Crystallization of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol

Abstract

This invention provides a novel crystal form of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol, which exhibits superior selective inhibition of adenylate cyclase 1 and can be used to prepare a treatment for neuropathic pain and / or inflammatory pain.

Description

Technical Field

[0001] This invention relates to a polymorph of a pharmaceutical compound, specifically to a polymorph of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol, which selectively inhibits adenylate cyclase 1 and can be used to treat neuropathic pain and inflammatory pain. Background Technology

[0002] Pain can be categorized based on its cause into inflammatory pain, neuropathic pain, nociceptive pain, and psychogenic pain. Inflammatory pain is caused by inflammation resulting from external noxious mechanical, thermal, or chemical stimuli. It is known that in inflammatory pain, not only the site of inflammation but also inflammatory cytokines and cyclooxygenases in the spinal cord play important roles. Neuropathic pain is pathological pain caused by abnormal function of the peripheral or central nervous system. Nociceptive pain is caused by damage to normal tissues or by noxious stimuli that may damage normal tissues; it is further divided into somatic pain and visceral pain.

[0003] As treatments for inflammatory pain, cyclooxygenase (COX) inhibitors such as indomethacin, COX-II inhibitors such as celecoxib, centrally acting analgesics such as tramadol, and antipyretic analgesics such as acetaminophen can be used. However, prolonged use of COX inhibitors can sometimes cause gastrointestinal disturbances as a side effect. Furthermore, there are reports of COX-II inhibitors causing gastric ulcers, and recently, their side effects on the cardiovascular system, such as myocardial infarction and cerebral infarction, have also become a concern.

[0004] As a treatment for neuropathic pain, morphine and other papaverine-based analgesics, as well as anticonvulsants such as gabapentin and pregabalin, can be used. However, it is known that with prolonged use, the dosage sometimes needs to be increased and side effects such as sedation may occur. Currently, there are no drugs that are safe to use without side effects.

[0005] Patent Document 1 (WO2007 / 041863) describes 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol as selectively inhibiting adenylate cyclase 1, which can be used to treat neuropathic pain and inflammatory pain. However, Patent Document 1 does not provide a method for preparing 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol suitable for industrial application, nor does it provide a crystal form suitable for pharmaceutical preparation. Furthermore, different preparation and crystallization methods require consideration of avoiding the use of Class II or higher organic solvents harmful to mammals in the final product, thus reducing the adverse effects of residual organic solvents on users.

[0006] For drug polymorphs, different polymorphs can possess different chemical and physical properties, including melting point, chemical stability, apparent solubility, dissolution rate, optical and mechanical properties, vapor pressure, and density. These properties directly or indirectly affect the processing or production of active pharmaceutical ingredients and formulations, and can influence the stability, solubility, and bioavailability of formulations. Therefore, drug polymorphs are of significant importance to the quality, safety, and efficacy of drug formulations. For 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol, there is a need in the art for a preparation method suitable for industrial production and a polymorph with excellent physicochemical properties.

[0007] Existing technical documents

[0008] Patent document 1WO2007 / 041863 Summary of the Invention

[0009] Drug polymorphism is one of the important factors affecting drug quality and clinical efficacy. Although patent document 1 (WO2007 / 041863) describes that 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol can selectively inhibit adenylate cyclase 1 and can be used to treat neuropathic pain and / or inflammatory pain, it does not provide a crystallized form of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol that can be used in clinical applications.

[0010] Therefore, the object of the present invention is to provide a crystalline form of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol, which is excellent in terms of efficacy and safety in the treatment of neuropathic pain and / or inflammatory pain.

[0011] In addition, through extensive research, the inventors have surprisingly discovered a synthetic route for 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol and obtained a new crystal form, successfully solving the problems existing in the prior art. The new crystal form has the advantages of excellent physicochemical properties, good stability, and is more suitable for industrial preparation.

[0012] This invention provides crystals of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol, which have the following powder X-ray data:

[0013] The crystallization was measured using Cu-Ka, and characteristic peaks were observed at interplanar spacings d of 13.8°±0.2°, 16.0°±0.2°, 17.1°±0.2°, 21.6°±0.2°, 22.1°±0.2°, 22.5°±0.2°, 23.7°±0.2°, 24.7°±0.2°, and 31.7°±0.2° in powder X-ray diffraction.

