Crystal forms of 20(s)-protopanaxadiol, preparation method therefor and use thereof

Abstract

The present invention relates to the field of medicine, and specifically relates to a crystal form A and crystal form B of 20(S)-protopanaxadiol, a preparation method therefor and the use thereof. The crystal form A and crystal form B provided in the present invention are different from the existing known crystal forms of 20(S)-protopanaxadiol, each have a distinct profile, can be perfectly reproduced, have good bulk density, fluidity and stability, and good characteristics in terms of fighting depression, anxiety and cognitive disorder, preventing and / or treating Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and treating diseases such as tumors or cardiovascular diseases, and have broad application prospects in the drug development of 20(S)-protopanaxadiol.

Description

Crystal form, preparation method and application of 20(S)-protopanaxadiol

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023114247117, filed on October 30, 2023, entitled “Crystal form, preparation method and application of 20(S)-protopanaxadiol,” the entire disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of medicine, and in particular to a crystal form, a preparation method and application of 20(S)-protopanaxadiol. Background Art

[0004] 20(S)-Protopanaxdiol (20(S)-PPD) is a plant ingredient with many pharmacological activities, including inhibiting cancer cell growth, anti-depression, anti-anxiety, anti-cognitive impairment, preventing and / or treating Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and anti-cardiovascular disease. Its molecular formula is C 30 H 52 O3, the structural formula is:

[0005] Currently, there is little research on the crystal forms of 20(S)-protopanaxadiol. Patent publication number CN115991729A discloses two crystal forms of 20(S)-protopanaxadiol, which are prepared by heating and dissolving in acetone and acetonitrile, followed by cooling and crystallization.

[0006] In addition, existing research has also disclosed several methods for preparing 20(S)-protopanaxadiol. Most of these methods use plant saponins (such as ginseng, Panax notoginseng, red ginseng, American ginseng, and Gynostemma pentaphyllum) as raw materials. 20(S)-protopanaxadiol is obtained through hydrolysis and other techniques. Column chromatography is then used to separate and purify 20(S)-protopanaxadiol using alcohol as the primary elution solvent, ultimately yielding a white powdered 20(S)-protopanaxadiol. However, the 20(S)-protopanaxadiol products (including amorphous crystalline 20(S)-protopanaxadiol) obtained by these methods still suffer from disadvantages such as low bulk density, low solubility, and poor mechanical stability.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present invention provides a new crystal form of 20(S)-protopanaxadiol, a preparation method, and its application. The details are as follows:

[0009] In a first aspect, the present invention provides a crystalline form A of 20(S)-protopanaxadiol.

[0010] In the present invention, the X-ray powder diffraction pattern of the crystalline form A expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 6.5°, 4.2° and 15.7°.

[0011] Preferably, the X-ray powder diffraction pattern of the crystalline form A expressed at a diffraction angle of 2θ±0.2° is at least:

[0012] (1) At least one of 17.1°, 18.6°, and 13.1° exhibits a characteristic diffraction peak; and / or

[0013] (2) At least one of 17.9°, 10.6°, and 20.7° exhibits a characteristic diffraction peak.

[0014] In a preferred embodiment, the X-ray powder diffraction pattern of the crystalline form A expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 6.5°, 4.2°, 15.7°, 17.1°, 18.6°, 13.1°, 17.9°, 10.6°, and 20.7°.

[0015] In a further preferred embodiment, the crystalline form A of 20(S)-protopanaxadiol of the present invention has a powder X-ray diffraction pattern substantially as shown in FIG1 .

[0016] The crystal form A is represented by the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d, and its data are basically as shown in Table 1:

[0017] Table 1

[0018] In the present invention, the melting point of the crystalline form A is 219-221°C.

[0019] When differential scanning calorimetry analysis is performed, Form A shows an endothermic signal when heated to around 133°C, an exothermic signal when heated to around 175°C, and begins to melt when heated to around 220°C. Its DSC graph is shown in FIG2 .

[0020] The 20(S)-protopanaxadiol crystal form A provided by the present invention has a clear outline and can be perfectly reproduced. It has the characteristics of high bulk density and strong fluidity, and exhibits superior properties in terms of mechanical stability and solid-state stability. It shows higher solubility in FaSSIF (artificial intestinal fluid in the fasting state) and has broad application prospects in the drug development of 20(S)-protopanaxadiol.

[0021] In a second aspect, the present invention provides a method for preparing the crystalline form A.

[0022] In the present invention, the preparation method of the crystal form A includes: dissolving the crude 20(S)-protopanaxadiol in a positive solvent to obtain a 20(S)-protopanaxadiol solution, adding an antisolvent to the 20(S)-protopanaxadiol solution until a colloid is formed, and subjecting the colloid to at least one temperature cycle.

[0023] Among them, the crude 20(S)-protopanaxadiol described in the present invention is an existing known compound, which can be obtained through various channels.

[0024] In an optional embodiment of the present invention, the crude 20(S)-protopanaxadiol can be selected from the "protopanaxadiol" prepared by the preparation method described in patent publication number CN1252082C (Preparation Method of Protopanaxadiol and Protopanaxatriol); or, the "Crystal Form A" described in patent publication number CN115991729A (20(S)-Protopanaxadiol Crystal Form A, Crystal Form B, and Preparation Method Thereof). Testing and characterization revealed that the crystal forms of the aforementioned "protopanaxadiol" and "Crystal Form A" in CN115991729A are actually the "Crystal Form D" described in the present invention (see "Comparative Examples and Test Examples" described later herein).

[0025] In a more specific embodiment provided by the present invention, the crude 20(S)-protopanaxadiol is preferably prepared by the method provided in Example 3 of the invention patent publication number CN1252082C (Preparation method of protopanaxadiol and protopanaxadiol).

