Nemorubicin co-crystal and preparation method thereof

Abstract

The invention relates to a nemorubicin co-crystal and a preparation method thereof. The absolute configuration of a chiral center in a molecule of the nemorubicin co-crystal is {C17 (S), C19 (S), C22 (R), C23 (S), C24 (S), C25 (S) and C30 (S)}. The problem of structure confirmation of the compound in the development process is solved, and a powerful support basis is provided for absolute configuration confirmation of a terminal product PNU-159682. Moreover, the preparation method of the nemorubicin crystal form provided by the invention is simple to operate, the prepared crystal form is high in purity and uniform in particle size distribution, the production cost is reduced, and the purity of the nemorubicin is improved.

Description

Technical Field

[0001] This invention belongs to the field of single crystal preparation technology, specifically relating to a nemoroxib star co-crystal and its preparation method. Background Technology

[0002] Antibody drug conjugates (ADCs) are a novel class of antitumor drugs. Their mechanism of action involves attaching a cytotoxin to an antibody. The antibody recognizes specific antigens on the surface of cancer cells, and the cytotoxin enters the cancer cells via endocytosis, thus delivering the cytotoxin to the target site for targeted therapy of malignant tumors. Compared to traditional small-molecule antitumor drugs, ADCs are more specific and effective due to the targeted recognition of antibodies and the high activity of the cytotoxin. ADCs consist of three parts: an antibody, a cytotoxin, and a linker. The antibody delivers the cytotoxin to tumor tissue, achieving targeted delivery and thus exerting antitumor activity.

[0003] PNU-159682 is a metabolite of the anthracycline neomycin and a Topoisomerase II (TOPO II) inhibitor with excellent cytotoxicity, widely used in the design and synthesis of drug-adjuvant conjugates (ADCs). Currently, commercially available PNU-159682 is an amorphous solid, and no literature or patents have reported its crystal structure and absolute configuration. The synthesis of PNU-159682 involves a long process, the starting materials often have low purity, and the presence of multiple chiral centers makes absolute configuration control difficult. Low chiral purity leads to challenges in absolute configuration characterization, affecting the safety and efficacy of drug conjugates. Nemorubicin is the starting material for PNU-159682 and forms the cornerstone of its molecular skeleton's absolute configuration. Therefore, improving the purity of the starting materials and determining the absolute configuration of their chiral carbons is crucial for confirming the configuration of PNU-159682. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the first objective of this invention is to provide a nemorubicin eutectic.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a nemorubicin co-crystal, the absolute configuration of the chiral center in its molecule being: {C17(S), C19(S), C22(R), C23(S), C24(S), C25(S), C30(S)}.

[0006] Ideally, it belongs to the orthorhombic crystal system, space group P212121.

[0007] Furthermore, its unit cell parameters are

[0008] Ideally, it is a solvent crystal form, and its crystals are chloroform solvates.

[0009] Another objective of this invention is to provide a method for preparing the above-mentioned nemorubicin co-crystal, wherein nemorubicin is cultured as a single crystal using a slow solution evaporation method, a gas-liquid diffusion method, or a slow cooling method.

[0010] Ideally, the nemorubicin sample is mixed with a solvent to form a solution of a certain concentration, the filtrate is collected by filtration and sealed, and a small hole is set at the sealing opening after sealing; the solvent is allowed to evaporate slowly at room temperature; the solvent is a single solvent or a mixed solvent, and the mixed solvent is a mixture of multiple components selected from halogenated hydrocarbons, alcohols, alkanes, ethers and acetonitrile.

[0011] Furthermore, the concentration of the nemorubicin sample in the solvent is 1 mg / ml to 50 mg / ml.

[0012] Another object of the present invention is to provide an application of the above-mentioned nemorubicin co-crystal as a standard or reference.

[0013] Compared with existing technologies, the present invention has the following advantages: The nemorubicin co-crystal of the present invention is the first in the industry to confirm the absolute configuration of nemorubicin, solving the problem of structural confirmation of this compound during the research and development process, and providing strong support for the confirmation of the absolute configuration of the end product PNU-159682; moreover, the preparation method of the nemorubicin crystal form provided by the present invention is simple to operate, the prepared crystal form has high purity and uniform particle size distribution, reduces production costs, and improves the purity of nemorubicin. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating the synthesis process of the nemorubicin co-crystallized product of the present invention.

