Tanshinone IIA sodium sulfonate related substance and refining method thereof

Abstract

The invention discloses a tanshinone IIA sodium sulfonate related substance and a refining method thereof. The invention relates to a refining method of a compound as shown in a formula II, which comprises the following steps of: firstly neutralizing a water phase in residual mother liquor for preparing tanshinone IIA sodium sulfonate by using alkali, and then carrying out gradient elution by using a preparative chromatography, wherein the preparative chromatography comprises a mobile phase A and a mobile phase B; the mobile phase A is a purified aqueous solution with the volume percent of triethylamine being 0.1%; and the mobile phase B is a methanol solution in which the volume percent of triethylamine is 0.1%. The method is simple and convenient, the obtained compound shown in the formula II can be used as a reference substance for quantitative and qualitative analysis of tanshinone IIA sodium sulfonate production, and the quality standard of tanshinone IIA sodium sulfonate is further improved.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a tanshinone IIA sodium sulfonate related substance and a purification method thereof. Background Art

[0002] Danshen is a perennial herbaceous plant of the genus Salvia in the family Lamiaceae. It contains a variety of active ingredients. Tanshinone ⅡA is the most abundant and representative fat-soluble component in Danshen. Pharmacological experiments have shown that Tanshinone ⅡA has a wide range of pharmacological effects, such as natural antioxidant effects, cardiovascular pharmacological effects, anti-infection effects, anti-tumor effects, protective effects on nerve cells, protective effects on the liver, kidneys, and lungs, enhancing the body's immune function, analgesia, anti-aging, etc. Since Tanshinone ⅡA has poor water solubility and slow intestinal absorption, some people sulfonate it into a water-soluble sodium salt - Tanshinone ⅡA sodium sulfonate for clinical and animal experiments.

[0003] Tanshinone IIA sulfonate sodium can reduce atherosclerosis, dilate coronary arteries, increase coronary blood flow, and improve myocardial hypoxia; it can also inhibit platelet aggregation and lower blood lipids. Therefore, it is an important cardiovascular drug for the treatment of coronary heart disease, angina pectoris, and myocardial infarction.

[0004] With the improvement of drug registration requirements in my country in recent years, the preparation of high-purity sodium tanshinone IIA sulfonate and the quality research of related substances are particularly important. Therefore, it is of great significance to obtain these extremely low-content standard products of related substances and to establish standards for the content control of related substances in sodium tanshinone IIA sulfonate. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects in the prior art that the types of substances that affect the quality of sodium tanshinone IIA sulfonate contained in sodium tanshinone IIA sulfonate are not disclosed, and there is no refining and purification method for the substance, resulting in the inability to determine its structure, lack of reference substances, and thus the inability to perform qualitative analysis on related substances, and provide a sodium tanshinone IIA sulfonate related substance and a refining method thereof. The method is simple and convenient, and the obtained compound as shown in formula II can be used as a reference substance for the quantitative and qualitative analysis of the compound shown in formula II in the production of sodium tanshinone IIA sulfonate, further improving the quality standard of sodium tanshinone IIA sulfonate.

[0006] The present invention provides a compound as shown in formula II,

[0007] .

[0008] The present invention also provides a method for refining the compound shown in formula II, which comprises the following steps:

[0009] The waste liquid from the preparation of sodium tanshinone IIA sulfonate was purified by preparative chromatography to obtain a compound as shown in formula II, wherein:

[0010] The waste liquid for preparing sodium tanshinone IIA sulfonate comprises a compound as shown in formula II;

[0011] In the preparative chromatography:

[0012] The chromatographic column is a C18 reverse phase preparative column;

[0013] The mobile phase is a mixture of mobile phase A and mobile phase B, wherein the mobile phase A is a triethylamine aqueous solution with a volume percentage of 0.05% to 0.1%, and the mobile phase B is a mixture of triethylamine and methanol with a volume percentage of 0.05% to 0.1% of triethylamine;

[0014] The elution method of the preparative chromatography is a four-stage gradient elution, and the four-stage gradient elution conditions are: (where % represents volume percentage)

[0015]

[0016] Among them, T1 is 1-5; T2 is 5-25; T3 is 5-25; T4 is 30-50;

[0017] X1 is 100-90; X2 is 90-85; X3 is 85-80; X4 is 25-20;

[0018] Wherein, the T represents the duration of each stage;

[0019] In the purification method, in the preparative chromatography, the eluate with a retention time of 15-30 minutes is collected.

[0020] In one embodiment, the retention time may be 15-18 min, 25-30 min or 15-20 min; for example, 25-30 min.

[0021] Preferably, the waste liquid for preparing sodium tanshinone IIA sulfonate contains: one or more of a sodium tanshinone IIB sulfonate derivative with a purity of 34% to 40% (e.g., 37.1%), a compound as shown in formula II with a purity of 18.0% to 24.0% (e.g., 21.0%), sodium tanshinone I sulfonate with a purity of 3.0% to 5.0% (e.g., 4.1%), sodium tanshinone A sulfonate with a purity of 5.0% to 7.0% (e.g., 6.3%), and sodium tanshinone IIA sulfonate with a purity of 5.0% to 7.0% (e.g., 6.2%).