[0026] The preparation method of the crystal form A of the present invention first dissolves the crude 20(S)-protopanaxadiol in a positive solvent to obtain a 20(S)-protopanaxadiol solution.

[0027] The positive solvent can be selected from solvents known to dissolve 20(S)-protopanaxadiol well, preferably at least one of methyl isobutyl ketone, anisole or chloroform, and more preferably methyl isobutyl ketone.

[0028] In order to further control the solubility of the solute, the ratio of the crude 20(S)-protopanaxadiol to the positive solvent is preferably 10-30 mg: 0.5-1.0 mL, and more preferably 18-22 mg: 0.6-0.8 mL.

[0029] In the preparation method provided by the present invention, after obtaining the 20(S)-protopanaxadiol solution, the 20(S)-protopanaxadiol solution is preferably further filtered using a filter membrane with a pore size of 0.4-0.5 μm.

[0030] In the present invention, the above-mentioned filtration operation can not only achieve conventional impurity removal, but also effectively eliminate the influence of impurities on the formation of the microstructure of the crystal during the subsequent gas-liquid permeation crystallization process, thereby promoting the smoother precipitation of crystal form A from the crude 20(S)-protopanaxadiol solution.

[0031] The pore size of the filter membrane of the present invention is preferably 0.44-0.46 μm, more preferably 0.45 μm.

[0032] The present invention does not particularly limit the material of the filter membrane, and any conventional commercial source in the art can be used.

[0033] In a more specific embodiment provided by the present invention, the filter membrane is selected from a polytetrafluoroethylene filter membrane.

[0034] In the preparation method provided by the present invention, the obtained 20(S)-protopanaxadiol solution is preferably stirred to obtain a clear solution. Preferably, the stirring speed is 500-2000 r / min, more preferably 1000 r / min.

[0035] Furthermore, in the present invention, an antisolvent is added dropwise to the 20(S)-protopanaxadiol solution until a jelly is formed; and then the jelly is subjected to at least one temperature cycle.

[0036] The anti-solvent can be selected from solvents that are not conducive to 20(S)-protopanaxadiol, and is preferably at least one of n-heptane, n-hexane, or n-pentane, and more preferably n-heptane.

[0037] In the present invention, the ratio of the anti-solvent to the positive solvent is preferably 5 mL:0.5-1.0 mL, more preferably 5 mL:0.6-0.8 mL, to better promote the formation of crystals.

[0038] In the preparation method provided by the present invention, temperature control is one of the key factors in the formation of high-quality crystals. The solubility state of 20(S)-protopanaxadiol in the positive solvent and antisolvent of the present invention will be affected to a certain extent by temperature. Appropriate temperature control is very beneficial to the crystal growth and crystallization of 20(S)-protopanaxadiol.

[0039] Preferably, the temperature cycle comprises: heating the colloid to 45-55°C, maintaining the temperature for 1-3 hours, cooling the temperature to 4-6°C at a rate of 0.05-1°C / min, and growing the crystal at the temperature for 1-3 hours. Preferably, the heating is: uniformly heating to 45-55°C over 10-40 minutes; and the cooling rate is preferably 0.1-0.5°C / min.

[0040] In a preferred embodiment of the present invention, the temperature cycle comprises: heating the colloid to 50°C within 0.3-0.5 hours, maintaining the temperature for 2 hours, then cooling the temperature to 5°C at a rate of 0.1-0.5°C / min, and growing the crystal at the temperature for 2 hours.

[0041] In the preparation method provided by the present invention, while the temperature cycle is being performed on the colloid, the colloid is preferably stirred except for the constant temperature crystal growing stage (i.e., the heating, constant temperature of heating to 45-55°C, and cooling steps are all carried out under stirring); preferably, the stirring speed is 800-1200 r / min; more preferably 1000 r / min.

[0042] The present invention does not particularly limit the specific operation mode of stirring in each step, and conventional stirring operations in the art, such as magnetic stirring, etc., can be used.

[0043] In the present invention, the number of cycles of the temperature cycle is preferably 2 to 4. After the temperature cycle is completed, a precipitated solid crystal form A can be obtained.

[0044] In the present invention, the precipitated solid crystal form A is preferably filtered and dried. The filtering and drying steps can be performed by conventional methods in the art and are not particularly limited in the present invention.

[0045] As the best embodiment of the present invention, the preparation method preferably comprises the following steps:

[0046] Approximately 20 units (by weight) of crude 20(S)-protopanaxadiol solid was weighed and placed in a glass bottle. 0.6 units (by volume) of methyl isobutyl ketone (MIK) was added to dissolve the crude 20(S)-protopanaxadiol solid. The sample solution was filtered through a 0.45 μm PTFE filter into a new glass bottle. The solution was then magnetically stirred (at approximately 1000 rpm) to obtain a clear solution. Five units (by volume) of n-heptane were then added dropwise to obtain a colloidal sample. The sample was then transferred to a temperature cycle for crystal growth and crystallization.

[0047] In the preparation method of the present invention, unless otherwise specified, when the mass unit is mg, the corresponding volume unit is mL.

[0048] The temperature cycle conditions are as follows:

[0049] First cycle: raise the temperature to 50°C at a constant rate over 0.3 hours and maintain it at 50°C for 2 hours; reduce the temperature to 5°C at a rate of 0.5°C / min and maintain it at 5°C for 2 hours.

[0050] Second cycle: raise the temperature to 50°C at a constant rate over 0.4 hours and maintain it at 50°C for 2 hours; lower the temperature to 5°C at a rate of 0.3°C / min and maintain it at 5°C for 2 hours.