[0015] Figure 2 This is a structural diagram of the nemorubicin eutectic of the present invention;

[0016] Figure 3 This is the HPLC chromatogram of the nemorubicin co-crystallized product of the present invention;

[0017] Figure 4 This is the MS spectrum of the nemorubicin eutectic of the present invention;

[0018] Figure 5 The ¹H NMR spectrum of the nemorubicin co-crystallized crystal of this invention;

[0019] Figure 6This is a PLM diagram of the nemorubicin eutectic of the present invention;

[0020] Figure 7 Table of approximate room temperature solubility of nemorubicin samples from batch LN405-98;

[0021] Figure 8 PLM images of needle-like crystal samples of the present invention: (a) PLM image of the single crystal sample obtained by cultivation; (b) Micrograph of the single crystal sample measured on the diffractometer loop.

[0022] Figure 9 The crystallographic structure data and structural refinement parameter diagram of the nemorubicin chloroform solvate single crystal of the present invention are shown below.

[0023] Figure 10 A schematic diagram of the asymmetric unit of the single-crystal structure model of the present invention obtained by analysis;

[0024] Figure 11 This is an ORTEP diagram of the atomic thermal vibration ellipsoid of the compound Nemoroboron star in the crystal structure of this invention.

[0025] Figure 12 This is a stereochemical structure diagram of nemorubicin of the present invention;

[0026] Figure 13 This is a schematic diagram of the single-crystal structure model of the nemorubicin chloroform solvate of the present invention.

[0027] Figure 14 This is a diagram showing the connection between the nemorubicin molecule and the adjacent chloroform molecule in this invention;

[0028] Figure 15 This is a summary diagram of hydrogen bond information in the single crystal structure of this invention;

[0029] Figure 16 This is a schematic diagram of the molecular packing structure in the single crystal structure of the present invention (viewed along the crystallographic a-axis direction);

[0030] Figure 17 This is a schematic diagram of the molecular packing structure in the single crystal structure of the present invention (viewed along the crystallographic b-axis direction);

[0031] Figure 18 This is a schematic diagram of the molecular packing structure in the single crystal structure of the present invention (viewed along the crystallographic c-axis direction);

[0032] Figure 19 The calculated XRPD diagram of the single-crystal structure model of the nemorubicin chloroform solvate of this invention is shown below.

[0033] Figure 20 This is an XRPD image of the nemorubicin starting sample of this invention. Detailed Implementation

[0034] This invention discloses a nemorubicin co-crystal with the absolute configuration of its chiral center in the molecule as follows: {C17(S), C19(S), C22(R), C23(S), C24(S), C25(S), C30(S)}. This is the first time in the industry that the absolute configuration of nemorubicin has been confirmed, solving the problem of structural confirmation during the development of this compound and providing strong support for the absolute configuration confirmation of the end product PNU-159682. Furthermore, the method for preparing the nemorubicin crystal form provided by this invention is simple to operate, produces crystals with high purity and uniform particle size distribution, reduces production costs, and improves the purity of nemorubicin.

[0035] It belongs to the orthorhombic crystal system, space group P212121. Its unit cell parameters are... It is a solvent crystal form, and its crystal is a chloroform solvate (i.e., the asymmetric structural unit of the nemorubicin co-crystal consists of one nemorubicin molecule and a total of about 1.5 chloroform solvent molecules, indicating that the nemorubicin crystal is a chloroform solvate).

[0036] The above-mentioned method for preparing nemorubicin co-crystals involves cultivating nemorubicin into single crystals using a slow solution evaporation method, a gas-liquid diffusion method, or a slow cooling method.

[0037] Ideally, the nemorubicin sample is mixed with a solvent to form a solution of a certain concentration, the filtrate is collected by filtration and sealed, and a small hole is set at the sealing opening after sealing; the solvent is allowed to evaporate slowly at room temperature; the solvent is a single solvent or a mixed solvent, and the mixed solvent is a mixture of multiple components selected from halogenated hydrocarbons, alcohols, alkanes, ethers and acetonitrile.