[0014] The present invention provides a synthetic route for 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol as follows:

[0015]

[0016] This invention also provides a pharmaceutical product containing the novel crystals of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol provided by this invention. The 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol provided by this invention and the 5-[2-[(6-amino)-9H-purin-9-yl]ethylamino]-1-pentanol described in Japanese Priority Application JP2016- are different representations of the same compound.

[0017] The drug provided by this invention is an inhibitor of adenylate cyclase 1.

[0018] The drug provided by this invention is an analgesic.

[0019] The medicine provided by this invention is a medicine for treating neuropathic pain and / or inflammatory pain.

[0020] The drug provided by this invention is a drug for treating neurogenic pain.

[0021] The drug provided by this invention is a drug for treating anxiety caused by neuropathic pain and anxiety and depression related to other diseases.

[0022] The medicine provided by this invention is a medicine for treating chronic visceral pain and related anxiety and depression.

[0023] The present invention also provides the use of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol crystals in the preparation of medicaments for treating neuropathic pain and / or inflammatory pain.

[0024] The present invention also provides the use of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol crystals in the preparation of medicaments for treating anxiety caused by neuropathic pain and anxiety and depression associated with other diseases.

[0025] The present invention also provides the use of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol crystals in the preparation of medicaments for treating chronic visceral pain and related anxiety and depression.

[0026] The present invention also provides the use of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol crystals in the treatment of neuropathic pain and / or inflammatory pain.

[0027] The present invention also provides the use of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol crystals in the treatment of anxiety and depression caused by neuropathic pain and other diseases.

[0028] The present invention also provides the use of 5-((2-(6-amino-9H-purine-9-yl)ethyl)amino)pentane-1-pentanol crystals in the treatment of chronic visceral pain and its associated anxiety and depression. Attached Figure Description

[0029] Figure 1 It is the crystalline XRPD involved in the embodiments of the present invention.

[0030] Figure 2 This is a photograph of the crystals involved in the embodiments of the present invention under a polarizing microscope.

[0031] Figure 3 This is a TGA diagram of the crystals involved in the embodiments of the present invention.

[0032] Figure 4 This is a DSC diagram of crystallization involved in the embodiments of the present invention.

[0033] Figure 5 This is cycle 1 of the adsorption-desorption curve of the crystal involved in the embodiment of the present invention, which shows the water adsorption kinetics of the crystal at 25°C.

[0034] Figure 6 The adsorption / desorption isothermal cycle of the crystal involved in the embodiments of the present invention shows the water adsorption kinetics of the crystal at 25°C.

[0035] Figure 7 This is a graph showing the results of pain behavior testing in rats after modeling. Detailed Implementation

[0036] The 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol of the present invention has the following structure:

[0037]

[0038] In this invention, the compound is designated as NB001.

[0039] The compound NB001 of the present invention can be synthesized and purified by the following methods:

[0040]

[0041] Compound 1 is 9-(2-hydroxyethyl)adenine, with the English name 2-(6-Aminopurin-9-yl)ethanol, which is a commercially available product, such as products of Bailingwei Technology Co., Ltd., Shanghai Mairui Chemical Technology Co., Ltd., or INTATRADE GmbH of Germany. In the embodiments of this invention, compound 1 was purchased from Shanghai Kehua Biotechnology Co., Ltd.

[0042] Compound 3 is 5-amino-1-pentanol, such as products of Wako Kogyo Co., Ltd. or HBCChem, Inc. In this embodiment of the invention, compound 3 was purchased from TCI.

[0043] Compound 2 was prepared by substitution reaction using compound 1 and thionyl chloride according to the method described above. The substitution reaction was carried out by conventional methods or in a solvent that would not affect the reaction.

[0044] Examples of solvents that do not affect the reaction include ethers such as dioxane, tetrahydrofuran, and 1,2-dimethoxyethane; halogenated hydrocarbons such as chloroform; aromatic hydrocarbons such as toluene; amides such as N,N-dimethylformamide; and sulfoxides such as dimethyl sulfoxide. Two or more of these solvents can be mixed in appropriate proportions. The amount of these solvents relative to compound 1 is, for example, 1 to 100 times the volume.