[0051] The third cycle: the temperature was raised to 50°C at a constant rate over 0.5 hours and maintained at 50°C for 2 hours; the temperature was lowered to 5°C at a rate of 0.1°C / min and maintained at 5°C for 2 hours.

[0052] In the above three cycles, except for the crystal growth at a constant temperature of 5°C for 2 hours, the other cycles were carried out under magnetic stirring.

[0053] After a total of three cycles, solid precipitated, and the obtained solid was separated by centrifugation to obtain Form A.

[0054] The preparation method provided by the present invention can perfectly reproduce the 20(S)-protopanaxadiol crystal form A, and the product quality is stable and reliable, which plays an important role in the subsequent development and application of the 20(S)-protopanaxadiol crystal form A.

[0055] FIG3 shows a powder X-ray diffraction pattern of crude 20(S)-protopanaxadiol obtained in Example 3 of the invention patent publication No. CN1252082C (Preparation Method of Protopanaxadiol and Protopanaxadiol).

[0056] As can be seen from Figure 3, the X-ray powder diffraction pattern of the crude 20(S)-protopanaxadiol expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at 5.0°, 5.8°, 12.6°, 17.5°, 7.3°, 17.0°, 10.0°, 16.6°, and 15.3°.

[0057] The crude 20(S)-protopanaxadiol product shown in FIG3 is represented by the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d. The data are basically as shown in Table 2:

[0058] Table 2

[0059] The crude 20(S)-protopanaxadiol has a melting point of 215-217°C.

[0060] When differential scanning calorimetry analysis was performed, the crude 20(S)-protopanaxadiol exhibited an endothermic signal when heated to approximately 50°C, an exothermic signal when heated to approximately 159°C, and began to melt when heated to approximately 216°C. The DSC graph is shown in FIG4 .

[0061] The crude 20(S)-protopanaxadiol can also be represented by crystal form D in the present invention. By comparing the powder X-ray diffraction pattern (i.e., Figure 3) and DSC pattern (i.e., Figure 4) of the crude 20(S)-protopanaxadiol (crystal form D) with the powder X-ray diffraction pattern (i.e., Figure 1) and DSC pattern (i.e., Figure 2) of the 20(S)-protopanaxadiol crystal form A claimed in the present invention, it can be seen that the crystal form A obtained in the present invention is different from the currently known 20(S)-protopanaxadiol crystal forms.

[0062] In a third aspect, the present invention provides another crystalline form B of 20(S)-protopanaxadiol.

[0063] In the present invention, the X-ray powder diffraction pattern of the crystalline form B expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 7.5°, 14.4° and 15.4°.

[0064] Preferably, the X-ray powder diffraction pattern of the crystalline form B expressed at a diffraction angle of 2θ±0.2° is at least:

[0065] (1) at least one of 4.6°, 3.8° and 16.9° shows a characteristic diffraction peak; and / or

[0066] (2) At least one of 18.1°, 6.7°, and 13.1° exhibits a characteristic diffraction peak.

[0067] In a preferred embodiment, the X-ray powder diffraction pattern of the crystalline form B expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at at least 7.5°, 14.4°, 15.4°, 4.6°, 3.8°, 16.9°, 18.1°, 6.7°, and 13.1°.

[0068] In a further preferred embodiment, the crystalline form B of 20(S)-protopanaxadiol of the present invention has a powder X-ray diffraction pattern substantially as shown in FIG5 .

[0069] The crystal form B is represented by the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d, and its data are basically as shown in Table 3:

[0070] Table 3

[0071] In the present invention, the melting point of the crystalline form B is 218-220°C.

[0072] When differential scanning calorimetry analysis was performed, Form B began to melt when heated to around 219° C., and its DSC graph is shown in FIG6 .

[0073] The 20(S)-protopanaxadiol crystal form B provided by the present invention has a clear outline and can be perfectly reproduced. It has the characteristics of high bulk density, strong fluidity and good solid-state stability.

[0074] In a fourth aspect, the present invention provides a method for preparing the crystalline form B.

[0075] In the present invention, the preparation method of the crystal form B comprises: first heating the crude 20(S)-protopanaxadiol to 160-180° C. and maintaining the temperature for 2-5 minutes; then cooling the temperature to 25-35° C. to obtain the crystal form B.

[0076] Among them, the crude 20(S)-protopanaxadiol is also an existing known compound (crystalline form D). In a more specific embodiment provided by the present invention, the crude 20(S)-protopanaxadiol is prepared by the method provided in Example 3 of the invention patent announcement No. CN1252082C (Preparation method of protopanaxadiol and protopanaxadiol).

[0077] In the method for preparing Form B of the present invention, the heating is preferably carried out in an inert gas environment, wherein the inert gas is preferably N2.

[0078] In the present invention, the heating rate is preferably 8-15°C / min, more preferably 10-12°C / min. The cooling rate is preferably 25-35°C / min, more preferably 30°C / min.

[0079] As the best embodiment of the present invention, the preparation method preferably comprises the following steps:

[0080] The crude solid of 20(S)-protopanaxadiol was placed in a DSC aluminum pan. Under nitrogen purge, the temperature was increased to 170°C at a heating rate of 10-15°C / min, maintained at a constant temperature for 3 minutes, and then cooled to 30°C at a cooling rate of 30°C / min, and the solid was collected.

[0081] By comparing the powder X-ray diffraction pattern (i.e., Figure 3) and DSC pattern (i.e., Figure 4) of the crude 20(S)-protopanaxadiol with the powder X-ray diffraction pattern (i.e., Figure 5) and DSC pattern (i.e., Figure 6) of the 20(S)-protopanaxadiol crystal form B claimed in the present invention, it can be seen that the crystal form B obtained in the present invention is different from the existing known 20(S)-protopanaxadiol crystal forms.

