 per cage. The room number where the animals were placed throughout the experiment was recorded in the experimental record. The mouse padding is sawdust subjected to high-temperature treatment (hot pressing treatment, Shanghai SIPPR-BK laboratory animals Co., Ltd.), and the padding is replaced twice per week. The room in which the experimental mice were housed was located in the animal house, and the number of filtration, ventilation and air exchange was 15 to 25 per hour in this area. The temperature is maintained between 20-25 deg.C (68-77 deg.F) and the relative humidity is 40-70%. The lighting conditions were 12 hours (08:00-20:00) day of daylight lamp illumination and 12 hours of darkness. Experimental mice received unlimited amounts of rodent chow (sterilized by irradiation, Shanghai Silake laboratory animals Co., Ltd.). Each batch of animal feed received is accompanied by a certificate of analysis of the corresponding batch provided by the supplier. The certificate of analysis should be kept archived. During the whole experiment, municipal tap water sterilized at high temperature was obtained from experimental mice in an unlimited amount. Within foreseeable limits, the known levels of contaminants in animal food and drinking water will not affect the purpose or implementation of the experiment.
Each animal was assigned a unique number. Animal hair was dyed with picric acid solution for identification of its number. Before animals are grouped, the squirrel cage labels are labeled with item number, animal species/strain, gender, cage number, and animal number. After the animals are grouped, the squirrel cage is marked with the group information and the information by using a label. The grouping situation is recorded in a random grouping file. The cages were layered to reduce the effect of environmental factors on the experiment.
Second, Experimental methods
Sodium carboxymethylcellulose 0.5% will be used as the test vehicle and vehicle control.
1. Animal grouping:
mice were randomly divided into 4 groups of 8 mice by weight, and the grouping was as shown in table 1.
TABLE 1 grouping scheme
The drug effect, acute toxicity, long-term toxicity and other experiments of animals are administrated 1 time per day, and the frequency is maintained in the experiment. To observe the drug effect more fully, the drug administration is planned to be continuous for 5 days, and the observation is performed once a day.
2. Preparing a reagent:
preparing 0.5% CMC-Na:
adding 200mL ddH into a beaker2And O, heating to boil, stopping heating, adding 1.0g of sodium carboxymethylcellulose, stirring until the sodium carboxymethylcellulose is completely dissolved, and taking the supernatant to obtain a 0.5% CMC-Na solution.
Preparing a compound shown in a formula I: it is prepared before use. Prepared 1 time a day.
1.25mg/ml (12.5mg/kg) 38mg (98% purity) was added 29.79ml of 0.5% CMC and sonicated to give a homogeneous suspension.
0.5mg/ml (5mg/kg) 6ml of 1.25mg/ml solution is taken, 0.5% CMC is added, the volume is adjusted to 15ml, and the mixture is mixed evenly.
0.025mg/ml (0.25mg/kg) taking 0.3ml of 1.25mg/ml solution, adding 0.5% CMC, diluting to 15ml, and mixing.
3. Administration:
the compound of formula I and 0.5% CMC-Na were administered by gavage in a volume of 10mL/kg body weight 1 time per day.
4. And (4) observation:
general condition observation of animals. The appearance, behavior, stool characteristics and the like of each mouse were observed about 0.5 to 2 hours after each administration (the specific observation indication was according to the "research technical guidance on toxicity of single drug administration" promulgated by the national drug administration). All abnormal appearance and behavioral activities were recorded in the observation table.
4 days after dosing, animals underwent CO2The anatomy was sacrificed and the dirty changes observed.
Animal body weight and mortality. Animal weights were recorded once before and after the experiment. Animal mortality was recorded daily.
TABLE 2 animal observations indications (the table is used only as a reference for evaluation of the results analysis, and other scientific, rational analyses are acceptable; observations of all or part of the indications listed in the table may be required)
TABLE 2
5. Statistical analysis:
the results are expressed as: mean ± standard deviation. Pairwise comparisons between groups using the t-test, p <0.05 will be considered as statistically significant differences.
Third, experimental results
1. General observations of animals:
the behavior, activity and the like of the animals in the blank control group are not obviously different before and after administration.
After the high dose 12.5mg/kg group was administered, 8 mice all exhibited significant decreased activity, ataxia, low-lying, prone and other symptoms, and had significant decreased food intake.
After the group with the medium dose of 5mg/kg, 8 mice all showed obvious symptoms of reduced activity and low-voltage, and the food intake was slightly reduced.
After the low dose of 0.25mg/kg group, 2 mice showed obvious symptoms of reduced activity, low voltage and the like, but no ataxia appeared.
Each group of mice recovered normal activity about 1-2 hours after administration. ' Qiyi
2. Animal weight change:
the body weight changes of the animals in each group are shown in Table 3 and FIG. 1.
The body weight of the mice increased slightly during the administration of the blank control group, but there was no statistical difference.
The low dose of 0.25mg/kg body weight did not change significantly.
In the 5mg/kg and 12.5mg/kg groups, a significant decrease in the volume of the mice occurred 4 days after administration.
TABLE 3 mouse mean body weight Change Table (g)
| | Day | 0 | |
| Blank control group | 22.16±0.99 | 22.66±1.25 | |
| 0.25mg/kg | 22.70±0.93 | 22.74±0.67 | |
| 5mg/kg | 22.20±1.0 | 21.94±0.81 | |
| 12.5mg/kg | 22.29±0.41 | 21.63±0.37 |
Based on the above similar method, the mice were examined for weight change using pirfenidone with a compound of formula II having an HPLC purity of 95% and pirfenidone with a compound of formula II having an HPLC purity of 98% (converted to the above dose groups with a compound of formula I having an HPLC purity of 5% and 2%, respectively), and the weight loss of the mice in the low purity 95% group was significantly higher than that in the 98% group.