[0045] The reaction temperature is typically from about 20°C to about 250°C, preferably from 20°C to 120°C.

[0046] The reaction time is typically from about 0.5 to about 36 hours.

[0047] Compound 2 obtained in this way can be separated and purified using known separation and purification methods, such as concentration, vacuum concentration, solvent extraction, crystallization, recrystallization, phase transfer, and chromatography. It should be noted that compound 2 can be used directly in subsequent reactions without separation.

[0048] Next, compound 2 and compound 3 undergo a condensation reaction to obtain compound NB001 of the present invention.

[0049] The reaction was carried out using conventional methods in a solvent that would not affect the reaction.

[0050] Examples of solvents that do not affect the reaction include hydrocarbons such as hexane; alcohols such as methanol; ethers such as tetrahydrofuran; esters such as ethyl acetate; halogenated hydrocarbons such as chloroform; aromatic hydrocarbons such as toluene; amides such as N,N-dimethylformamide; and sulfoxides such as dimethyl sulfoxide. Two or more of these solvents can be mixed in appropriate proportions. The amount of these solvents relative to compound 2 is, for example, 1 to 100 times the volume.

[0051] The reaction temperature is typically from about 20°C to about 250°C, preferably from 20°C to 120°C.

[0052] The reaction time is typically from about 0.5 to about 24 hours.

[0053] The resulting compound NB001 can be separated and purified using well-known methods, such as concentration, vacuum concentration, solvent extraction, crystallization, recrystallization, phase transfer, and chromatography. It should be noted that compound NB001 can be used directly in subsequent experiments without further separation.

[0054] Examples of solvate crystallization of compound NB001 include, for example, alcohol solvate crystallization such as methanol solvate crystallization, ethanol solvate crystallization, etc.; organic solvent hydrate crystallization having water and organic solvent (e.g., alcohol solvate hydrate crystallization, such as methanol hydrate crystallization, ethanol hydrate crystallization, etc.), etc.

[0055] The crystallization of the present invention can be produced by transforming the amorphous compound NB001, or other crystals of compound NB001, into a crystal form. This crystal form transformation is a phenomenon where the crystal structure changes when the temperature or pressure exceeds a certain level.

[0056] Examples of methods for crystal transformation include those known per se, such as crystallization by solution (e.g., concentration, slow cooling, reaction (diffusion, electrolysis), hydrothermal growth, fluxing), crystallization by vapor (e.g., vaporization (sealed tube, gas flow), gas-phase reaction, chemical transport), crystallization by melt (e.g., conventional freezing (lifting, temperature gradient, Bridgman), zone melting (zone homogenization, floating zone), special growth methods (VLS, liquid phase epitaxy), stream fog (where crystals are dissolved in a solvent and, after filtration, the solvent is evaporated under atmospheric conditions), slurry method (where crystals are added to a solvent such that excess solid is retained therein to obtain a suspension, which is stirred at room temperature or under heating or cooling conditions, and the solid is collected by filtration), and methods such as vacuum drying, grinding, pulverizing, pressurizing, etc.

[0057] To obtain the crystals of the present invention, a slurry method is particularly preferred in the above-described method. Specifically, the following method is preferred: adding the crystals of compound NB001 to a solvent, such that excess solid is retained therein, to obtain a suspension; stirring the suspension; and collecting the solid by filtration. Solvents used include, for example, aromatic hydrocarbons (e.g., benzene, toluene, xylene, etc.), halogenated hydrocarbons (e.g., dichloromethane, chloroform, etc.), saturated hydrocarbons (e.g., hexane, heptane, cyclohexane, etc.), ethers (e.g., diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, etc.), nitriles (e.g., acetonitrile, etc.), ketones (e.g., acetone, etc.), sulfoxides (e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide, etc.), esters (e.g., ethyl acetate, etc.), alcohols (e.g., methanol, ethanol, 2-propanol, etc.), and water. These solvents can be used alone or in mixtures of two or more in appropriate proportions (e.g., 1:1 to 1:100). Alcohols (e.g., 2-propanol), ketones (e.g., methyl ethyl ketone), and esters (e.g., ethyl acetate) are preferred, and ketones (e.g., methyl ethyl ketone) are even more preferred.

