CN114656527A

CN114656527A - 4- (dihydroxyacetyl-L-serine) -goserelin, preparation method and application thereof

Info

Publication number: CN114656527A
Application number: CN202210300430.XA
Authority: CN
Inventors: 付婷婷; 白景; 贺晓艳; 薛英
Original assignee: Shandong Luye Pharmaceutical Co Ltd
Current assignee: Shandong Luye Pharmaceutical Co Ltd
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-06-24

Abstract

The invention provides a new compound and a preparation method and application thereof; in particular to a novel compound which is 4- (dihydroxyacetyl-L-serine) -goserelin, a goserelin long-acting preparation containing less than 1 weight percent of 4- (dihydroxyacetyl-L-serine) -goserelin, and an application of the 4- (dihydroxyacetyl-L-serine) -goserelin as a reference substance in impurity detection of the goserelin long-acting preparation.

Description

4- (dihydroxyacetyl-L-serine) -goserelin and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel compound, a preparation method and application thereof, wherein the novel compound is 4- (dihydroxyacetyl-L-serine) -goserelin.

Background

Goserelin (Goserelin) is a synthetic, luteinizing hormone-releasing hormone analog that, upon prolonged use, inhibits the secretion of luteinizing hormone by the pituitary gland, causing a decrease in male serum testosterone and female serum estradiol, which is reversible upon withdrawal. The testosterone concentration of male patients can be reduced to the castration level about 21 days after the first medication, and in the treatment process of 1 medication every 28 days, the testosterone concentration is always kept in the castration concentration range, and the testosterone inhibition can make most prostate tumors regress and improve symptoms. Female patients are inhibited in serum estradiol concentrations about 21 days after initial administration and maintained at postmenopausal levels every 28 days thereafter. This inhibition is associated with hormone-dependent breast cancer, endometriosis.

At present, the marketed goserelin long-acting injection is a long-acting implant, and the action time can be maintained for one month or three months. The blood concentration of the long-acting injection of goserelin is relatively stable, but the injection of the implant needs special equipment and professional personnel for administration. Second, the implant is more traumatic to the patient and therefore less compliant. Therefore, the development of goserelin long-acting microsphere preparation and other improved long-acting injection is necessary, and related domestic product research and declaration exists.

Although the goserelin long-acting preparation is clinically used for years, various side effects such as rash and occasional slight congestion at an injection part are still common; male patients may have reactions such as flushing, decreased libido, swollen and tender breasts, temporary exacerbation of skeletal pain, urethral obstruction, spinal cord compression, etc.; female patients have flushing, hyperhidrosis, decreased libido, headache, depression, vaginal dryness, hemorrhage, and breast size change; patients with endometriosis may develop irreversible amenorrhea after administration.

The adverse reaction generated by the medicine in clinical use is not only related to the pharmacological activity of the medicine, but also has a great relationship with impurities in the medicine. Therefore, the research on impurities is carried out in a standardized way, and the quality and the safety of the medicines on the market are directly related. Management of impurities of pharmaceutically active substances can be greatly enhanced by knowing the chemical structure and synthetic route of the impurities and by identifying parameters that affect the content of impurities in the final product. Establishing quality standard for impurity monitoring requirement of medicinal active substance, and determining proper separation and detection conditions to control impurities; in the quality standard, the currently generally adopted impurity detection methods are mainly liquid chromatography and the like.

The goserelin long-acting injection usually adopts polylactide-co-glycolide (PLGA) as a long-acting release auxiliary material, and PLGA is approved by drug administration as a carrier material of a drug delivery system for a human body, and is widely used for the research of the slow-release long-acting injection. In the preparation of long-acting preparations and long-term storage periods of goserelin, the research on the generation of related impurities of goserelin after auxiliary materials are added and during the storage period of the goserelin is lacked, and the research on impurity components is urgently needed to discover in clinical use of related products.

Disclosure of Invention

The invention aims to provide a novel compound, a preparation method and application thereof. In particular to a novel compound which is 4- (dihydroxyacetyl-L-serine) -goserelin.