[0022] The sodium tanshinone IIB sulfonate derivative is

[0023] More preferably, the waste liquid for preparing sodium tanshinone IIA sulfonate contains one or more of the following substances:

[0024]

[0025]

[0026]

[0027] Among them, the retention time of each substance in the above table is obtained by testing the waste liquid of preparing sodium tanshinone IIA sulfonate under the following conditions:

[0028] Purity analysis was performed using Shimadzu LC-2030C Plus HPLC. Chromatographic conditions: ZORBAX SB-C18 column (4.6×150 mm, 5-micton), mobile phase A: 20 mM KH 2 PO 4 +0.5% triethylamine (pH 4.0)-methanol (60:40), mobile phase B: 20 ​​mM KH 2 PO 4 +0.5% triethylamine (pH 4.0)-methanol (20:80); gradient elution program: flow rate 1.0 mL / min, column temperature 25°C, detection wavelength 271 nm, injection volume 10 μl.

[0029] The waste liquid for preparing sodium tanshinone IIA sulfonate can be obtained by the following method, which comprises the following steps:

[0030] S1: using a mixed solution of tanshinone IIA, acetic anhydride and an organic solvent as feed solution A, and a mixed solution of sulfuric acid and acetic acid as feed solution B, adding the feed solution A and the feed solution B into a first microreactor to perform a continuous sulfonation reaction to obtain a reaction solution D;

[0031] S2: adding the reaction solution D and the sodium salt aqueous solution into a second microreactor to perform a continuous salt-forming reaction to obtain a reaction solution E;

[0032] S3: filtering the reaction solution E and taking the filtrate;

[0033] In S1, the retention volume of the first microreactor is 40-60 mL (e.g., 50 mL); the flow rate of the feed liquid A pumped into the first microreactor is 8-10 mL / min (e.g., 9 mL / min); the flow rate of the feed liquid B pumped into the first microreactor is 0.5-1.5 mL / min (e.g., 1 mL / min); the residence time of the continuous sulfonation reaction is 2-8 min (e.g., 5 min); the molar ratio of tanshinone IIA to acetic anhydride in the feed liquid A is 1:(6-10), e.g., 1:8; the reaction molar ratio of tanshinone IIA to the sulfuric acid is 1:(4-8), e.g., 1:6; the temperature of the continuous sulfonation reaction is 20-30°C (e.g., 25°C); the feed liquids A and B are simultaneously pumped into the first microreactor by the first pump and the second pump, mixed, and the continuous sulfonation reaction is carried out; the material of the first pump is one or more of Hastelloy, stainless steel, PEEK, and modified polytetrafluoroethylene; the material of the second pump is PEEK and / or modified polytetrafluoroethylene;

[0034] In S2, the retention volume of the second microreactor is 5~15mL (for example, 10mL); the flow rate of the sodium salt aqueous solution pumped into the second microreactor is 5~15mL / min (for example, 10mL / min); the temperature of the continuous salt-forming reaction is 20~30°C (for example, 25°C); the sodium salt aqueous solution is pumped into the second microreactor by a third pump to perform a continuous salt-forming reaction with the reaction liquid D; the material of the third pump is one or more of Hastelloy, stainless steel, PEEK and modified polytetrafluoroethylene.

[0035] In S1, the organic solvent is a conventional organic solvent in the art that can dissolve tanshinone IIA and acetic anhydride at the same time, such as dichloromethane;

[0036] In S1, the molar ratio of sulfuric acid to acetic acid in the feed liquid B is a conventional molar ratio for such reactions in the art, such as 1:(3-5), for example 1:3.9.

[0037] In S1, the sulfuric acid is concentrated sulfuric acid, for example, a concentrated sulfuric acid aqueous solution with a concentration of 15-20 mol / L, and the concentration of the acetic acid is 15-20 mol / L, for example, 17.5 mol / L.

[0038] In S1, the first pump is preferably a horizontal flow pump.

[0039] In S1, the material of the first pump is preferably modified polytetrafluoroethylene.

[0040] In S1, the second pump is preferably a horizontal flow pump.

[0041] In S1, the material of the second pump is preferably modified polytetrafluoroethylene.

[0042] In S2, the sodium salt in the sodium salt aqueous solution is conventional in the art, preferably an inorganic acid sodium salt and / or an organic acid sodium salt; more preferably an inorganic acid sodium salt; for example, sodium chloride.

[0043] In S2, the concentration of the sodium salt aqueous solution is preferably greater than 0.7 mol / L, for example, the concentration of the sodium salt is prepared into a saturated aqueous solution.

[0044] In S2, the residence time of the continuous salt-forming reaction is a conventional parameter in the art, preferably 0.3-1.2 min, such as 0.5 min.

[0045] In S2, the third pump is preferably a horizontal flow pump; the material of the third pump is preferably modified polytetrafluoroethylene.

[0046] Preferably, in S3, after the filtrate is separated, the aqueous phase is taken to adjust the pH to 5-6, and the pH is preferably adjusted using alkali metal carbonates, alkali metal bicarbonates or alkali metal hydroxides, such as sodium carbonate, and further such as saturated sodium carbonate aqueous solution; preferably, when adjusting the pH, the temperature of the aqueous phase can be 0-20°C, preferably 0-5°C.

[0047] In the purification method, the chromatographic column is preferably a dynamic axial compression column.

[0048] In the refining method, the filler of the chromatographic column can be C18 super water-resistant filler, preferably C18 Aq super water-resistant filler, and more preferably UniSil 10-100 C18 Aq super water-resistant filler, such as UniSil 10-100 C18 Aq super water-resistant filler purchased from Suzhou Nanomicrobiology Technology Co., Ltd.

[0049] In the purification method, the pore size of the filler of the chromatographic column may be 8-12 nm, for example, 10 nm.