[0058] The amount of solvent used, relative to the crystallization of compound NB001 (1g), is typically about 5 mL to about 65 mL, preferably about 5 mL to about 25 mL.

[0059] The suspension is preferably stirred at room temperature or about 30°C to about 60°C, more preferably about 30°C to about 60°C. In this specification, room temperature refers to about 15°C to about 30°C. The stirring time at about 30°C to about 60°C is typically about 30 minutes to about 4 hours, preferably about 2 hours to about 4 hours. The cooling temperature is room temperature. The stirring time under cooling conditions is typically about 30 minutes to about 24 hours, preferably about 30 minutes to about 2 hours. Crystals in the suspension can be separated by methods known per se, such as filtration. The filtration temperature is room temperature, preferably about 20°C to about 30°C.

[0060] Alternatively, the suspension can be stirred at about 0°C to about 10°C, and then the crystals can be collected by filtration at about 0°C to about 10°C.

[0061] The crystals of the present invention can be obtained by drying the obtained crystals using methods known per se. The drying can be carried out by reducing pressure or by ventilation. The drying temperature is preferably not higher than about 60°C, more preferably about 45°C to about 55°C.

[0062] The crystals other than those of the present invention can be prepared by other known methods.

[0063] X-ray diffraction crystallography is typically used to analyze the obtained crystals. In addition, crystal orientation can also be determined by mechanical or optical methods (such as FT-Raman spectroscopy, solid-state NMR spectroscopy, etc.).

[0064] The peaks in the spectra obtained by the above analytical methods will inevitably have a certain degree of measurement error. Crystallization with spectral peaks within the error range is also included in the crystallization of this invention. For example, "±0.2" in the interplanar spacing (d) of powder X-ray diffraction means that this error is acceptable.

[0065] Example

[0066] Example 1: Synthesis of compound NB001

[0067] Compound NB001 was synthesized according to the following synthetic route.

[0068]

[0069] (1) Synthesis and purification of intermediate 2:

[0070] Compound 1 (20.00 g, 111.62 mmol, 1.00 eq) was dissolved in dioxane (600.00 mL), and then SOCl2 (26.56 g, 223.24 mmol, 16.20 mL, 2.00 eq) was slowly added to the above reaction solution. The mixture was stirred at 100 °C for 4 hours. LCMS analysis showed that the starting material reacted completely and the desired product was formed. The solvent in the reaction solution was removed under reduced pressure using a water pump. The gray residue was added to 100 mL of ethanol and stirred for 10 minutes. The mixture was filtered through a sintered glass funnel. 100 mL of saturated sodium carbonate solution was added to the filtered solid, and the mixture was stirred for 20 minutes. The solid was then filtered again through a sintered glass funnel. The solid was evaporated to dryness under reduced pressure using a water pump to obtain crude intermediate 2 (19.60 g, 98.59 mmol, yield 88.32%, purity 99.4%), which was directly proceeded to the next step without further purification.

[0071] (2) Synthesis and purification of NB001:

[0072] Compound 2 (19.60 g, 99.18 mmol, 1.00 eq) was dissolved in n-butanol (390.00 mL), and then compound 3 (30.69 g, 297.54 mmol, 3.00 eq) was added to the above reaction solution. The mixture was stirred at 110 °C for 18 hours. LC-MS analysis showed that the starting material reacted completely and the desired product was formed. The solvent was removed by pumping water under reduced pressure, and the product was concentrated to obtain a yellow crude product. 196 mL of DMF was added to the yellow crude product, and the mixture was stirred at -40 °C for 1 hour. The mixture was then filtered through a sintered glass funnel, and 200 mL of ethyl acetate was added to the filtered solid. The mixture was filtered again to obtain a grayish-white solid NB001 (19.74 g, 71.38 mmol, yield 71.97%, purity 95.587%).

[0073] Example 2: Preparation of a new crystal form of NB001

[0074] Weigh 2.0 mg of the active pharmaceutical ingredient (API) into a 7 mL vial. Add appropriate amounts of methanol, ethanol, isopropanol, n-butanol, acetonitrile, acetone, ethyl acetate, 2-methyltetrahydrofuran, tetrahydrofuran, dioxane, n-pentanol, toluene, isopropanol + water (3:1 mass ratio), acetonitrile + water (3:1 mass ratio), acetone + water (3:1 mass ratio), and ethanol + water (3:1 mass ratio). Shake rapidly to dissolve the compound until the solution becomes clear. The approximate solubility of the API in organic solvents and mixed solvents is shown in Table 1.