[0082] In a fifth aspect, the present invention also provides a pharmaceutical composition containing the above-mentioned 20(S)-protopanaxadiol crystal form A or crystal form B, and the specific pharmaceutical composition includes but is not limited to powder injection, tablets, capsules, microspheres, self-microemulsification composition, dry suspension, lyophilized powder or infusion, etc.

[0083] The composition of the present invention may further contain a pharmaceutically acceptable excipient; more preferably, the pharmaceutically acceptable excipient includes at least one of a carrier, a diluent, and an excipient. For example, in the case of a lyophilized powder, the pharmaceutically acceptable carrier may be an excipient; in the case of a tablet, the pharmaceutically acceptable carrier may be a disintegrant, an excipient, or the like.

[0084] The 20(S)-protopanaxadiol crystal form A or crystal form B described in the present invention has the same chemical formula structure as the crude 20(S)-protopanaxadiol (only the crystal form is different), and it also exhibits ideal therapeutic effects in anti-depression, anti-anxiety, anti-cognitive impairment, prevention and / or treatment of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), anti-tumor or anti-cardiovascular disease.

[0085] Since the 20(S)-protopanaxadiol crystal form A or crystal form B obtained in the present invention has ideal physical and chemical properties, those skilled in the art can foresee that after preparing it into various pharmaceutical preparations, the stability and efficacy of the pharmaceutical preparations can be further improved, and the determination of the specific dosage form and its prescription is within the control of those skilled in the art.

[0086] In addition, the present invention further claims protection for any of the following applications of the above-mentioned 20(S)-protopanaxadiol crystal form A or crystal form B:

[0087] (1) For the preparation of antidepressant, antianxiety, anti-cognitive disorder, anti-tumor or anti-cardiovascular disease drugs;

[0088] (2) Antidepressant, anti-anxiety, anti-cognitive disorder, anti-tumor or anti-cardiovascular disease;

[0089] (3) For the preparation of drugs for preventing and / or treating Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS).

[0090] The 20(S)-protopanaxadiol crystal form A or crystal form B of the present invention exhibits excellent medicinal activity in anti-depression, anti-anxiety, anti-cognitive impairment, anti-tumor and anti-cardiovascular disease, prevention and / or treatment of Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), etc.; and the 20(S)-protopanaxadiol crystal form A or crystal form B of the present invention exhibits better crystal characteristics such as bulk density, fluidity and stability than the crude 20(S)-protopanaxadiol, which has advantages in the drug development of 20(S)-protopanaxadiol. Beneficial effects:

[0091] The present invention provides novel 20(S)-protopanaxadiol crystalline forms A and B, as well as corresponding preparation methods and applications. The novel crystalline forms provided by the present invention are clearly defined and perfectly reproducible, exhibiting excellent properties in terms of bulk density, fluidity, and stability, and have broad application prospects in the development of 20(S)-protopanaxadiol pharmaceuticals. The preparation method provided by the present invention is simple and easy to operate, facilitating widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG1 is an X-ray powder diffraction pattern of 20(S)-protopanaxadiol crystal form A of the present invention.

[0093] FIG2 is a differential scanning calorimetry analysis diagram of 20(S)-protopanaxadiol crystal form A of the present invention.

[0094] FIG3 is an X-ray powder diffraction pattern of the crude 20(S)-protopanaxadiol (crystal form D) used in the present invention.

[0095] FIG4 is a differential scanning calorimetry analysis diagram of the crude 20(S)-protopanaxadiol (crystalline form D) used in the present invention.

[0096] FIG5 is an X-ray powder diffraction pattern of 20(S)-protopanaxadiol crystal form B of the present invention.

[0097] FIG6 is a differential scanning calorimetry analysis diagram of 20(S)-protopanaxadiol crystal form B of the present invention.

[0098] FIG7 is a dynamic water adsorption diagram of 20(S)-protopanaxadiol crystal form A of the present invention.

[0099] FIG8 is a dynamic water adsorption diagram of 20(S)-protopanaxadiol crystal form B of the present invention.

[0100] FIG9 is a dynamic water adsorption diagram of the crude 20(S)-protopanaxadiol (crystalline form D) used in the present invention.

[0101] FIG10 is a PLM diagram of 20(S)-protopanaxadiol crystal form A of the present invention.

[0102] FIG11 is a PLM diagram of 20(S)-protopanaxadiol crystal form B of the present invention.

[0103] FIG12 is a PLM diagram of the crude 20(S)-protopanaxadiol (crystalline form D) used in the present invention. DETAILED DESCRIPTION

[0104] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Obviously, the described embodiments are merely some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are intended to fall within the scope of protection of the present invention.

[0105] The abbreviations used in the present invention are explained as follows:

[0106] XRPD: X-ray powder diffraction

[0107] DSC: Differential Scanning Calorimetry

[0108] TGA: Thermogravimetric analysis

[0109] DVS: Dynamic Water Sorption

[0110] The X-ray powder diffraction patterns of the present invention were collected on a PANalytical Empyrean or X'Pert3 X-ray powder diffractometer. The X-ray powder diffraction method parameters of the present invention are as follows:

[0111] X-ray source: Cu, Kα

[0112] 1.54060; 1.54439

[0113] Kα2 / Kα1 intensity ratio: 0.50

[0114] Voltage: 45 kilovolts (kV)

[0115] Current: 40 milliamperes (mA)

[0116] Scanning range: from 3.0 to 40.0 degrees

[0117] The differential scanning calorimetry (DSC) graphs of the present invention were collected on a TA Discovery 2500. The method parameters of the differential scanning calorimetry (DSC) of the present invention are as follows:

