Method for removing organic solvent residue in chlorine photosensitizer bulk drug

Abstract

The invention discloses a method for removing solvent residues in a dihydroporphin photosensitizer bulk drug, which relates to the field of medicine, and is characterized in that a synthesized dihydroporphin photosensitizer crude product is subjected to silica gel column chromatography purification, concentration, redissolution, concentration and reduced pressure drying to obtain the dihydroporphin photosensitizer bulk drug. The detection shows that the residual solvents acetone and methanol of the obtained dihydroporphin photosensitizer raw material drug do not exceed 0.1%, the residual solvents dichloromethane do not exceed 0.01%, and the residual solvents are far lower than the residual solvent limit values specified in the Chinese pharmacopoeia: the acetone is less than or equal to 0.5%, the dichloromethane is less than or equal to 0.06%, and the methanol is less than or equal to 0.3%, the dihydroporphin photosensitizer raw material drug is powder with uniform color, the purity is high, the moisture is low, and the product quality is obviously improved. According to the method, through mild conditions and simple operation, efficient removal of organic solvent residues is achieved, the chemical stability of the dihydroporphin photosensitizer is not affected, and the cost is low.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a method for removing residual solvent of a chlorin photosensitizer raw material. Background Art

[0002] Photosensitizer, also known as sensitizer, sensitizer, and photocrosslinker, is one of the three elements of photodynamic therapy (PDT) and plays a vital role in the field of PDT. The quality of photosensitizer will directly affect the safety of the patients and the efficacy of PDT. Chlorin photosensitizers are derivatives of chlorophyll a with good light absorption properties and can be used as a good type of photosensitizer. Currently, many chlorin photosensitizers have been approved for marketing or entered the clinical research stage at home and abroad.

[0003] Chlorin photosensitizers have a chlorin structural core, and their chemical properties are mostly unstable, and they are prone to degradation reactions under various conditions such as light and high temperature. Therefore, light and high temperature should be avoided during the preparation of such drugs or intermediates. The existing methods are limited by the drying temperature, resulting in excessive residual organic solvents, which affects the stability and drug safety of chlorin photosensitizers.

[0004] How to dry chlorin photosensitizers while ensuring their quality has become an urgent problem to be solved in the current pharmaceutical industry, especially in chlorin photosensitizer manufacturers. After searching, no method for removing organic solvent residues from chlorin photosensitizers has been reported.

[0005] Based on the defects and shortcomings of the prior art, it is necessary to provide a drying method for dihydrochlorin photosensitizers that has a simple process, can be operated continuously, and is suitable for industrial production to control the solvent residue of the product, thereby obtaining high-quality dihydrochlorin photosensitizer raw materials. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for removing organic solvent residues in dihydrochlorin photosensitizer raw materials. Through mild conditions and simple operations, efficient removal of organic solvent residues is achieved without affecting the chemical stability of dihydrochlorin photosensitizers and at a low cost.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for removing organic solvent residues from chlorin-type photosensitizer raw materials, wherein the chlorin-type photosensitizer refers to a photosensitizer drug containing chlorin in the chemical structure and the mechanism of action of which is mainly photodynamic therapy, including chlorin e6, chlorin e6-15-ethyl ester and analogs thereof, comprising the following steps:

[0008] Step A: Purify the crude product of the dihydrochlorin photosensitizer by separation gradient or isocratic elution via silica gel column chromatography, and collect the target fractions.

[0009] The crude product of the chlorin photosensitizer described in the present invention can be prepared by methods disclosed in the prior art, such as the preparation methods described in patent documents such as CN102068428A, CN113461697A, CN111943954A, CN103396419A and CN107987081A. These methods usually use chlorophyll a as a raw material to prepare crude products of chlorin photosensitizers through chemical reactions such as hydrolysis, esterification, and reduction. However, such crude products often contain a high amount of residual organic solvents during the preparation process, such as acetone, methanol, and dichloromethane, and these residual solvents may affect the stability and drug safety of the photosensitizer. To this end, the present invention provides an improved purification method to effectively remove these residual solvents. The "crude product of the chlorin photosensitizer prepared according to the prior art" described in claim 1 of the present invention refers to a chlorin photosensitizer prepared by the method described in the above patent documents or a method known in the art.

[0010] Step B, concentrating the fraction obtained in step A to dryness under reduced pressure to obtain a primary dry product of the dihydrochlorin photosensitizer.

[0011] Step C, adding the primary dry product of the dihydrochlorin photosensitizer obtained in step B into an aqueous organic solvent for redissolution.

[0012] Step D, the reconstituted solution obtained in step C is concentrated to dryness under reduced pressure again to obtain a secondary dry product of the dihydrochlorin photosensitizer.

[0013] Step E, drying the secondary dried product of the chlorin photosensitizer obtained in step D under reduced pressure to obtain a chlorin photosensitizer API with low residual solvent.