The invention provides specific compounds as follows: the compound is 4- (dihydroxyacetyl-L-serine) -goserelin, and the product sequence is as follows: H-Glp-His-Trp-Ser (glycol-glycol) -Tyr-D-Ser (tBu) -Leu-Arg-Pro-AzaGly-NH₂Having the formula:

the compound can be prepared by the following method:

step 1: weighing goserelin, adding glycolide into the goserelin, adding a DMF solvent, and reacting at room temperature.

Step 2: separating the reaction liquid in the step 1 by preparative high performance liquid chromatography, taking phosphoric acid aqueous solution-acetonitrile as a mobile phase, performing gradient elution with the acetonitrile ratio of 20-40%, collecting chromatographic peaks with specific retention time, and retaining related eluents.

And step 3: and (3) taking reverse phase silica gel, uniformly mixing with methanol, stirring, homogenizing, filling into a column, replacing the methanol with purified water, adding the eluent obtained in the step (2) into a reverse phase silica gel chromatographic column by using a peristaltic pump, adjusting the rotating speed of the peristaltic pump, and closing the peristaltic pump after all the eluent obtained in the step (2) passes through the chromatographic column. The peristaltic pump was turned on, the column was eluted with methanol, and all of the methanol solution passed through the column was collected. Rotary evaporation yielded a liquid.

And 4, step 4: and (4) freeze-drying the liquid obtained in the step (3) to obtain white solid powder.

In the method, the relevant chromatographic conditions of the preparative high performance liquid chromatography in the step 2 are as follows; a chromatographic column: phenomenex, luna 5 μm, 250X 21.20mm, S/N: 443257-2, flow rate 20ml/min, detection wavelength 220 nm.

In the method, the phosphoric acid aqueous solution-acetonitrile mobile phase in the step 2 is 0.5% phosphoric acid aqueous solution-acetonitrile as a mobile phase.

The specific retention time collected in step 2 of the above method means that the retention time is 14.03 min.

In the method, the rotating speed of the peristaltic pump is adjusted in the step 3, namely the liquid outflow speed of the chromatographic column is controlled to be 1200ml/h

In the method, the rotary evaporation is carried out in the step 3 under the conditions that the temperature is 26-28 ℃ and the vacuum degree is less than or equal to 0.6 Mpa.

The abbreviations correspond to the chinese names, DMF: n, N-dimethylformamide.

The compound provided by the invention is applied as a reference substance in impurity detection of a goserelin long-acting preparation.

The invention provides a method for determining impurity content of a goserelin long-acting preparation, which adopts a liquid chromatography analysis method and adopts the compound as a reference substance.

The method for determining the impurity content of the goserelin long-acting preparation comprises the steps of dissolving the compound in a solution to prepare a reference solution, dissolving the goserelin long-acting preparation in the solution to prepare a test solution, analyzing by adopting a liquid chromatography to obtain liquid chromatograms of the reference solution and the test solution, comparing the peak-off time in the liquid chromatograms of the reference solution and the test solution to determine that the test solution contains the compound, and determining the weight percentage of the compound in the goserelin long-acting preparation according to an external standard method.

The compounds of the invention are at least 80% pure, preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure, as determined by HPLC or UPLC.

The invention also provides a goserelin implant or microsphere preparation and other long-acting preparations, wherein the goserelin implant or microsphere preparation and other long-acting preparations contain less than 1% of the compound by weight.

Drawings

FIG. 1 is a H-NMR spectrum of a compound

FIG. 2 is a C-NMR spectrum of a compound

FIG. 3 is a compound first mass spectrum (MS plot).

FIG. 4 is a compound secondary mass spectrum (MSMS graph)

FIG. 5 is a chromatogram of a control solution

FIG. 6 is a comparison of the peak time chromatograms of the test solutions.

FIG. 7 is a chromatogram of a test sample of a Gorgorelin microsphere preparation accelerated for 0 month

FIG. 8 is a chromatogram of experimental sample for accelerating the Gorgorelin microsphere preparation for 3 months

FIG. 9 is a chromatogram of a test sample of a Gorgorelin microsphere preparation accelerated for 6 months

Detailed Description

The present invention is further illustrated by the following examples and test examples, but is not limited thereto in any way.

Example 14 preparation of (dihydroxyacetyl-L-serine) -goserelin

Step 1: to a 10ml vial of penicillin, 500mg of goserelin, 50mg of glycolide and 5ml of DMF solvent were added and the mixture was reacted at room temperature for 24 hours.