[0075] Table 1 Approximate solubility of the active pharmaceutical ingredient in organic solvents and mixed solvents.

[0076]

[0077] Weigh 50 mg of the active pharmaceutical ingredient according to Table 2 and place it in a 2 mL vial. Add appropriate amounts of acetonitrile, tetrahydrofuran, acetone, isopropanol, ethyl acetate, and ethanol to form a suspension. Stir the suspension in a homogenizer at 40°C for two days. Centrifuge the suspension and dry it at 40°C. Methanol, ethanol + water (mass ratio 3:1), isopropanol + water (mass ratio 3:1), and acetonitrile + water (mass ratio 3:1) were dissolved completely and allowed to evaporate naturally for 3 days. Perform XRPD on the resulting solids and compare them with the active pharmaceutical ingredient.

[0078] The X-ray powder diffractometer (XRPD) used was a Panaco Rheinland from the Netherlands, with the following parameters: tube: copper: K-Alpha Generator: Voltage: 40kV, Current: 40mA; Scanning range: 4–40 degrees; Sample rotation speed: 15 rpm; Scanning speed: 10 degrees / min; Results as follows Figure 1 As shown.

[0079] Table 2. Proportions of various solutions used in the crystallization of active pharmaceutical ingredients.

[0080] 1 Acetonitrile 52.2 1.0 40℃ 2 Tetrahydrofuran 51.7 1.0 40℃ 3 acetone 50.5 1.0 40℃ 4 methanol 51.2 1.0 40℃ 5 Isopropanol 52.4 1.0 40℃ 6 Ethyl acetate 51.8 1.0 40℃ 7 ethanol 51.2 1.0 40℃ 8 ethanol + water 50.9 1.0 40℃ 9 Acetonitrile + water 51.8 1.0 40℃ 10 Isopropanol + Water 50.1 1.0 40℃

[0081] Table 3 Powder X-ray Diffraction Data

[0082] 13.8°±0.2° 6.41 17.4 16.0°±0.2° 5.53 12.8 17.1°±0.2° 5.17 14.3 21.6°±0.2° 4.10 20.6 22.1°±0.2° 4.02 47.7 22.5°±0.2° 3.95 100.0 23.7°±0.2° 3.75 17.6 24.7°±0.2° 3.59 27.6 31.7°±0.2° 2.81 25.2

[0083] Example 3: Other physical properties of the new NB001 crystal form

[0084] Physical characterization of the active pharmaceutical ingredient was performed using PLM, TGA, DSC, and DVS. Results are shown in [Figure number missing]. Figures 1 to 5 .

[0085] (1) Polarizing microscope (PLM)

[0086] The solid sample was dispersed in silicone oil and observed under a polarized light microscope using a 10x eyepiece and 20 / 50x objectives. The microscope used was a Nikon Polarized Light Microscope-Nikon Eclipse LV 100POL; the image under the 20x objective is shown below. Figure 2 As shown.

[0087] (2) Thermogravimetric analysis (TGA) method

[0088] Place 2–5 mg of sample in a sample dish and heat from room temperature to 300 °C at a rate of 10 °C / min. Results are as follows: Figure 3 As shown.

[0089] (3) Differential Scanning Calorimetry (DSC) method

[0090] DSC method: An appropriate amount of sample is placed in a specific aluminum pan and heated from 25°C to 300°C at a rate of 10°C / min. The results are as follows. Figure 4 As shown.

[0091] (4) Dynamic vapor adsorption (DVS)

[0092] Take approximately 20 mg of sample onto the sample tray and place it into the instrument for testing.

[0093] The parameters are as follows:

[0094] Temperature: 25℃

[0095] Equilibrium: dm / dt: 0.01% / min (shortest: 10min, longest: 180min)

[0096] Drying: Dry at 0% RH for 120 min

[0097] RH (%) test step: 10%

[0098] RH (%) test range: 0%-90%-0%

[0099] The results of dynamic vapor adsorption are as follows Figure 5 and Figure 6 As shown.