[0118] Scan rate: 10℃ / min

[0119] Shielding gas: N2

[0120] The thermogravimetric analysis (TGA) graph of the present invention was collected on a TA Discovery 5500. The method parameters of the thermogravimetric analysis (TGA) of the present invention are as follows:

[0121] Scan rate: 10℃ / min

[0122] Shielding gas: N2

[0123] The high performance liquid chromatography (HPLC) purity data in this invention were collected from a Thermo Fisher Scientific Vanquish Core using a diode array detector (DAD). The HPLC method parameters for purity testing described in this invention are as follows:

[0124] 1. Chromatographic column: Xselect CSH C18, 150×4.6mm, 3.5μm

[0125] 2. Mobile phase:

[0126] A: water

[0127] B: Acetonitrile

[0128] The elution gradient is as follows:

[0129] 3. Flow rate: 1.0 mL / min

[0130] 4. Injection volume: 5 μL

[0131] 5. Detection wavelength: 203nm

[0132] 6. Column temperature: 40°C

[0133] 7. Injector temperature: room temperature

[0134] 8. Diluent: acetonitrile

[0135] Unless otherwise specified, the following examples were all performed at room temperature.

[0136] Example 1

[0137] Preparation of crude 20(S)-protopanaxadiol:

[0138] The crude product of 20(S)-protopanaxadiol was prepared by the method of Example 3 of invention patent 200410018038.8 (Preparation method of protopanaxadiol and protopanaxadiol).

[0139] Using Cu-Kα radiation, the X-ray powder diffraction pattern of the crude 20(S)-protopanaxadiol is shown in FIG3 .

[0140] The crude 20(S)-protopanaxadiol shown in FIG3 is represented according to the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d, and the data are shown in Table 2.

[0141] When differential scanning calorimetry analysis was performed, the crude 20(S)-protopanaxadiol showed an endothermic signal when heated to around 50°C, an exothermic signal when heated to around 159°C, and began to melt when heated to around 216°C. The DSC graph is shown in Figure 4.

[0142] Example 2

[0143] Preparation of 20(S)-protopanaxadiol Crystal Form A:

[0144] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as starting material, approximately 20 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 20 mL glass vial. 0.6 mL of methyl isobutyl ketone was added to dissolve the crude 20(S)-protopanaxadiol solid. The sample solution was filtered through a 0.45 μm pore size polytetrafluoroethylene filter into a new 20 mL glass vial. The solution was then magnetically stirred (at approximately 1000 rpm) to obtain a clear solution. 5 mL of n-heptane was then added dropwise to obtain a colloidal sample. The sample was then transferred to a crystallization stage under temperature cycling conditions (except for the constant temperature crystallization stage, all other stages were performed under magnetic stirring at approximately 1000 rpm).

[0145] The temperature cycle conditions are as follows:

[0146] First cycle: raise the temperature to 50°C at a constant rate over 0.3 hours and maintain it at 50°C for 2 hours; reduce the temperature to 5°C at a rate of 0.5°C / min and maintain it at 5°C for 2 hours.

[0147] Second cycle: raise the temperature to 50°C at a constant rate over 0.4 hours and maintain it at 50°C for 2 hours; lower the temperature to 5°C at a rate of 0.3°C / min and maintain it at 5°C for 2 hours.

[0148] The third cycle: the temperature was raised to 50°C at a constant rate over 0.5 hours and maintained at 50°C for 2 hours; the temperature was lowered to 5°C at a rate of 0.1°C / min and maintained at 5°C for 2 hours.

[0149] After a total of 3 cycles, solid precipitated, and the obtained solid was separated by centrifugation to obtain Form A.

[0150] Using Cu-Kα radiation, the X-ray powder diffraction pattern of the 20(S)-protopanaxadiol crystal form A is shown in FIG1 .

[0151] The 20(S)-protopanaxadiol crystal form A is represented by the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d, and its data are shown in Table 1.

[0152] When differential scanning calorimetry analysis is performed, Form A shows an endothermic signal when heated to around 133°C, an exothermic signal when heated to around 175°C, and begins to melt when heated to around 220°C. The DSC graph is shown in Figure 2.

[0153] Example 3

[0154] Preparation of 20(S)-protopanaxadiol Crystal Form A:

[0155] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as a raw material, approximately 19.8 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 20-ml glass vial. 0.8 ml of anisole was added to dissolve the crude 20(S)-protopanaxadiol solid. The sample solution was filtered into a new 20-ml glass vial using a 0.45-μm pore polytetrafluoroethylene filter, followed by magnetic stirring at a speed of approximately 1000 rpm to obtain a clarified solution, to which 5 ml of n-pentane was dropwise added to obtain a colloidal sample. The sample was then transferred to a temperature cycle for crystal growth (except for the constant temperature crystal growth stage, all other steps were performed under magnetic stirring at a speed of approximately 1000 rpm).

[0156] The temperature cycle program is as follows:

[0157] First cycle: raise the temperature to 50°C at a constant rate over 0.3 hours, maintain the temperature at 50°C for 2.5 hours; reduce the temperature to 5°C at a rate of 0.5°C / min, maintain the temperature at 5°C for 2.5 hours.

[0158] Second cycle: raise the temperature to 50°C at a constant rate over 0.4 hours and maintain it at 50°C for 2.5 hours; then reduce the temperature to 5°C at a rate of 0.3°C / min and maintain it at 5°C for 2.5 hours.

[0159] The third cycle: the temperature was raised to 50°C at a constant rate over 0.5 hours and maintained at 50°C for 2.5 hours; the temperature was lowered to 5°C at a rate of 0.1°C / min and maintained at 5°C for 2.5 hours.

[0160] After a total of three cycles, solid precipitated, and the solid crystals obtained by centrifugation were tested to be Form A.