[0050] In the purification method, the particle size of the filler of the chromatographic column may be 90-110 μm, for example, 100 μm.

[0051] In the preparation method, the length of the chromatographic column may be 580-620 mm, for example, 600 mm.

[0052] In the preparation method, the diameter of the chromatographic column may be 45-55 mm, for example, 50 mm.

[0053] In a preferred embodiment of the present invention, the length of the chromatographic column is 600 mm, the diameter of the chromatographic column is 50 mm, and the filler of the chromatographic column is UniSil 10-100 C18 Aq super water-resistant filler, the pore size of the filler is 10nm, and the particle size of the filler is 100μm.

[0054] In the purification method, the system used in the preparative chromatography method can be an LC-20AP high pressure liquid phase preparation system.

[0055] In the refining method, the chromatographic column in the preparative chromatography is a dynamic axial compression column, the equipment model of which is DAS50*600 and the serial number is SF2310101.

[0056] In the purification method, the operating pressure of the chromatographic column is preferably 0-10 MPa, such as 5 MPa.

[0057] In the purification method, the column temperature of the chromatographic column is preferably 5-60°C, such as 25°C.

[0058] In the purification method, the mobile phase A may be a 0.1% by volume aqueous solution of triethylamine or a 0.05% by volume aqueous solution of triethylamine, for example, a 0.1% by volume aqueous solution of triethylamine.

[0059] In the purification method, the mobile phase B may be a mixture of triethylamine and methanol at a volume percentage of 0.1% or a mixture of triethylamine and methanol at a volume percentage of 0.05%, for example, a mixture of triethylamine and methanol at a volume percentage of 0.1%.

[0060] In the refining method, the mobile phase is a mixture of mobile phase A and mobile phase B, the mobile phase A is a 0.1% by volume triethylamine aqueous solution, and the mobile phase B is a 0.1% by volume mixture of triethylamine and methanol;

[0061] Alternatively, the mobile phase A is a 0.05% by volume triethylamine aqueous solution, and the mobile phase B is a 0.05% by volume mixture of triethylamine and methanol.

[0062] In the purification method, the flow rate of the mobile phase can be 90-110 mL / min or 40-60 mL / min.

[0063] In the purification method, the flow rate of the mobile phase can be 100 mL / min or 50 mL / min.

[0064] In the purification method, the detection method of the preparative chromatography can be ultraviolet detection (the detector can be Shimadzu LC-20AP); the detection wavelength of the preparative chromatography can be 271 nm.

[0065] In the purification method, in the preparative chromatography, the injection volume is preferably 300-600 mL, for example 400 mL.

[0066] In the purification method, in the preparative chromatography, the following gradient elution conditions are preferably used:

[0067] When the gradient elution is a four-stage gradient elution, in the first stage, the volume percentage of the mobile phase A is 90%, and the volume percentage of the mobile phase B is 10%;

[0068] And / or, in the second stage, the volume percentage of the mobile phase A is 85%, and the volume percentage of the mobile phase B is 15%;

[0069] And / or, in the third stage, the volume percentage of the mobile phase A is 80%, and the volume percentage of the mobile phase B is 20%;

[0070] And / or, in the fourth stage, the volume percentage of the mobile phase A is 20%, and the volume percentage of the mobile phase B is 80%;

[0071] And / or, when the gradient elution is a four-stage gradient elution, in the first stage, the elution time is preferably 3 min or 5 min, for example 3 min;

[0072] And / or, in the second stage, the elution time is preferably 10 min or 20 min, for example 10 min;

[0073] And / or, in the third stage, the elution time is preferably 10 min or 20 min, for example 10 min;

[0074] And / or, in the fourth stage, the elution time is preferably 37 min or 45 min, for example 10 min;

[0075] Preferably, in the purification method, in the preparative chromatography, the flow rate is 90-110 mL / min, for example 100 ml / min;

[0076] In the first stage, the elution time is 2 to 4 minutes;

[0077] In the second stage, the elution time is 8 to 12 minutes;

[0078] In the third stage, the elution time is 8 to 12 minutes;

[0079] In the fourth stage, the elution time is 35~40min.

[0080] Preferably, in the purification method, in the preparative chromatography, the flow rate is 40-60 mL / min, for example 50 ml / min;

[0081] In the first stage, the elution time is 4 to 6 minutes;

[0082] In the second stage, the elution time was 18-22 min;

[0083] In the third stage, the elution time was 18-22 min;

[0084] In the fourth stage, the elution time is 40 to 50 minutes.

[0085] In the purification method, in the preparative chromatography, the following four-stage gradient elution conditions are further preferably used:

[0086] Condition 1:

[0087]

[0088] ;or,

[0089] Condition 2:

[0090] .

[0092] Preferably, in the purification method, in the preparative chromatography, when the gradient elution condition is condition 1, the mobile phase A is a 0.1% by volume aqueous solution of triethylamine, and the mobile phase B is a 0.1% by volume mixture of triethylamine and methanol, and the eluent with a retention time of 15-20 min (e.g., 15-18 min) is collected. Preferably, the injection volume is 340~460 mL (e.g., 400 mL).

[0093] Preferably, in the purification method, in the preparative chromatography, when the gradient elution condition is condition 2, mobile phase A is a 0.1% by volume aqueous solution of triethylamine, and mobile phase B is a 0.1% by volume mixture of triethylamine and methanol, the eluate with a retention time of 20-30 min (e.g., 25-30 min) is collected, and preferably, the injection volume is 340-460 mL (e.g., 400 mL).