3. Dirty observation of animals:
the lungs of the mice in the administered group showed varying degrees of congestion, with the medium and high dose groups showing severe congestion in the lower dose group, as shown in figure 2.
4. Pathological section observation:
the group administered with the high dose of 12.5mg/kg showed more severe congestion compared to the control group, as shown in FIG. 3.
5. Animal mortality observation:
none of the animals in each group died.
Fourth, conclusion
Pirfenidone is an oral drug with anti-fibrotic and anti-inflammatory effects, which is marketed in several countries, and the common side effects of patients are: (1) gastrointestinal discomfort, nausea, vomiting, and the like. (2) Light-sensitive rashes. (3) Liver function impairment may occur: liver function is impaired by elevation of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), etc., and liver failure may occur, and liver function is periodically examined. (4) The nervous system: drowsiness, dizziness and unstable walking.
In this example, 5-methyl-1- ((6-oxo-1-phenyl-1, 6-dihydropyridin-3-yl) methyl) pyridin-2 (1H) -one, which is a main impurity contained in pirfenidone, was administered to mice at three doses, high, medium, and low, respectively, and the toxicity of the impurity to the mice was observed to study the potential relationship between the drug side effects of pirfenidone capsules and the impurity.
Comparing the response of mice in the control blank group and the high dose group (12.5mg/kg), the medium dose group (5mg/kg), and the low purity group (2.5mg/kg), the responses of mice in each group were observed as the dose increased: (1) the activity of the mouse is obviously reduced, and the animal has symptoms such as low voltage, prostrate and the like; (2) the body weight is obviously reduced; (3) the lungs of mice with the traditional Chinese medicine composition are congested to different degrees, and the congestion of the middle-dose combined high-dose group is serious; (4) after the high-dose mice are administrated, ataxia, reduced food intake and the like appear, which shows that the toxicity of the central nervous system and the side reaction of the gastrointestinal tract are actually related to the impurities contained in the medicine.
Experimental observation and data show that the toxicity of impurities and the toxic and side effects of the pirfenidone capsule have an obvious relationship, and the toxic and side effects have an obvious enhancement trend along with the increase of the concentration of the impurities. Therefore, the aim of reducing the side effect of the medicine can be achieved by improving the purity of the pirfenidone.
Example 3
Purification of pirfenidone
150g of drinking water is put into a reaction bottle, stirred and heated to 90-95 ℃, 10g of crude product (obtained according to the method described in US 39764281 or US 8680123) is added, the temperature is kept, stirred and dissolved for 30 minutes, 2g of medicinal activated carbon (granular) is added, the mixture is stirred for 1 hour under the condition of heat preservation, sampling and analysis are carried out, and the mixture is put into a filter when the mixture is qualified. Filtering while hot, filtering to remove active carbon, placing the filtrate into another clean reaction bottle while keeping the temperature, stirring, cooling to 10-15 deg.C, keeping the temperature for 1 hr, centrifuging, filtering to obtain white solid crystal, and drying to obtain the final product.
FIG. 4 is a liquid chromatogram of crude pirfenidone, wherein 5.144min is a compound of formula I.
FIG. 5 is a liquid chromatogram of the impurity of formula I after initial purification (without preparative column purification), wherein 5.127min is the compound of formula I, corresponding to the time of the peak appearance of the impurity of formula I in the crude pirfenidone product of FIG. 4.
FIG. 6 is a liquid chromatogram of a purified pure product of pirfenidone showing that the impurities of formula I have been completely removed.
Example 4
The compound of formula I is an impurity of pirfenidone
The mother liquor remaining after pirfenidone purification (according to the purification method provided in example 3) was further purified by column chromatography (ethyl acetate/methanol 20:1), TLC large plate crude purification (ethyl acetate/methanol 10:1) to give a crude product, preparative chromatography (i.e. the purification method in example 1) to give a compound with 98% purity, LCMS, hplc, and the like,1HNMR、13A series of structures such as CNMR are identified as formula I. Wherein in the mother liquor liquid mass diagram (figure 7) remaining after the purification of pirfenidone, the 1.160min molecular ion peak 293 is the molecular ion peak of formula I and the crude product liquid mass diagram (figure 8) of formula I, and (I) pure product liquid mass diagram (Fig. 9) corresponds to a molecular ion peak of 293.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.
Claims (3)
1. A compound with a structure shown as a formula I,
2. a method for preparing a compound having a structure represented by formula i, comprising the steps of:
(1) carrying out bromination reaction on the compound with the structure shown in the formula II to obtain a compound with the structure shown in the formula III;
(2) mixing a compound with a structure shown in a formula III and 5-methylpyridine-2 (1H) -ketone to obtain a compound with a structure shown in a formula I;
3. the application of a compound with a structure shown as a formula I is used for controlling the quality of pirfenidone or pirfenidone medicaments; and/or as an impurity reference substance during the inspection of the pirfenidone and/or pirfenidone drug related substances;
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| CN112645862B (en) * | 2020-12-09 | 2022-04-01 | 南京华威医药科技集团有限公司 | Pirfenidone photodegradation impurities and preparation method thereof |
| CN114113397A (en) * | 2021-11-30 | 2022-03-01 | 江苏知原药业股份有限公司 | Content determination method for active ingredients of pirfenidone tablets |
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| CN101842355A (en) * | 2007-06-20 | 2010-09-22 | 奥斯拜客斯制药有限公司 | Substituted n-aryl pyridinones as fibrotic inhibitors |
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