[0161] Example 4

[0162] Preparation of 20(S)-protopanaxadiol Crystal Form A:

[0163] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as raw material, approximately 19.8 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 20 mL glass vial. 0.8 mL of chloroform was added to dissolve the crude 20(S)-protopanaxadiol solid. The sample solution was filtered through a 0.45 μm pore size polytetrafluoroethylene filter into a new 20 mL glass vial. The solution was then magnetically stirred (at approximately 1000 rpm) to obtain a clear solution. 5 mL of n-hexane was then added dropwise to obtain a colloidal sample. The sample was then transferred to a temperature cycle for crystal growth (except for the constant temperature crystal growth stage, all other stages were performed under magnetic stirring at approximately 1000 rpm).

[0164] The temperature cycle program is as follows:

[0165] First cycle: raise the temperature to 50°C at a constant rate over 0.3 hours and maintain it at 50°C for 1.5 hours; reduce the temperature to 5°C at a rate of 0.5°C / min and maintain it at 5°C for 1.5 hours.

[0166] Second cycle: raise the temperature to 50°C at a constant rate over 0.4 hours and maintain it at 50°C for 1.5 hours; then reduce the temperature to 5°C at a rate of 0.3°C / min and maintain it at 5°C for 1.5 hours.

[0167] The third cycle: the temperature was raised to 50°C at a constant rate over 0.5 hours and maintained at 50°C for 1.5 hours; the temperature was lowered to 5°C at a rate of 0.1°C / min and maintained at 5°C for 1.5 hours.

[0168] After a total of three cycles, solid precipitated, and the solid crystals obtained by centrifugation were tested to be Form A.

[0169] Example 5

[0170] Preparation of 20(S)-protopanaxadiol Crystal Form B:

[0171] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as starting material, approximately 15 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in an aluminum DSC pan. Under a nitrogen purge, the temperature was raised to 170°C at a heating rate of 10°C / min. The temperature was maintained for 3 minutes, and then cooled to 30°C at a cooling rate of 30°C / min. A total of 17 batches were set up in parallel. All solids were collected to obtain Form B.

[0172] Using Cu-Kα radiation, the X-ray powder diffraction pattern of the 20(S)-protopanaxadiol crystal form B is shown in FIG5 .

[0173] The 20(S)-protopanaxadiol crystal form B is represented by the spectral line position (Bragg angle 2θ, expressed in degrees), relative intensity (%) and interplanar spacing d, and its data are shown in Table 3.

[0174] When differential scanning calorimetry analysis was performed, Form B began to melt when heated to around 219°C, and its DSC graph is shown in FIG6 .

[0175] Comparative Example 1

[0176] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as raw material, 19.7 mg of the crude 20(S)-protopanaxadiol was weighed and placed in a 2 mL glass vial. 0.3 mL of tetrahydrofuran / n-heptane (volume ratio 1:1) was added to obtain a suspension. The suspension was magnetically stirred at 50°C (approximately 1000 rpm) for approximately two hours, then sealed and cooled from 50°C to 5°C at a rate of 0.1°C / min. The suspension was then allowed to stand at 5°C for approximately one day to obtain solid crystals. Testing and characterization confirmed that the solid crystals obtained in this comparative example were Form D.

[0177] Comparative Example 2

[0178] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as raw material, 20.1 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 3 mL glass vial. 1.0 mL of acetonitrile was added to obtain a suspension. The suspension was magnetically stirred at 50°C (approximately 1000 rpm) for approximately two hours. After sealing, the suspension was cooled from 50°C to 5°C at a rate of 0.1°C / min and then maintained at 5°C for approximately one day to obtain solid crystals. Testing and characterization confirmed that the solid crystals obtained in this comparative example were Form D.

[0179] Comparative Examples 3-6

[0180] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as a raw material, approximately 20 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 3 ml glass vial. A corresponding volume of solvent was added, and the 3 ml glass vial was sealed with a sealing film. Four small holes were pierced in the sealing film, and the solution was allowed to evaporate at room temperature until solid precipitated.

[0181] The detailed test conditions involved in the above comparative examples are shown in Table 4.

[0182] Table 4

[0183] After testing and characterization, the solid crystals obtained in Comparative Examples 3-6 were all of Form D.

[0184] Comparative Example 7

[0185] Using the crude 20(S)-protopanaxadiol prepared in Example 1 as raw material, approximately 20 mg of the crude 20(S)-protopanaxadiol solid was weighed and placed in a 20 mL glass vial. 0.6 mL of methyl isobutyl ketone was added to dissolve the crude 20(S)-protopanaxadiol solid. Magnetic stirring (at approximately 1000 rpm) was then applied to obtain a clear solution. The solvent was allowed to evaporate naturally and slowly, precipitating a solid. The resulting solid was centrifuged and characterized, confirming that the solid crystals obtained in this comparative example were Form D.

[0186] Test Example 1: Moisture absorption test

[0187] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the hygroscopicity of each test sample was tested.

[0188] Dynamic moisture adsorption / desorption (DVS) curves of each sample were tested and plotted, and the experimental results are shown in Table 5. The DVS graph of Form A described in Example 2 is shown in Figure 7, the DVS graph of Form B described in Example 5 is shown in Figure 8, and the DVS graph of the crude 20(S)-protopanaxadiol described in Example 1 is shown in Figure 9.

[0189] Table 5

[0190] According to the definition of drug hygroscopicity in the "Chinese Pharmacopoeia 2020 Edition", the crystal form A, crystal form B and 20(S)-protopanaxadiol crude product described in the present invention are all slightly hygroscopic and can still maintain stable properties under high humidity conditions. During production, storage and use, they can remain stable without specific humidity control conditions, and can well meet the requirements of drug production and use.