[0014] Preferably, the silica gel column chromatography in step A is gradient eluted with a mixed solvent of dichloromethane-methanol-acetone in a volume ratio of 100:0:0 to 100:50:50, and monitored by thin layer chromatography or a 400nm ultraviolet detector.

[0015] Preferably, the reduced pressure concentration in step B and step D is carried out in a rotary evaporator, the water bath temperature is 20-35° C., and the pressure is -0.1 to -0.08 MPa.

[0016] Preferably, the aqueous organic solvent in step C is a mixed solvent consisting of water, dichloromethane and methanol in a certain proportion. Furthermore, the amount of water used is 10%-20% of the weight of the first dry product, the volume of dichloromethane used is 20-30 times the weight of the first dry product, and the volume of methanol used is 2-3 times the weight of the first dry product.

[0017] Preferably, the reduced pressure drying in step E is carried out at a pressure of -0.1 to -0.08 MPa and a temperature of 20 to 35° C. for 3 to 5 hours, with nitrogen or air being introduced for replacement 2 to 3 times during the drying process.

[0018] The numerical ranges of the concentration and drying temperatures are obtained by the inventors of the present application through a large number of experiments, which fully consider the solvent selected for the purification of the crude product of the dihydrochlorin photosensitizer and do not affect the quality of the dihydrochlorin photosensitizer.

[0019] Due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention makes full use of the physical property that the boiling point of water is higher than that of organic solvents such as dichloromethane, methanol, acetone, etc., and finds a method for effectively removing the above residual solvents based on a large number of experiments: the silica gel column effluent of the crude dihydrochlorin photosensitizer is concentrated to dryness, and then a certain proportion of water-containing organic solvent is added to dissolve it again, and then concentrated again and dried under reduced pressure to obtain the dihydrochlorin photosensitizer raw material medicine, and the whole process is simple to operate, low in implementation cost, and mild in conditions; after testing, the residual solvents of acetone and methanol in the obtained dihydrochlorin photosensitizer raw material medicine are both less than 0.1%, and the dichloromethane is less than 0.01%, which are far lower than the residual solvent limit values ​​specified in the Chinese Pharmacopoeia: acetone≤0.5%, dichloromethane≤0.06%, methanol≤0.3%, and the appearance is a powder with uniform color, high purity, low water content, and the product quality is significantly improved.

[0020] The present invention realizes efficient removal of organic solvent residues through mild conditions and simple operations without affecting the chemical stability of the chlorin photosensitizer. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] The present invention adopts the solvent residual determination method according to the 2020 edition of the Chinese Pharmacopoeia and the ICH Q3C guidelines.

[0023] The details are as follows:

[0024] ①Chromatographic conditions

[0025] A. Chromatographic conditions

[0026]

[0027] B. Headspace Conditions

[0028]

[0029] ②Solution preparation

[0030] Blank solution: Pipette 5 ml of dimethyl sulfoxide (DMSO) into a 20 ml headspace bottle as a blank solution.

[0031] Reference substance stock solution: Take 60 mg of dichloromethane (DCM), 300 mg of methanol (MeOH), 500 mg of ethanol (EtOH), and 500 mg of acetone (Acetone), accurately weigh them, place them in a 50 ml volumetric flask, and dilute to the scale with dimethyl sulfoxide (DMSO) as the reference substance stock solution.

[0032] Reference solution: Accurately measure 2.0 ml of the reference stock solution and place it in a 100 ml volumetric flask, dilute to the mark with DMSO, shake well, and use it as the reference solution. The concentrations of each solvent are: dichloromethane (24 μg / ml), methanol (120 μg / ml), ethanol (200 μg / ml), acetone (200 μg / ml). Accurately measure 5 ml of the reference solution and place it in the empty bottle, and tighten the cap.

[0033] Test solution: Take about 200 mg of the chlorin photosensitizer API, weigh accurately, place in an empty bottle, accurately add 5.0 ml of dimethyl sulfoxide, press the bottle cap, dissolve and shake well, as the test solution. Prepare 2 portions in parallel.

[0034] ③System applicability

[0035] The RSD value of the peak area of ​​each residual solvent peak in the chromatograms of 5 consecutive reference solution should not be greater than 5%; the separation between each solvent peak should not be less than 1.5, and the number of theoretical plates should not be less than 2000.

[0036] ④Testing process

[0037] After the chromatographic system is stable, the residual solvents dichloromethane, methanol, ethanol and acetone in the dihydrochlorin photosensitizer raw materials are determined according to the following procedures:

[0038] Step 1: Take blank solvent and inject until there is no interference;

[0039] Step 2: Take the reference solution and inject 5 times continuously;

[0040] Step 3: Take the test sample solution and inject 1 injection each time.