[0094] Preferably, in the purification method, in the preparative chromatography, when the gradient elution condition is the condition 1, the mobile phase A is a 0.05% by volume aqueous solution of triethylamine, and the mobile phase B is a 0.05% by volume mixture of triethylamine and methanol, and the eluent with a retention time of 15-25 min (e.g., 15-20 min) is collected. Preferably, the injection volume is 340~460 mL (e.g., 400 mL).

[0095] In the refining method, the refining method may further include: performing a second elution on the eluate obtained by the above-mentioned preparative chromatography;

[0096] Preferably, in the chromatographic conditions of the second elution, after the organic phase is concentrated and removed, the eluent A is diluted 1-2 times (eg, 1 time), and the preparative chromatography is used to adjust the flow rate to 50 mL / min for the second elution.

[0097] After the second elution is completed, concentration and / or drying may also be included; the concentration is preferably reduced pressure concentration.

[0098] The drying operation can be conventional in the art, such as freeze drying, which is drying by a freeze dryer; the freeze drying step is pre-freezing at -40°C for 4 hours, heating to 30°C at 1°C / 8 minutes, and keeping warm for 36 hours to obtain a red solid.

[0099] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain the preferred embodiment of the present invention.

[0100] The reagents and raw materials used in the present invention are commercially available.

[0101] The positive and progressive effect of the present invention is that the present invention prepares the related substance of sodium tanshinone IIA sulfonate (such as the compound shown in formula II) by reverse phase. The refining method is simple and convenient, and the purity of the obtained impurity reference substance can reach more than 98%. It is applied to the quantitative and qualitative analysis of the compound in the production of sodium tanshinone IIA sulfonate, which can further improve the quality standard of sodium tanshinone IIA sulfonate. DETAILED DESCRIPTION

[0102] The crude product of the sodium tanshinone IIB sulfonate derivative (compound shown in formula II) used in the following examples was prepared by the following method:

[0103] In the following examples, the structure of the related substance of sodium tanshinone IIA sulfonate is: .

[0104] In the microreactor, the connecting pipe is a 1 / 8 pipe made of modified polytetrafluoroethylene, and the three pumps are all horizontal flow pumps made of modified polytetrafluoroethylene. The retention volume of the first microreactor D is 50mL, and the retention volume of the second microreactor E is 10mL. Tanshinone IIA (0.068mol) and acetic anhydride (0.55mol) were dissolved in dichloromethane and diluted to 1L as feed liquid A; 18mol / L concentrated sulfuric acid (1.8mol) and 17.5mol / L acetic acid (about 7mol) were diluted to 500mL as feed liquid B; saturated sodium chloride solution was used as feed liquid C. The flow rate of the first horizontal flow pump A was 9.0mL / min, the flow rate of the second horizontal flow pump B was 1.0mL / min, and the flow rate of the third horizontal flow pump C was 10mL / min. The molar ratio of tanshinone IIA to acetic anhydride is about 1:8, and the molar ratio of tanshinone IIA to sulfuric acid is 1:6. The temperatures of the sulfonation reaction and the salt-forming reaction are both 25°C. The residence time of the continuous sulfonation reaction is 5 minutes, and the residence time of the continuous salt-forming reaction is 0.5 minutes. The feed liquids A and B are pumped into the first microreactor D by the first feed horizontal flow pump A and the second feed horizontal flow pump B respectively, and the feed liquid C is pumped into the second microreactor E by the third feed horizontal flow pump C. The three pumps are operated simultaneously, and the reaction liquid flows through the first microreactor D and the second microreactor E in turn, and finally flows into the receiver F through the control of the multi-channel switching valve, and the filtration is completed, and the filtrate is collected to obtain a reaction liquid containing the sodium tanshinone IIB sulfonate derivative shown in formula II. In the reaction liquid, the sodium tanshinone IIB sulfonate derivative (the substance corresponding to peak number 4 in Table 1, the structure is as follows ) with a purity of 37.1%, the related substance of sodium tanshinone IIA sulfonate shown in formula II (the substance corresponding to peak number 3 in Table 1, the structure is as follows ) has a purity of 21.0%, the purity of sodium tanshinone I sulfonate (the substance corresponding to peak number 14 in Table 1) is 4.1%; the purity of sodium tanshinone A sulfonate (the substance corresponding to peak number 16 in Table 1) is 6.3%; the purity of sodium tanshinone IIA sulfonate (the substance corresponding to peak number 20 in Table 1) is about 6.2%. The substance information in the reaction solution is shown in the following table:

[0105] Table 1

[0106]

[0107]

[0108]

[0109] The data in the above table and the following examples were analyzed for purity using Shimadzu LC-2030C Plus high performance liquid chromatography. Chromatographic conditions: ZORBAX SB-C18 column (4.6×150 mm, 5-micton), mobile phase A: 20 mM KH2 PO 4 +0.5% triethylamine (pH 4.0)-methanol (60:40), mobile phase B: 20 ​​mM KH 2 PO 4 +0.5% triethylamine (pH 4.0)-methanol (20:80). Gradient elution program: flow rate 1.0 mL / min, column temperature 25°C, detection wavelength 271 nm, injection volume 10 μl.