Step 2: separating the reaction liquid in the step 1 by preparative high performance liquid chromatography (chromatographic column: phenomenex, luna 5 μm, 250X 21.20mm, S/N: 443257-2, flow rate 20ml/min, detection wavelength 220nm), gradient eluting with 0.5% phosphoric acid water solution-acetonitrile as mobile phase, acetonitrile ratio 20% -40%, collecting chromatographic peak with retention time 14.03min, and collecting 750ml of eluent.

And step 3: and (3) taking reverse phase silica gel, uniformly mixing with methanol, stirring, homogenizing, filling into a column, replacing the methanol with purified water, adding the eluent obtained in the step (2) into a reverse phase silica gel chromatographic column by using a peristaltic pump, adjusting the rotating speed of the peristaltic pump, controlling the liquid outflow speed of the chromatographic column to be about 1200ml/h, and closing the peristaltic pump after all the eluent obtained in the step (2) passes through the chromatographic column. Measuring 600ml of methanol, opening a peristaltic pump, eluting the chromatographic column with methanol, and collecting all methanol liquid passing through the chromatographic column. And (3) performing rotary evaporation (the temperature is 26-28 ℃, and the vacuum degree is less than or equal to 0.6Mpa) to obtain 2ml of liquid.

And 4, step 4: the liquid obtained in step 3 was freeze-dried to obtain 120mg of a white solid powder.

According to the structural characteristics, a plurality of analysis technical means are adopted for structure confirmation, wherein MS is used for confirming the molecular weight of the product, MS/MS is used for confirming the sequence of the product, NMR is used for confirming the connection condition of corresponding functional groups of the product and the primary structure of the corresponding functional groups of the product, and amino acid component analysis is used for confirming the ratio of each amino acid. Compounds in DMSO-d using nuclear magnetic resonance assay₆In solution¹H and¹³c NMR spectra were assigned, and NMR data are shown in Table 1, wherein FIG. 1 is an H-NMR nuclear magnetic resonance spectrum, and FIG. 2 is a C-NMR nuclear magnetic resonance spectrum.

The primary mass spectrum is shown in figure 3, and the secondary mass spectrum is shown in figure 4.

TABLE 1 NMR spectra attribution

The nuclear magnetic resonance structure is numbered as follows

Sequence analysis

The amino acid sequence of the product is determined by an Agilent Q-TOF mass spectrometer, ion detection is carried out on a quadrupole, and corresponding fragment ions are obtained by collision induced dissociation after parent ions are screened. Obtaining a secondary mass spectrum of selected ions by TOF analysis, wherein the secondary mass spectrum comprises b ion fragment information and y ion fragment information, matching corresponding fragment ions with a sequence, and testing the sequence of a sample to be' H-Glp-His-Trp-Ser (glycol-glycol) -Tyr-D-Ser (tBu) -Leu-Arg-Pro-AzaGly-NH₂", consistent with theory. See table below for sequence analysis.

TABLE 2 sequence analysis Table

Amino acid ratio

The amino acid ratio of the product is measured by weighing about 1mg of the product, placing the product into a vessel, placing the product into a cracking bottle with 2ml of 6mol/L hydrochloric acid solution (containing 0.1% phenol), charging nitrogen, sealing the bottle, hydrolyzing at 110 ℃ for 24 hours, cooling, unsealing, drying at 105 ℃ for about 1 hour till the product is dry, adding 1000ul of water for dissolving to be used as a sample solution, measuring according to a proper amino acid analysis method, taking one sixth of the sum of the molar concentrations of histidine, tyrosine, arginine, leucine, proline, serine and glutamic acid as 1, and calculating the relative proportion of each amino acid.