[0038] The alcohol solvent is a C1-C5 monohydric alcohol, preferably any one of methanol, ethanol, n-propanol, and isopropanol; the ether solvent is a C1-C7 ether, preferably any one of diethyl ether, 2-methyltetrahydrofuran, and methyl tert-butyl ether; the halogenated hydrocarbon solvent is preferably dichloromethane, chloroform, etc. The selected solvent is preferably dichloromethane or chloroform. The concentration of the nemorubicin sample in the solvent is 1 mg / ml to 50 mg / ml, preferably 10 to 40 mg / ml.

[0039] The above-mentioned nemorubicin co-crystals are used as standards or reference materials.

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0041] Example 1

[0042] This embodiment provides a method for synthesizing nemorubicin samples for single-crystal culture, such as... Figure 1 As shown, the details are as follows:

[0043] (a)N em -1 synthesis

[0044] In a 30L reactor, 600g of doxorubicin (Adr) (1 eq, 1.03 mol) was dissolved in MeOH (7.2L). While stirring at an internal temperature of 20–25℃, 114.8g of acetylhydrazine (2 eq, 2.07 mol) and TFA (0.2 eq, 12.8 mL) were added. The temperature was raised to 50–55℃ and the reaction proceeded for 4–8 hours. HPLC analysis confirmed complete conversion. The reaction system was then cooled to 25–30℃ to obtain N. em For solutions containing -1, the reaction system proceeds directly to the next step.

[0045] (b)N em -3 synthesis

[0046] The N obtained in the previous step em -1 The reaction solution was cooled to about 5-10℃, and compound N was added. em A dichloromethane solution (1088 g, 8.0 eq, 8.2 mol, with 4 L of dichloromethane added and stirred until homogeneous) was added to the reaction system. NaCNBH3 (4 eq, 4.1 mol, 260.66 g) was added while maintaining the reaction vessel temperature at 5–10 °C. After the reaction was completed according to HPLC monitoring, post-processing was performed. 5 L of saturated NaHCO3 was added dropwise to quench the reaction. 4 L each of water and dichloromethane were added, and the mixture was stirred, extracted, and separated. The aqueous phase was extracted twice with 5 L of dichloromethane each time. The organic phases were combined, concentrated, and evaporated to dryness to obtain 1425 g of crude Nem-3.

[0047] (c) Nemorubici n Sample synthesis

[0048] 1425 g of the crude Nem-3 obtained in the previous step was dissolved in a mixed solvent of acetonitrile (2122 mL) and water (2122 mL). AcOH (1.2 L) was added while maintaining the reaction temperature at 10–15 °C. The reaction was maintained at 10–15 °C for 2–6 h. HPLC analysis confirmed the reaction was complete, followed by post-processing.

[0049] Post-processing: Quench the reaction system with 4L saturated NaHCO3 solution, adjust the pH of the system to 7-8, add 2L DCM to the system, filter with a Buchner funnel, separate the filtrate, take the lower layer, wash the filter cake three times with 2L dichloromethane each time, use the filtrate from each wash to back-extract the aqueous phase, combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate to obtain 1022g of crude product.

[0050] Fine purification by normal phase column chromatography:

[0051] The crude product was dissolved in 2L of dichloromethane and mixed with 600g of 100-200 mesh silica gel. 4.5kg of 300-400 mesh silica gel was added to the sample column. The flow rate was 300ml / min, wavelengths were 234nm and 254nm, and the mobile phase was DCM and MEOH in a ratio of 100:1 to 10:1, using gradient elution. The pure product was collected and concentrated to obtain 306g of Nemorubicin (batch number LN405-98), with a total yield of 51.7% and an HPLC accuracy of 96.6% (e.g., as shown in the image). Figure 3 (As shown). LCMS: 643.64, Detection result: 644.57 [M+H] + (like Figure 4 and Figure 5 (As shown).

[0052] Preparation experiments for culturing Nemorubicin crystal samples:

[0053] like Figure 6 As shown, macroscopically it appears as a brick-red solid powder. Under a polarizing microscope, the sample particles appear as brick-red, flat, rod-shaped crystals, and birefringence can be observed in the particles under the polarizing microscope. LN405-98 batch Nemorubicin n The approximate room temperature solubility of the sample is as follows: Figure 7 As shown in the table (this table measures Nemorubici), n The sample's approximate room temperature solubility in 20 commonly used solvents (this approximate solubility was used in the next step of single-crystal cultivation experiments).