[0110] The following examples use Shimadzu LC-20AP high pressure liquid phase preparation system for preparative separation, and the chromatographic column is a high performance liquid reverse phase preparative column (dynamic axial compression column) with independent filler; the equipment model is DAS50*600, the serial number is SF2310101, and the filler of the chromatographic column is purchased from Suzhou Nano-Microbiology Technology Co., Ltd. UniSil 10-100 C18 Aq ultra-water-resistant filler, the pore size of the filler is 10 nm, the particle size is 100 μm, the length is 600 mm, and the diameter is 50 mm; the detection wavelength is 271 nm;

[0111] Wherein, the operating pressure of the chromatographic column is 5MPa;

[0112] Wherein, the operating temperature of the chromatographic column is 25 o C;

[0113] Example 1

[0114] Purification of Related Substances of Tanshinone IIA Sulfonate Sodium

[0115] Take 1200 mL of the reaction solution containing the related substances of sodium tanshinone IIA sulfonate, separate the liquid to obtain 400 mL of aqueous phase, add an appropriate amount of sodium carbonate aqueous solution at 0-5°C to neutralize to pH = 5-6. According to the volume of the liquid after pH adjustment, add methanol, shake well, and prepare a 10% methanol solution. Use the loading system to load the crude product, then elute from the reverse phase chromatographic column, and collect the eluate with a retention time of 15-18 minutes.

[0116] The chromatographic conditions are:

[0117] Mobile phase A is a 0.1% by volume triethylamine aqueous solution, and mobile phase B is a 0.1% by volume mixture of triethylamine and methanol;

[0118] The flow rate was 100 mL / min; the detection wavelength was 271 nm; the injection volume was 400 mL;

[0119] The column temperature of the chromatographic column was 25°C;

[0120] The elution gradient is (the times in the following table are time points):

[0121]

[0122] After obtaining the eluate, the methanol was removed by concentration and diluted with purified water by one half, and then all the diluted eluate was re-adsorbed into the chromatographic column filler, and the above chromatographic conditions were continued, only the flow rate was adjusted to 50 mL / min for the second elution. After obtaining the eluate, the sample was concentrated under reduced pressure to remove methanol, and lyophilized to obtain a red solid. HPLC analysis was performed, and the main peak retention time was 1.715 min. The area normalization method showed that the liquid phase purity was 98.192%. The red solid obtained in this embodiment is a high-resolution mass spectrum C of a related substance of sodium tanshinone IIA sulfonate as shown in Formula II 19 H 16 NaO 10 S 2 (M-Na) - = 491.0101. 1 HNMR and 13 C NMR is as follows: the prepared red solid (compound shown in Formula II) was added to the sodium tanshinone IIA sulfonate raw material for HPLC chromatographic analysis, and the chromatogram was superimposed with the chromatogram of the sodium tanshinone IIA sulfonate raw material without the addition of the compound shown in Formula II. It was found that the sodium tanshinone IIA sulfonate raw material contained an impurity with a retention time of 1.7 min (the retention time of the main peak of the compound shown in Formula II was 1.7 min), and the impurity peak with a retention time of 1.7 min increased significantly. Based on the superposition results, it was inferred that the related substance existed in the sodium tanshinone IIA sulfonate raw material.

[0123] 1 H NMR (600 MHz, Methanol-d 4 ) δ 7.82 (d, J = 7.8 Hz, 1H), 7.75 (d, J =7.8 Hz, 1H), 4.43 (dd, J = 7.8, 2.4 Hz, 1H), 3.45 (dt, J = 19.2, 6.6 Hz, 1H), 3.25 (dt, J = 19.2, 6.6 Hz, 1H), 2.47 (s, 3H), 2.33 – 2.23 (m, 2H), 1.42 (s, 3H), 1.40 (s, 3H). 13 C NMR (151 MHz, Methanol-d 4) δ 184.0, 177.0, 161.6,153.0, 150.7, 144.5, 135.2, 128.4, 127.6, 122.4, 121.3, 121.0, 82.5, 40.5,30.3, 26.5, 24.5, 24.3, 9.7.

[0124] Example 2

[0125] Purification of Related Substances of Tanshinone IIA Sulfonate Sodium

[0126] Take 1000 mL of the reaction solution of the sodium tanshinone IIA sulfonate related substance shown in formula II, separate the liquid to obtain 300 mL of the aqueous phase, add an appropriate amount of saturated sodium carbonate aqueous solution at 0-5°C to neutralize to pH = 5-6. According to the volume of the liquid after pH adjustment, add methanol, shake well, and then prepare a 10% methanol solution, use the loading system to load the crude product, and then elute from the reverse phase chromatographic column, and collect the eluate with a retention time of 25-30 minutes.

[0127] The chromatographic conditions are:

[0128] Mobile phase A is a 0.1% by volume triethylamine aqueous solution, and mobile phase B is a 0.1% by volume mixture of triethylamine and methanol;

[0129] The flow rate was 50 mL / min; the detection wavelength was 271 nm; the injection volume was 400 mL;

[0130] The column temperature was 25 o C;

[0131] The elution gradient is (the times in the following table are time points):

[0132]

[0133] After obtaining the eluate, the methanol was removed by concentration and then diluted with purified water by half. All the diluted eluate was re-adsorbed into the chromatographic column filler and the above chromatographic conditions were continued, only the flow rate was adjusted to 50 mL / min for the second elution. After obtaining the eluate, the sample was concentrated under reduced pressure to remove methanol and freeze-dried to obtain a red solid. HPLC analysis showed that the retention time of the main peak was 1.717 min and the liquid phase purity was 98.556% by area normalization method.