[0041] ⑤Calculation

[0042] The residual solvent content was calculated by external standard method according to the following formula:

[0043]

[0044] Where: Aspl: peak area of ​​known solvent in sample solution;

[0045] Astd: average of the known solvent peak areas in the reference solution;

[0046] Wstd: the amount of known solvent in the reference stock solution (mg);

[0047] Wspl: sample weight (mg).

[0048] Example 1

[0049] Take 10g of crude product of dihydrochlorin e6-15-ethyl ester, add 20g of silica gel and mix well, use BiotageIsolera One rapid preparative chromatograph, use 330g of silica gel (300-400 mesh) as stationary phase. The mobile phase is solvent A: dichloromethane; solvent B: methanol: acetone = 1:1 (V / V) mixed solvent. Flow rate: 60ml / min, detection wavelength 400nm, gradient elution program and liquid volume are as follows:

[0050]

[0051] By TLC detection, it was confirmed that the target compound eluate was collected and concentrated under reduced pressure (rotary evaporator, 25°C water bath, -0.1MPa) to dryness to obtain 6g of dihydrochlorin e6-15-ethyl ester primary dry product. The primary dry product was placed in a flask, and 120ml of dichloromethane, 12ml of methanol and 0.6ml of purified water were added in sequence, and stirred for 10min until the solid was completely dissolved to obtain a reconstituted solution. The obtained reconstituted solution was concentrated under reduced pressure (rotary evaporator, 25°C water bath, -0.1MPa) to dryness and then scraped out and dried in a vacuum drying oven for 3 hours (drying conditions -0.1MPa, 25°C, nitrogen was introduced 3 times in the middle) to obtain a dark green powder dihydrochlorin e6-15-ethyl ester raw material drug, the content of which was 99.1%, the water content was 0.8%, and the residual solvent met the Chinese Pharmacopoeia standards, as shown in Table 1.

[0052] Table 1 Example 1 Chlorin e6-15-ethyl ester API residual solvent test results

[0053]

[0054] Example 2

[0055] Take 20g of crude product of dihydrochlorin e6-15-ethyl ester, add 40g of silica gel and mix well, use BiotageIsolera One rapid preparation chromatograph, use 330g of silica gel (300-400 mesh) as stationary phase. The mobile phase is solvent A: dichloromethane; solvent B: methanol: acetone = 1:1 (V / V) mixed solvent. Flow rate: 60ml / min, detection wavelength 400nm, gradient elution program and liquid volume are as follows:

[0056]

[0057] By TLC detection, it was confirmed that the target compound eluate was collected and concentrated under reduced pressure (rotary evaporator, 25°C water bath, -0.1MPa) to dryness to obtain 13g of dihydrochlorin e6-15-ethyl ester primary dry product. The primary dry product was placed in a flask, and 325ml of dichloromethane, 35ml of methanol and 2ml of purified water were added in sequence, and stirred for 10min until the solid was completely dissolved to obtain a reconstituted solution. The obtained reconstituted solution was concentrated under reduced pressure (rotary evaporator, 25°C water bath, -0.1MPa) to dryness and then scraped out and dried in a vacuum drying oven for 3.5 hours (drying conditions -0.1MPa, 30°C, nitrogen was introduced 3 times in the middle) to obtain a dark green powder dihydrochlorin e6-15-ethyl ester raw material drug, the content of which was 99.0%, the water content was 0.6%, and the residual solvent met the Chinese Pharmacopoeia standards, as shown in Table 1.

[0058] Table 2 Example 2 Chlorin e6-15-ethyl ester API residual solvent test results

[0059]

[0060] Example 3

[0061] Take 100g of crude dihydrochlorin e6, add 200g of silica gel and mix well, use as the upper layer, use CHEMBOX chromatograph, use 7000g of silica gel (300-400 mesh) as the stationary phase. Perform isocratic elution at a pressure of 10MPa and a flow rate of 1.5L / h. The mobile phase is a mixed solvent of dichloromethane: methanol: acetone = 100:3:3 (V / V / V). By TLC detection, collect about 15L of the eluate containing dihydrochlorin e6-15-ethyl ester, and concentrate under reduced pressure (rotary evaporator, 30℃ water bath, -0.1MPa) to dryness to obtain 66g of dihydrochlorin e6 primary dry product.

[0062] The obtained primary dry product was placed in a flask, and 1650 ml of dichloromethane, 165 ml of methanol and 8 ml of purified water were added in sequence, and the solution was obtained after stirring for 10 min until the solid was completely dissolved. The obtained solution was concentrated under reduced pressure (rotary evaporator, 30°C water bath, -0.1 MPa) until dry, and then scraped out and dried in a vacuum drying oven for 4 hours (drying conditions -0.1 MPa, 30°C, nitrogen was introduced for replacement 3 times in the middle) to obtain a dark green powder of dihydrochlorin e6 raw material drug, the content of which was 98.8%, the water content was 0.9%, and the residual solvent met the Chinese Pharmacopoeia standard, as shown in Table 3.