Amino acids	Theoretical value	Standard of merit	Measured value
				Histidine
	1	0.9～1.1	1.0
				Tyrosine	1	0.9～1.1	1.0
Arginine	1	0.9～1.1	1.0
				Leucine	1	0.9～1.1	1.0
Proline	1	0.9～1.1	1.0
				Serine	2	1.6～2.2	1.6
Glutamic acid	1	0.9～1.1	1.0

Test example 14 application of (dihydroxyacetyl-L-serine) -goserelin as a control in the content of impurities in goserelin microsphere formulations

Taking about 100mg (about equivalent to 4mg of goserelin) of goserelin acetate sustained-release microspheres for injection, precisely weighing, placing in a 10ml measuring flask, adding 2ml of acetonitrile, performing ultrasonic treatment until no agglomeration exists, adding 4.7ml of 0.1% acetic acid water (taking 1ml of glacial acetic acid and diluting with water to 1000ml), performing ultrasonic treatment for 10min, adding 0.1% acetic acid water for dilution to a scale, shaking uniformly, taking a proper amount to place in a centrifuge tube for centrifugation, and taking supernatant as a sample solution; accurately weighing an appropriate amount of impurity reference substances, and adding acetonitrile: 0.1% acetic acid solution (2:8) was dissolved and quantitatively diluted to give about 0.02mg per 1ml of solution as a control solution.

Chromatographic conditions are as follows:

chromatography column (Waters acquisition UPLC Peptide CSH C1815 cm × 2.1mm, 1.7 μm,) using octadecyl silica gel as packing; taking 0.5% phosphoric acid water solution (5 ml of phosphoric acid is taken and diluted to 1000ml by water) as a mobile phase A, taking 0.5% phosphoric acid acetonitrile solution (5 ml of phosphoric acid is taken and diluted to 1000ml by acetonitrile) as a mobile phase B, and carrying out gradient elution according to the following table; the column temperature is 50 ℃; flow rate: 0.3ml/min, the detection wavelength is 220nm, and the theoretical plate number is not less than 5000 in terms of goserelin. Injecting 5 μ l of each of the reference solution or the sample solution into an ultra high performance liquid chromatograph.

Mobile phase elution gradient

Time(min)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
10	82	18
			30	82	18
40	79	21
			55	60	40
55.5	90	10
			65	90	10

And (3) detecting the content of the goserelin microsphere preparation sample, wherein the attached figure 5 is a chromatogram of a reference solution, and the attached figure 6 is a chromatogram of a test solution compared with the peak time. The weight percentage of 4- (dihydroxyacetyl-L-serine) -goserelin related to 3 batches of microsphere preparations is below 1% calculated by an external standard method.

Test example 2 accelerated test of goserelin microsphere preparation to determine impurity content

According to the method and the requirements of relevant accelerated experiments on the 'guiding principle of raw material drug and preparation stability test' in 'China pharmacopoeia 2020 edition', the accelerated experiments are carried out on the goserelin acetate sustained-release microspheres, and the goserelin acetate sustained-release microspheres are placed for 6 months under the conditions of 25 ℃ and 2 ℃ and 60% relative humidity and 5%. The equipment can control the temperature of 2 ℃ and the relative humidity of 5 percent and can monitor the real temperature and the real humidity. Samples were taken at the end of each of 0, 1, 2, 3 and 6 months during the test period and examined according to stability stress examination. And the change of the related impurities is inspected, and the detection method of the related impurities comprises the following steps:

taking about 100mg (about equivalent to 4mg of goserelin) of goserelin acetate sustained-release microspheres for injection, precisely weighing, placing in a 10ml measuring flask, adding 2ml of acetonitrile, performing ultrasonic treatment until no agglomeration exists, adding 0.1% acetic acid water (taking 1ml of glacial acetic acid, diluting with water to 1000ml)4.7ml of ultrasonic treatment for 10min, adding 0.1% acetic acid water for dilution to scale, shaking up, taking a proper amount of the mixture, placing in a centrifuge tube for centrifugation, and taking supernatant as a sample solution; accurately weighing an appropriate amount of impurity reference substances, and adding acetonitrile: 0.1% acetic acid solution (2:8) was dissolved and quantitatively diluted to give about 0.02mg per 1ml of solution as a control solution.

Chromatographic conditions are as follows:

chromatography column (Waters acquisition UPLC Peptide CSH C1815 cm × 2.1mm, 1.7 μm,) using octadecyl silica gel as packing; gradient elution was performed using 0.5% phosphoric acid aqueous solution (5 ml phosphoric acid, diluted to 1000ml with water) as mobile phase a and 0.5% phosphoric acid acetonitrile solution (5 ml phosphoric acid, diluted to 1000ml with acetonitrile) as mobile phase B according to the following table; the column temperature is 50 ℃; flow rate: 0.3ml/min, the detection wavelength is 220nm, and the theoretical plate number is not less than 5000 in terms of goserelin. 5 mul of each of the reference solution or the test solution is injected into an ultra high performance liquid chromatograph.