[0134] Example 3

[0135] Take 1200 mL of the reaction solution containing the compound shown in Formula II, separate the liquid to obtain 400 mL of the aqueous phase, add an appropriate amount of saturated sodium carbonate aqueous solution at 0-5°C to neutralize to pH = 5-6. Add methanol according to the volume of the liquid after pH adjustment, shake well, and then prepare a 10% methanol solution, use the loading system to load the crude product, and then elute from the reverse phase chromatographic column, and collect the eluate with a retention time of 15-20 minutes.

[0136] The chromatographic conditions are:

[0137] Mobile phase A is a 0.05% by volume triethylamine aqueous solution, and mobile phase B is a 0.05% by volume mixture of triethylamine and methanol;

[0138] The flow rate was 100 mL / min; the detection wavelength was 271 nm; the injection volume was 400 mL;

[0139] The column temperature of the chromatographic column was 25°C;

[0140] The elution gradient is (the times in the following table are time points):

[0141]

[0142] After obtaining the eluate, the methanol was removed by concentration and then diluted by half with purified water. All the diluted eluate was re-adsorbed into the chromatographic column filler and the above chromatographic conditions were continued, only the flow rate was adjusted to 50 mL / min for the second elution. After obtaining the eluate, the sample was concentrated under reduced pressure to remove methanol and freeze-dried to obtain a red solid. The HPLC was used for detection. The retention time of the main peak of purity was 1.721 min. The liquid phase purity was 90.402% by area normalization method.

[0143] Comparative Example 1

[0144] Take 1200 mL of the reaction solution containing the compound shown in Formula II, separate the liquid to obtain 400 mL of the aqueous phase, add an appropriate amount of saturated sodium carbonate aqueous solution at 0-5°C to neutralize to pH = 5-6. Add methanol according to the volume of the liquid after pH adjustment, shake well, and then prepare a 10% methanol solution, use the loading system to load the crude product, and then elute from the reverse phase chromatographic column, and collect the eluate with a retention time of 15-20 minutes.

[0145] The chromatographic conditions are:

[0146] Mobile phase A was water, and mobile phase B was methanol;

[0147] The flow rate was 100 mL / min; the detection wavelength was 271 nm; the injection volume was 400 mL;

[0148] The column temperature was 25 oC;

[0149] The elution gradient is (the times in the following table are time points):

[0150]

[0151] After obtaining the eluate, the methanol was removed by concentration and then diluted with purified water by half. All the diluted eluate was then re-adsorbed onto the chromatographic column filler and the above chromatographic conditions were continued, only the flow rate was adjusted to 50 mL / min for the second elution. After obtaining the eluate, the sample was concentrated under reduced pressure to remove methanol and freeze-dried to obtain a red solid. The HPLC analysis showed that the retention time of the main peak was 1.719 min and the liquid phase purity was 73.585% by the area normalization method.

Claims

1. A method for refining related substances of sodium tanshinone IIA sulfonate, comprising the following steps: The waste liquid prepared from sodium tanshinone IIA sulfonate is separated by preparative chromatography to obtain the sodium tanshinone IIA sulfonate related substances, wherein the sodium tanshinone IIA sulfonate related substances are Wherein, the waste liquid for preparing sodium tanshinone IIA sulfonate includes the sodium tanshinone IIA sulfonate-related substances; In the preparative chromatography: The chromatographic column was a C18 reverse phase preparative column; The mobile phases are mobile phase A and mobile phase B for gradient elution, wherein the mobile phase A is a 0.1% by volume triethylamine aqueous solution, and the mobile phase B is a mixture of triethylamine and methanol with a triethylamine volume percentage of 0.1%; The elution of the preparative chromatography is a four-stage gradient elution, and the four-stage gradient elution conditions are: (where % represents volume percentage) Among them, T1 is 1-5; T2 is 5-25; T3 is 5-25; T4 is 30-50; X1 is 100-90; X2 is 90-85; X3 is 85-80; X4 is 25-20; Wherein, the T represents the duration of each stage; In the purification method, in the preparative chromatography, the eluate with a retention time of 15-30 minutes is collected.

2. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: The retention time is 15-18 min, 25-30 min or 15-20 min; for example 25-30 min.

3. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: The waste liquid for preparing sodium tanshinone IIA sulfonate contains one or more of the following substances: (1) a sodium tanshinone IIB sulfonate derivative having a purity of 34% to 40%, for example, a sodium tanshinone IIB sulfonate derivative having a purity of 37.1%; the sodium tanshinone IIB sulfonate derivative is (2) Tanshinone IIA sodium sulfonate related substances with a purity of 18.0% to 24.0%, for example, 21% of Tanshinone IIA sodium sulfonate related substances; (3) Tanshinone I sodium sulfonate with a purity of 3.0% to 5.0%, for example, 4.1% Tanshinone I sodium sulfonate; (4) sodium danshensulfonate with a purity of 5.0% to 7.0%, for example, sodium danshensulfonate with a purity of 6.3%; (5) Tanshinone IIA sodium sulfonate with a purity of 5.0% to 7.0%, for example, Tanshinone IIA sodium sulfonate with a purity of 6.2%; Preferably, the waste liquid for preparing sodium tanshinone IIA sulfonate contains one or more of the following substances: Among them, the retention time of each substance in the above table is obtained by testing the waste liquid of preparing sodium tanshinone IIA sulfonate under the following conditions: Purity detection and analysis were performed using Shimadzu LC-2030C Plus high performance liquid chromatography. Chromatographic conditions: ZORBAX SB-C18 column (4.6×150 mm, 5-micton), mobile phase A: 20 mM KH2PO4+0.5% triethylamine (pH 4.0)-methanol (60:40), mobile phase B: 20 ​​mM KH2PO4+0.5% triethylamine (pH 4.0)-methanol (20:80); gradient elution program: flow rate 1.0 mL / min, column temperature 25°C, detection wavelength 271 nm, injection volume 10 μl.

4. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: The sodium tanshinone IIA sulfonate waste liquid is obtained by the following method, which comprises the following steps: S1: using a mixed solution of tanshinone IIA, acetic anhydride and an organic solvent as feed solution A, and a mixed solution of sulfuric acid and acetic acid as feed solution B, adding the feed solution A and the feed solution B into a first microreactor to perform a continuous sulfonation reaction to obtain a reaction solution D; S2: adding the reaction solution D and the sodium salt aqueous solution into a second microreactor to perform a continuous salt-forming reaction to obtain a reaction solution E; S3: filtering the reaction solution E and taking the filtrate; In S1, the retention volume of the first microreactor is 40-60 mL; the flow rate of the feed liquid A pumped into the first microreactor is 8-10 mL / min; the flow rate of the feed liquid B pumped into the first microreactor is 0.5-1.5 mL / min; the residence time of the continuous sulfonation reaction is 2-8 min; the molar ratio of tanshinone IIA to acetic anhydride in the feed liquid A is 1:(6-10); the reaction molar ratio of tanshinone IIA to the sulfuric acid is 1:(4-8); the temperature of the continuous sulfonation reaction is 20-30°C; the feed liquids A and B are simultaneously pumped into the first microreactor by the first pump and the second pump, mixed and subjected to the continuous sulfonation reaction; the material of the first pump is one or more of Hastelloy, stainless steel, PEEK and modified polytetrafluoroethylene; the material of the second pump is PEEK and / or modified polytetrafluoroethylene; In S2, the retention volume of the second microreactor is 5-15 mL; the flow rate of the sodium salt aqueous solution pumped into the second microreactor is 5-15 mL / min; the temperature of the continuous salt-forming reaction is 20-30° C.; the sodium salt aqueous solution is pumped into the second microreactor by a third pump to perform a continuous salt-forming reaction with the reaction solution D; the material of the third pump is one or more of Hastelloy, stainless steel, PEEK and modified polytetrafluoroethylene; Preferably, it satisfies one or more of the following conditions: (1) In S1, the retention volume of the first microreactor is 50 mL; (2) In S1, the flow rate of the feed liquid A pumped into the first microreactor is 9 mL / min; (3) In S1, the feed liquid B is pumped into the first microreactor at a flow rate of 1 mL / min; (4) In S1, the residence time of the continuous sulfonation reaction is 5 min; (5) In S1, the molar ratio of tanshinone IIA to acetic anhydride in the feed solution A is 1:8; (6) In S1, the reaction molar ratio of the tanshinone IIA and the sulfuric acid is 1:6; (7) In S1, the temperature of the continuous sulfonation reaction is 25°C; (8) In S2, the retention volume of the second microreactor is 10 mL; (9) In S2, the flow rate of the sodium salt aqueous solution pumped into the second microreactor is 10 mL / min; (10) In S2, the temperature of the continuous salt-forming reaction is 25°C; (11) In S1, the organic solvent is an organic solvent that can dissolve tanshinone IIA and acetic anhydride at the same time, such as dichloromethane; (12) In S1, the molar ratio of sulfuric acid to acetic acid in the feed liquid B is 1:(3-5), for example, 1:3.9; (13) In S1, the sulfuric acid is concentrated sulfuric acid, for example, a concentrated sulfuric acid aqueous solution with a concentration of 15 to 20 mol / L, and the concentration of the acetic acid is 15 to 20 mol / L, for example, 17.5 mol / L; (14) In S1, the first pump is a horizontal flow pump; (15) In S1, the material of the first pump is modified polytetrafluoroethylene; (16) In S1, the second pump is a horizontal flow pump; (17) In S1, the material of the second pump is modified polytetrafluoroethylene; (18) In S2, the sodium salt in the sodium salt aqueous solution is an inorganic acid sodium salt and / or an organic acid sodium salt; preferably an inorganic acid sodium salt; for example, sodium chloride; (19) In S2, the concentration of the sodium salt aqueous solution is greater than 0.7 mol / L, for example, the sodium salt is prepared into a concentration of a saturated aqueous solution; (20) In S2, the residence time of the continuous salt-forming reaction is 0.3-1.2 min, for example, 0.5 min; (21) In S2, the third pump is a horizontal flow pump; the material of the third pump is preferably modified polytetrafluoroethylene; (22) In S3, after the filtrate is separated, the aqueous phase is taken to adjust the pH to 5-6. The pH is preferably adjusted using alkali metal carbonates, alkali metal bicarbonates or alkali metal hydroxides, such as sodium carbonate, and further such as saturated sodium carbonate aqueous solution. Preferably, when adjusting the pH, the temperature of the aqueous phase is 0-20°C, preferably 0-5°C.

5. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The chromatographic column is a dynamic axial compression column; (2) The filler of the chromatographic column is a C18 super water-resistant filler, preferably a C18 Aq super water-resistant filler, and more preferably a UniSil 10-100 C18 Aq super water-resistant filler, such as the UniSil 10-100 C18 Aq super water-resistant filler purchased from Suzhou Nanomicrobiology Technology Co., Ltd.; (3) The pore size of the filler of the chromatographic column is 8-12 nm, for example, 10 nm; (4) The particle size of the filler of the chromatographic column is 90-110 μm, for example, 100 μm; (5) The length of the chromatographic column is 580-620 mm, for example 600 mm; (6) The diameter of the chromatographic column is 45-55 mm, for example, 50 mm; (7) The system used in the preparative chromatography method is an LC-20AP high pressure liquid phase preparative system; (8) The chromatographic column used in the preparative chromatography method is a dynamic axial compression column, the equipment model of which is DAS50*600 and the serial number is SF2310101; (9) The operating pressure of the chromatographic column is 0-10 MPa, for example 5 MPa; (10) The column temperature of the chromatographic column is 5-60°C, for example 25°C; (11) The mobile phase A is a 0.1% by volume triethylamine aqueous solution or a 0.05% by volume triethylamine aqueous solution, for example, a 0.1% by volume triethylamine aqueous solution; (12) The mobile phase B is a mixture of triethylamine and methanol at a volume percentage of 0.1% or a mixture of triethylamine and methanol at a volume percentage of 0.05%, for example, a mixture of triethylamine and methanol at a volume percentage of 0.1%; (13) The flow rate of the mobile phase is 90-110 mL / min or 40-60 mL / min, for example, 100 mL / min or 50 mL / min; (14) The detection method of the preparative chromatography is ultraviolet detection (the detector is Shimadzu LC-20AP), and the detection wavelength of the preparative chromatography is 271 nm; (15) In the preparative chromatography method, the injection volume is preferably 300-600 mL, for example 400 mL; (16) In the first stage, the volume percentage of mobile phase A was 90% and the volume percentage of mobile phase B was 10%; (17) In the second stage, the volume percentage of mobile phase A is 85%, and the volume percentage of mobile phase B is 15%; (18) In the third stage, the volume percentage of mobile phase A is 80%, and the volume percentage of mobile phase B is 20%; (19) In the fourth stage, the volume percentage of mobile phase A is 20%, and the volume percentage of mobile phase B is 80%; (20) In the first stage, the elution time is preferably 3 min or 5 min, for example 3 min; (21) In the second stage, the elution time is preferably 10 min or 20 min, for example 10 min; (22) In the third stage, the elution time is preferably 10 min or 20 min, for example 10 min; (23) In the fourth stage, the elution time is preferably 37 min or 45 min, for example 10 min.

6. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: It satisfies any of the following conditions: Condition 1: In the preparative chromatography, the flow rate is 90-110 mL / min, for example, 100 ml / min; in the first stage, the elution time is 2-4 min; in the second stage, the elution time is 8-12 min; in the third stage, the elution time is 8-12 min; in the fourth stage, the elution time is 35-40 min; Preferably, the condition 1 is: ； Condition 2: In the preparative chromatography, the flow rate is 40-60 mL / min, for example, 50 ml / min; in the first stage, the elution time is 4-6 min; in the second stage, the elution time is 18-22 min; in the third stage, the elution time is 18-22 min; in the fourth stage, the elution time is 40-50 min; Preferably, the condition 2 is: 。 7. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 6, characterized in that: It meets one or more of the following conditions: (1) In the preparative chromatography, when the gradient elution condition is the condition 1, the mobile phase A is a 0.1% by volume triethylamine aqueous solution, and the mobile phase B is a 0.1% by volume mixture of triethylamine and methanol, the eluent with a retention time of 15-20 min, such as 15-18 min, is collected. Preferably, the injection volume is 340-460 mL, such as 400 mL. (2) In the preparative chromatography method, when the gradient elution condition is condition 2, mobile phase A is a 0.1% by volume aqueous solution of triethylamine, and mobile phase B is a 0.1% by volume mixture of triethylamine and methanol, the eluent with a retention time of 20-30 min, such as 25-30 min, is collected. Preferably, the injection volume is 340-460 mL, such as 400 mL; (3) In the purification method, in the preparative chromatography, when the gradient elution condition is condition 1, mobile phase A is a 0.05% by volume aqueous solution of triethylamine, and mobile phase B is a 0.05% by volume mixture of triethylamine and methanol, the eluent with a retention time of 15-25 min, for example, 15-20 min, is collected. Preferably, the injection volume is 340-460 mL, for example, 400 mL.

8. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: The method further comprises the following step: subjecting the eluate obtained by the preparation chromatography method to a second elution.

9. The method for refining the related substances of sodium tanshinone IIA sulfonate according to claim 1, characterized in that: It also includes the following steps: The eluate obtained by preparative chromatography is concentrated to remove the organic phase, and then diluted 1-2 times with water, preferably 1 time with water, and then eluted for the second time; Preferably, in the chromatographic conditions of the second elution, after the organic phase is concentrated and removed, the eluent A is diluted 1-2 times (for example, 1 time), and the preparative chromatography is used to adjust the flow rate to 50 mL / min for the second elution; Preferably, the second elution further comprises concentration and / or drying after completion; the concentration is preferably reduced pressure concentration; the drying is freeze drying; the freeze drying is drying by a freeze dryer; the freeze drying is preferably freeze drying according to a freeze drying curve, for example: pre-freezing at -40°C for 4 hours, heating to 30°C at 1°C / 8 minutes, and keeping warm for 36 hours.

10. A tanshinone IIA sodium sulfonate related substance, characterized in that: Its structural formula is: .

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