[0191] Regarding the description of hygroscopic characteristics and the definition of hygroscopic weight gain (Guidelines for Hygroscopicity Testing of Drugs in the Chinese Pharmacopoeia 2020 Edition):

[0192] Deliquescent: Absorbs enough water to form a liquid

[0193] Highly hygroscopic: weight gain due to moisture absorption is not less than 15%

[0194] Hygroscopic: Weight gain due to moisture absorption is less than 15% but not less than 2%

[0195] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%

[0196] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0197] Test Example 2 Solubility Test

[0198] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the solubility of each test sample was tested.

[0199] 50 mg of each test sample was weighed and prepared into suspensions with 5.0 mL of purified water, pH 1 buffer, SGF (simulated gastric fluid), FaSSIF (fasted artificial intestinal fluid), and FeSSIF (fed artificial intestinal fluid). After equilibration at 37°C for 1, 2, 4, and 24 hours, the suspensions were filtered to obtain clear solutions. The sample content in the saturated solutions was determined by HPLC. The test results are shown in Table 6.

[0200] The test results showed that the 24-hour solubilities of Form A, Form B, and crude 20(S)-protopanaxadiol were low in pure water and pH 1 buffer media. The 24-hour solubility of Form A was 0.13-0.15 μg / mL, while the solubilities of Forms B and D were less than the LOQ. There was no significant difference in the solubility of Form A, Form B, and crude 20(S)-protopanaxadiol (Form D) in SGF. In FaSSIF, the solubility of Form A and crude 20(S)-protopanaxadiol (Form D) was higher than that of Form B.

[0201] Table 6

[0202] LOQ1 is: 0.1 μg / mL. LOQ2 is: 0.3 μg / mL.

[0203] Test Example 3 Bulk density and tap density test

[0204] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the bulk density and tap density of each test sample were tested.

[0205] Approximately 800 mg of each sample was added to a 5 mL graduated cylinder and the resulting volume was recorded. The cylinder was tapped 200 times on the table and the resulting volume was recorded. Three parallel tests were performed. The results are shown in Table 7.

[0206] Table 7

[0207] Test Example 4: Angle of Repose Test

[0208] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the angle of repose of each test sample was tested.

[0209] Take an appropriate amount of each sample to be tested. Take a funnel and fix it on an iron stand so that it is placed perpendicular to the table. Add the taken Form A and Form B samples into the funnel respectively, and form a cone after free falling. Measure the height and diameter of the cone to calculate the angle of repose. Perform three tests in parallel. The test results are shown in Table 8. The results show that Form A has a smaller angle of repose than Form B and 20(S)-protopanaxadiol crude product, showing better fluidity.

[0210] Table 8

[0211] Test Example 5 Contact angle test

[0212] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the contact angle of each test sample was tested.

[0213] An appropriate amount of each sample was taken and its contact angle against water was measured. Two parallel tests were performed. The test results are shown in Table 9.

[0214] Table 9

[0215] Test Example 6 Mechanical stability test

[0216] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the mechanical stability of each test sample was tested.

[0217] An appropriate amount of each sample was placed in an agate mortar and ground manually for approximately 3 minutes before testing for crystal form. The results showed that Form A, Form B, and crude 20(S)-protopanaxadiol all essentially transformed into an amorphous form after grinding.

[0218] Appropriate amounts of each sample were added to a tablet press and compressed under a pressure of 200 MPa. The crystal forms were then tested. The results showed that Form A, Form B, and crude 20(S)-protopanaxadiol did not undergo any crystal transformation after tableting. However, the crystallinity of Form B and crude 20(S)-protopanaxadiol decreased, indicating that Form A was superior to Form B and crude 20(S)-protopanaxadiol.

[0219] Test Example 7 Thermodynamic stability test

[0220] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the thermodynamic stability of each test sample was tested.

[0221] An appropriate amount of Form A was placed in a 2 mL glass vial, and 0.5 mL of the corresponding solvent was added. The suspension was magnetically stirred at the appropriate temperature for approximately 5 hours to obtain a suspension. The suspension was then filtered through a 0.45 μm PTFE filter at room temperature or 50°C, respectively, and transferred to a new 2 mL glass vial. Approximately 5 mg each of Form A, Form B, and crude 20(S)-protopanaxadiol were weighed and added to the filtered filtrate, followed by magnetic stirring at the appropriate temperature. Test results show that Form A was obtained by stirring a mixture of Form A, Form B, and crude 20(S)-protopanaxadiol at room temperature and 50°C. The test results are shown in Table 10. These results indicate that Form A is more stable than Form B and crude 20(S)-protopanaxadiol within the range of room temperature to 50°C.

[0222] Table 10

[0223] Test Example 8 Solid-state stability test

[0224] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and the solid-state stability of each test sample was tested.

[0225] Appropriate amounts of each sample were weighed and subjected to stability tests at 80°C (closed) for one day, 25°C / 60% RH, and 40°C / 75% RH (open) for one month. Solid samples isolated under different conditions were tested for physical stability by XRPD analysis of the crystal form and chemical stability by HPLC analysis of the chemical purity. The results are summarized in Table 11. XRPD results showed that Form A, Form B, and crude 20(S)-protopanaxadiol did not undergo any crystal form transformation under all three conditions. HPLC results showed that the purity of crude 20(S)-protopanaxadiol decreased slightly after being stored closed at 80°C for one day, and after being stored open at 25°C / 60% RH and 40°C / 75% RH for one month. Form A and Form B showed no significant decrease in chemical purity under all three conditions, indicating that Forms A and B exhibited superior solid-state stability.