Mobile phase elution gradient

The accelerated test of the goserelin microsphere preparation shows that the content of the goserelin is detected at 0 month, 1 month, 2 months, 3 months and 6 months of the accelerated test, and the weight percentage of the related 4- (dihydroxyacetyl-L-serine) -goserelin in the microsphere preparation is below 1 percent according to the calculation of an external standard method, so that the requirement of the product stability is met.

Fig. 7 is a chromatogram of a test sample of a goserelin microsphere preparation accelerated for 0 month, fig. 8 is a chromatogram of a test sample of a goserelin microsphere preparation accelerated for 3 months, fig. 9 is a chromatogram of a test sample of a goserelin microsphere preparation accelerated for 6 months, and it can be seen from the drawings that the content of 4- (dihydroxyacetyl-L-serine) -goserelin in the accelerated test continuously increases along with the time increase, which requires that the content of 4- (dihydroxyacetyl-L-serine) -goserelin must be monitored during the production and sale of goserelin microsphere preparation products, particularly during the long-term storage, so as to ensure the reliability and stability of the products.

Claims

1. A compound having the formula:

2. a process for the preparation of a compound according to claim 1, characterized in that the process steps are as follows:

step 1: weighing a proper amount of goserelin, adding glycolide into the goserelin, adding a DMF (dimethyl formamide) solvent, and reacting at room temperature;

step 2: separating the reaction liquid in the step 1 by preparative high performance liquid chromatography, taking phosphoric acid aqueous solution-acetonitrile as a mobile phase, performing gradient elution with the acetonitrile ratio of 20-40%, collecting chromatographic peaks with specific retention time, and retaining related eluents;

and 3, step 3: and (3) taking reverse phase silica gel, uniformly mixing with methanol, stirring, homogenizing, filling into a column, replacing the methanol with purified water, adding the eluent obtained in the step (2) into a reverse phase silica gel chromatographic column by using a peristaltic pump, adjusting the rotating speed of the peristaltic pump, and closing the peristaltic pump after all the eluent obtained in the step (2) passes through the chromatographic column. The peristaltic pump was turned on, the column was eluted with methanol, and all of the methanol solution passed through the column was collected. Rotary evaporation to obtain liquid;

3. The method for preparing a compound according to claim 2, wherein the preparative high performance liquid chromatography of the step 2 is performed under the relevant chromatographic conditions; a chromatographic column: phenomenex, luna 5 μm, 250X 21.20mm, S/N: 443257-2, flow rate 20ml/min, detection wavelength 220 nm.

4. The method for preparing a compound according to claim 2, wherein the mobile phase of phosphoric acid aqueous solution-acetonitrile is 0.5% phosphoric acid aqueous solution-acetonitrile.

5. The method for producing a compound according to claim 2, wherein the collection-specified retention time is 14.03 min.

6. The method for preparing compounds according to claim 2, wherein the adjustment of the rotation speed of the peristaltic pump is performed to control the liquid outflow rate of the chromatographic column to 1200 ml/h; preferably, the rotary evaporation is carried out under the conditions that the temperature is 26-28 ℃ and the vacuum degree is less than or equal to 0.6 Mpa.

7. Use of a compound according to any one of claims 1 to 6 as a control in the detection of impurities in a goserelin depot.

8. A method for determining the impurity content of a goserelin depot by liquid chromatography, characterized in that a compound according to any one of claims 1 to 6 is used as a control.

9. The method according to claim 8, wherein the compound according to any one of claims 1 to 6 is dissolved in a solution to prepare a control solution, the goserelin depot is dissolved in a solution to prepare a test solution, a liquid chromatogram of the control solution and the test solution is obtained by analyzing the solutions by liquid chromatography, the test solution is determined to contain the compound according to any one of claims 1 to 6 by comparing the time of appearance of peaks in the liquid chromatogram of the control solution and the test solution, and the weight percentage of the compound according to any one of claims 1 to 6 in the goserelin depot is determined by an external standard method.

10. A goserelin depot characterized in that the goserelin depot comprises less than 1% by weight of a compound according to any one of claims 1 to 6.