[0063] Table 3 Example 3 Chlorin e6 API Residual Solvent Detection Results

[0064]

[0065]

[0066] Example 4

[0067] Take 300g of crude dihydrochlorin e6, add 600g of silica gel and mix well, use as the upper layer, use CHEMBOX chromatograph, use 10Kg silica gel (300-400 mesh) as the stationary phase. Perform isocratic elution at a pressure of 10MPa and a flow rate of 2L / h. The mobile phase is a mixed solvent of dichloromethane: methanol: acetone = 100:3:3 (V / V / V). By TLC detection, collect about 30L of the eluate containing dihydrochlorin e6, and concentrate under reduced pressure (rotary evaporator, 30℃ water bath, -0.1MPa) to dryness to obtain 202g of dihydrochlorin e6.

[0068] The obtained primary dry product was placed in a flask, and 6060 ml of dichloromethane, 106 ml of methanol and 28 ml of purified water were added in sequence, and the solution was obtained after stirring for 20 min until the solid was completely dissolved. The obtained solution was concentrated under reduced pressure (rotary evaporator, 30°C water bath, -0.1 MPa) until dry, and then scraped out and dried in a vacuum drying oven for 5 hours (drying conditions -0.1 MPa, 30°C, and replaced with air twice in the middle) to obtain a dark green powder of dihydrochlorin e6 raw material drug, which was tested to have a content of 97.6%, a moisture content of 1.0%, and a residual solvent that met the standards of the Chinese Pharmacopoeia, as shown in Table 4.

[0069] Table 4 Example 4 Chlorin e6 API Residual Solvent Detection Results

[0070]

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0072] Example 5

[0073] The results of residual solvent testing of the raw materials in Examples 1-4 are summarized in Table 5

[0074] Table 5 Summary of residual solvent test results of raw materials in Examples 1-4

[0075]

Claims

1. A method for removing organic solvent residues from chlorin-type photosensitizer APIs, characterized in that: The chlorin photosensitizer refers to a photosensitizer drug containing chlorin in the chemical structure and the mechanism of action of which is mainly photodynamic therapy, including chlorin e6, chlorin e6-15-ethyl ester and its analogs, comprising the following steps: Step A: Purify the crude product of the dihydrochlorin photosensitizer prepared according to the prior art by separation gradient or isocratic elution through silica gel column chromatography, and collect the target fractions. Step B, concentrating the fraction obtained in step A to dryness under reduced pressure to obtain a primary dry product of the dihydrochlorin photosensitizer. Step C, adding the primary dry product of the dihydrochlorin photosensitizer obtained in step B into an aqueous organic solvent for redissolution. Step D, the reconstituted solution obtained in step C is concentrated to dryness under reduced pressure again to obtain a secondary dry product of the dihydrochlorin photosensitizer. Step E, drying the secondary dried product of the chlorin photosensitizer obtained in step D under reduced pressure to obtain a chlorin photosensitizer API with low residual solvent.

2. The method for removing organic solvent residues from chlorin photosensitizer raw materials according to claim 1, characterized in that: In the silica gel column chromatography in step A, gradient elution is performed using a mixed solvent of dichloromethane-methanol-acetone in a volume ratio of 100:0:0 to 100:50:50, and monitoring is performed using thin layer chromatography or a 400 nm ultraviolet detector.

3. The method for removing organic solvent residues from chlorin photosensitizer raw materials according to claim 1, characterized in that: The reduced pressure concentration in step B and step D is carried out in a rotary evaporator, the water bath temperature is 20-35° C., and the pressure is -0.1 to -0.08 MPa.

4. The method for removing organic solvent residues from chlorin photosensitizer APIs according to claim 1, characterized in that: The aqueous organic solvent in step C is a mixed solvent composed of water, dichloromethane and methanol in a certain proportion, the amount of water used is 10%-20% of the weight of the first dry product, the volume of dichloromethane used is 20-30 times the weight of the first dry product, and the volume of methanol used is 2-3 times the weight of the first dry product.

5. The method for removing organic solvent residues from chlorin photosensitizer APIs according to claim 1, characterized in that: The reduced pressure drying in step E is carried out at a pressure of -0.1 to -0.08 MPa and a temperature of 20 to 35° C. for 3 to 5 hours, with nitrogen or air being introduced for replacement 2 to 3 times during the drying process.

6. The method for removing organic solvent residues from chlorin photosensitizer APIs according to claim 1, characterized in that: The chlorin photosensitizer is selected from chlorin e6, chlorin e6-15-ethyl ester or a combination thereof, or a pharmaceutically acceptable analogue thereof.

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