[0054] Nemorubici n Crystal preparation and cultivation scheme and implementation

[0055] Based on the solubility data from the previous step, three cultivation schemes were designed: slow evaporation method, gas-liquid diffusion method, and slow cooling method.

[0056] (1) Slow evaporation method:

[0057] Sample preparation method: Weigh a certain amount of the compound Nemorubicin. nThe solid was placed in a 3mL glass bottle, and the appropriate solvent was added. After appropriate sonication to promote solid dissolution, the solution was filtered. The filtrate was transferred to a 4mL single crystal growth bottle, sealed with a single crystal stopper, and a small hole was punched in the stopper. The single crystal bottle was then placed into a 20mL glass bottle pre-filled with 4mL of PEG-400. The bottle was sealed and placed in a 10℃ biochemical incubator for slow solvent evaporation. The solvent in the single crystal bottle evaporated, and its vapor was absorbed by PEG-400. As the solvent decreased, the crystals grew slowly in the single crystal bottle. A total of 30 slow evaporation experiments were designed, as shown in Tables 1 to 3 below.

[0058] Table 1. Single Crystal Cultivation Experiments Using the Slow Volatilization Method - 1

[0059] Slow Evaporation Method Single Crystal Cultivation Experiment - 1

[0060] Table 2. Single Crystal Cultivation Experiments Using the Slow Evaporation Method - 2

[0061] Table 3. Single Crystal Cultivation Experiments Using the Slow Volatilization Method - 3

[0062] Slow Evaporation Method Single Crystal Cultivation Experiment - 3

[0063]

[0064] (2) Gas-liquid diffusion method:

[0065] Sample preparation method: Weigh a certain amount of the compound Nemorubicin. n The sample was placed in a 3mL glass bottle and dissolved with a certain amount of the corresponding neutral solvent. The resulting sample solution was transferred to a clean 4mL single crystal bottle, which was then sealed with a PE stopper, and a small hole was punched in the stopper. The single crystal bottle was then placed into a 20mL glass bottle pre-filled with 3mL of antisolvent. Finally, the 20mL glass bottle was capped and sealed, and placed in a 10℃ biochemical incubator for gas-liquid diffusion culture experiments. A total of 12 gas-liquid diffusion experiments were designed, as shown in Table 4 below.

[0066] Table 4 Single Crystal Cultivation Experiments Using the Gas-Liquid Diffusion Method

[0067]

[0068] (c) Slow cooling method:

[0069] Sample preparation method: Weigh an appropriate amount of the compound Nemorubicin. nPlace the solid sample in a 3mL glass bottle and add a certain amount of the appropriate solvent. After appropriate ultrasonic agitation to promote solvent dissolution, filter the solution (if the sample dissolves completely, add an appropriate amount of sample to ensure that any undissolved solid sample remains in the system). Transfer the resulting filtrate to a clean 3mL glass bottle. Finally, place the bottle in a 25℃ biochemical incubator for slow cooling. Temperature program: A total of 6 slow cooling method experiments were designed, as shown in Table 5 below.

[0070] Table 5 Single Crystal Cultivation Experiment Using Slow Cooling Method

[0071] Single crystal growth experiment using slow cooling method

[0072]

[0073] Nemorubici n Crystal analysis:

[0074] Based on the experimental design in the previous step, a total of 48 single-crystal cultivation experiments were conducted (using three crystallization methods: slow evaporation, gas-liquid diffusion, and slow cooling). Observations revealed that some sample solutions (DCM solution and chloroform solution) obtained in the initial sample rough solubility determination experiment yielded nemorubicin after slow solvent evaporation. n In contrast, the needle-shaped single crystal sample in chloroform solution was subjected to a single crystal diffraction test, and the single crystal diffraction data was successfully collected.