[0226] Table 11

[0227] Test Example 9 Polarizing Microscope (PLM) Test

[0228] In this test example, the crude 20(S)-protopanaxadiol (crystal form D) prepared in Example 1, the 20(S)-protopanaxadiol crystal form A prepared in Example 2, and the 20(S)-protopanaxadiol crystal form B prepared in Example 5 were used as test samples, and PLM observations were performed on each test sample.

[0229] The PLM diagram of Form A described in Example 2 is shown in Figure 10, the PLM diagram of Form B described in Example 5 is shown in Figure 11, and the PLM diagram of the crude 20(S)-protopanaxadiol described in Example 1 is shown in Figure 12.

[0230] As shown in Figures 10, 11 and 12, it can be seen that under a polarizing microscope, Form A and Form B are mainly composed of irregular granular crystals, and Form D is mainly composed of irregular granular and needle-shaped / long plate-shaped crystals.

[0231] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Industrial Applicability

[0232] The present invention provides crystalline Form A and Form B of 20(S)-protopanaxadiol, as well as preparation methods and applications thereof. The crystalline Form A and Form B provided by the present invention differ from existing known crystalline forms of 20(S)-protopanaxadiol in that they each have distinct contours and are perfectly reproducible. They also exhibit excellent properties in terms of bulk density, fluidity, and stability, as well as in the treatment of depression, anxiety, cognitive impairment, tumors, or cardiovascular diseases. These properties demonstrate good economic value and application prospects in the development of 20(S)-protopanaxadiol drugs.

Claims

1. 20(S)-Protopanaxadiol crystalline form A, characterized in that: The X-ray powder diffraction pattern of the crystalline form A expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 6.5°, 4.2° and 15.7°; Preferably, the X-ray powder diffraction pattern of the crystalline form A represented by a diffraction angle of 2θ±0.2° is at least: (1) at least one of 17.1°, 18.6°, and 13.1° shows a characteristic diffraction peak; and / or (2) at least one of 17.9°, 10.6°, and 20.7° shows a characteristic diffraction peak; More preferably, the X-ray powder diffraction pattern of the crystalline form A represented by a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 6.5°, 4.2°, 15.7°, 17.1°, 18.6°, 13.1°, 17.9°, 10.6°, and 20.7°.

2. The crystalline form A of 20(S)-protopanaxadiol according to claim 1, characterized in that: The melting point of the crystalline form A is 219-221°C.

3. The method for preparing the crystalline form A of 20(S)-protopanaxadiol according to claim 1 or 2, characterized in that: Dissolving crude 20(S)-protopanaxadiol in a positive solvent to obtain a 20(S)-protopanaxadiol solution, adding an antisolvent dropwise to the 20(S)-protopanaxadiol solution until a jelly is formed, and subjecting the jelly to at least one temperature cycle; The temperature cycle comprises: raising the temperature of the colloid to 45-55° C., maintaining the temperature for 1-3 hours, reducing the temperature to 4-6° C. at a rate of 0.05-1° C. / min, and maintaining the temperature for 1-3 hours; Preferably, the temperature cycle is repeated 2-4 times.

4. The preparation method according to claim 3, characterized in that: Before adding the anti-solvent, the 20(S)-protopanaxadiol solution is filtered using a filter membrane with a pore size of 0.4-0.5 μm; and / or the positive solvent is selected from at least one of methyl isobutyl ketone, anisole or chloroform; the anti-solvent is selected from at least one of n-heptane, n-hexane or n-pentane. 5.20(S)-Protopanaxadiol crystalline form B, characterized in that: The X-ray powder diffraction pattern of the crystalline form B expressed at a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 7.5°, 14.4°, and 15.4°; Preferably, the X-ray powder diffraction pattern of the crystalline form B represented by a diffraction angle of 2θ±0.2° is at least: (1) at least one of 4.6°, 3.8°, and 16.9° shows a characteristic diffraction peak; and / or (2) at least one of 18.1°, 6.7°, and 13.1° shows a characteristic diffraction peak; More preferably, the X-ray powder diffraction pattern of the crystalline form B represented by a diffraction angle of 2θ±0.2° shows characteristic diffraction peaks at least at 7.5°, 14.4°, 15.4°, 4.6°, 3.8°, 16.9°, 18.1°, 6.7°, and 13.1°.

6. The crystalline form B of 20(S)-protopanaxadiol according to claim 5, characterized in that: The XRPD data of the crystalline form B are substantially as shown in Table 3; and / or, the XRPD pattern of the crystalline form B is substantially as shown in FIG5 .

7. The crystalline form B of 20(S)-protopanaxadiol according to claim 5 or 6, characterized in that: The melting point of the crystalline form B is 218-220°C.

8. The method for preparing the crystalline form B of 20(S)-protopanaxadiol according to any one of claims 5 to 7, characterized in that: The crude 20(S)-protopanaxadiol was first heated to 160-180°C and maintained for 2-5 minutes; then cooled to 25-35°C to obtain Form B; Preferably, the heating rate is 8-15°C / min; and / or the cooling rate is 25-35°C / min.

9. A pharmaceutical composition, characterized in that Contains a therapeutically effective amount of the crystalline form A according to any one of claims 1-2 or the crystalline form B according to any one of claims 5-7; preferably, the pharmaceutical composition also includes a pharmaceutically acceptable excipient.

10. Any one of the following uses of the crystalline form A according to any one of claims 1 to 2, or the crystalline form B according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 9: (1) For the preparation of antidepressant, antianxiety, anti-cognitive disorder, anti-tumor or anti-cardiovascular disease drugs; (2) Anti-depression, anti-anxiety, anti-cognitive disorder, anti-tumor or anti-cardiovascular disease; (3) Used for preparing drugs for preventing and / or treating Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS).

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