[0075] (a) Example of single-crystal cultivation for single-crystal analysis

[0076] The single-crystal sample of nemorubicin chloroform solvate used for single-crystal diffraction testing was obtained by slow solvent evaporation in a chloroform solution system. The specific experiment is as follows: 1.8 mg of nemorubicin (batch number: LN405-98) was weighed and placed in a 3 mL glass bottle. 0.1 mL of chloroform solvent was added, and appropriate ultrasonic vibration was used to completely dissolve the solid sample, forming a chloroform solution of the compound nemorubicin. The 3 mL glass bottle was then capped and placed in a fume hood at room temperature. After 5 days, the solvent in the bottle was observed to have evaporated, yielding needle-like crystal samples. The PLM image is shown below. Figure 8 (a).

[0077] (b) Single crystal testing and structural analysis

[0078] From the red needle-like crystal samples obtained above, a short rod-shaped single crystal with suitable diffraction quality was selected and cut out, such as... Figure 8 (b) Single-crystal X-ray diffraction characterization was performed, and its single-crystal structure was analyzed. The single-crystal X-ray diffraction results showed that the crystal belongs to the orthorhombic crystal system, space group P212121, and its unit cell parameters are: Figure 9 Other crystallographic data and structural refinement parameters for this crystal are listed.

[0079] Figure 10 The diagram shows the asymmetric unit of the single-crystal structure model obtained from the analysis. The asymmetric unit of this crystal structure consists of one nemorubicin molecule and approximately 1.5 chloroform solvent molecules. This indicates that the nemorubicin crystal is a chloroform solvate crystal form of nemorubicin. Figure 11 The image shows the atomic thermal vibration ellipsoid (ORTEP diagram) of the compound Nemorobetain in this crystal structure. Based on the single-crystal structure data of the chloroform solvate of Nemorobetain, the stereochemical structure of the compound Nemorobetain is confirmed as follows. Figure 12 As shown, the absolute configuration of the chiral center in its molecule is {C17(S), C19(S), C22(R), C23(S), C24(S), C25(S), C30(S)}, which is consistent with the expected target molecular structure. Figure 13 This is a unit cell schematic diagram of the single-crystal structure model of the nemorubicin chloroform solvate. Hydrogen bond information in this single-crystal structure is summarized in... Figure 15 The hydrogen bonds between adjacent nemorubicin molecules and the connections between nemorubicin molecules and adjacent chloroform molecules, such as Figure 14 As shown. Figure 16 , Figure 17 and Figure 18 This is a schematic diagram of the molecular packing structure in the crystal structure (observed along the crystallographic a-axis, b-axis, and c-axis directions, respectively). Figure 19 The image shown is the calculated XRPD plot of the single-crystal structure model of nemorubicin chloroform solvate. Figure 20 The image shown is an XRPD plot of the starting sample of Nemorubicin.

[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A nemorubicin eutectic, characterized in that, The absolute configuration of its chiral center in the molecule is: {C17(S), C19(S), C22(R), C23(S), C24(S), C25(S), C30(S)}.

2. The Nemorubicin eutectic according to claim 1, characterized in that: It belongs to the orthorhombic crystal system, space group P212121.

3. The Nemorubicin eutectic according to claim 2, characterized in that: Its unit cell parameters are 4. The Nemorubicin eutectic according to claim 1, characterized in that: It is a solvent crystal form, and its crystals are chloroform solvates.

5. A method for preparing the nemorubicin co-crystallized substance according to any one of claims 1 to 4, characterized in that: Nemorubicin was cultured as a single crystal using methods such as slow evaporation of solution, gas-liquid diffusion, or slow cooling.

6. The method for preparing the nemorubicin co-crystallized substance according to claim 5, characterized in that: The nemorubicin sample is mixed with a solvent to form a solution of a certain concentration. The filtrate is collected by filtration and sealed. A small hole is made at the sealing opening after sealing. The solvent is allowed to evaporate slowly at room temperature. The solvent can be a single solvent or a mixed solvent. The mixed solvent is a mixture of multiple components selected from halogenated hydrocarbons, alcohols, alkanes, ethers and acetonitrile.

7. The method for preparing the Nemorobetaine co-crystallization according to claim 6, characterized in that: The concentration of the nemorubicin sample in the solvent was 1 mg / ml to 50 mg / ml.

8. The use of the nemorubicin co-crystal as described in any one of claims 1 to 4 as a standard or reference.

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