[0100] Example 4: Stability of the NB001 crystal form

[0101] Tests on factors affecting the crystal form of the compound, and long-term and accelerated stability tests:

[0102] In accordance with the "Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Preparations" (Chinese Pharmacopoeia 2015, Part IV, General Chapter 9001), the stability of the crystal form of the compound was investigated under the conditions of high temperature (60℃), high humidity (92.5%RH), strong light (5Klx), 40℃ / 75%RH (accelerated test) and 25℃ / 60%RH (long-term test).

[0103] Take 10 mg of the crystalline sample involved in the embodiments of this invention, weigh it accurately, and place it at the bottom of a glass sample bottle, spreading it into a thin layer. For samples subjected to high temperature, high humidity, and long-term testing, seal the bottle opening with aluminum foil, making small holes in the foil to ensure sufficient contact between the sample and the environmental conditions; for samples subjected to strong light and accelerated testing, seal with a threaded cap. The placement conditions and time points are shown in Table 4. Samples placed under different conditions were analyzed on days 5, 10, and 30. The analysis results were compared with the initial detection results on day 0. The stability test results are shown in Table 5.

[0104] Table 4 shows the sampling conditions and time points for the crystallization stability test involved in the embodiments of the present invention.

[0105]

[0106] *Test item X: Properties, content and related substances.

[0107] Table 5. Results of the stability test of crystallization involved in the embodiments of the present invention.

[0108]

[0109] The above results show that the compound has good crystal stability under various test conditions.

[0110] Example 5: The therapeutic effect of NB001 crystals as an analgesic on cancer pain in rats.

[0111] (I) Method:

[0112] 1. Animals: 50 SD rats (180-220g) were kept in a quiet, warm (22℃) environment away from strong light, with free access to water and food.

[0113] 2. Cell Culture: The Walker 256 syngeneic cancer cell line from SD rats was preserved by the Animal Center of the Fourth Military Medical University; commercially available cell lines can also be used. RPMI 1640 medium (Gibco, USA, containing 10% fetal bovine serum, 100 U / ml each of penicillin and streptomycin) was used. Tumor cells were cultured in suspension at 37°C in a 5% CO2 incubator, with medium changed every other day and passaged every 2 days. Third-generation cells were collected at a concentration of 2 × 10⁶ cells / mL. 5 / 10μl.

[0114] 3. Establishment of a rat model of bone cancer pain: After anesthesia (sodium pentobarbital 40 mg / kg), a small incision was made at the left knee joint using aseptic surgical techniques. The muscles were gently dissected and the patellar ligament was cut to expose the distal femur. A 1 mm syringe needle was inserted vertically into the medullary cavity from the intercondylar fossa, with an insertion distance not exceeding 5 mm to prevent perforation. Then, a 20 μl microsyringe was used to slowly inject 10 μl of a suspension containing Walker 256 cells (each 10 μl contains 2 × 10⁻⁶ cells) into the medullary cavity. 5 (One cancer cell). After injection and a few minutes, the hole was quickly sealed with sterile bone wax and 75% alcohol was used to kill any tumor cells that had leaked out of the bone marrow cavity. The holes were then disinfected and sutured layer by layer. The sham surgery group underwent the same surgical procedure, but only the same volume of sterile saline was slowly injected into the bone marrow cavity.

[0115] 4. Imaging examination (X-ray): SD rats were anesthetized on the 7th, 14th and 21st days after surgery, and X-rays were taken of their left hind limbs to assess the degree of tumor-induced bone destruction and hyperplasia.

[0116] 5. Histopathological examination (HE staining): On the 21st day after rat modeling, the rats were euthanized after anesthesia. The left femur was fixed with 4% paraformaldehyde solution for 1 week, and then decalcified in formic acid-hydrochloric acid composite decalcification solution for 1 week. After routine dehydration, paraffin sections (Leica) were prepared, routine HE staining was performed, and the bone structure destruction was observed under a microscope (Olympus BX53).

[0117] 6. Drug Intervention: On the 18th day after surgery, the SD rats in the surgical group were randomly divided into 6 groups: model group; NB001 non-crystalline group (20 mg / kg, ig); the crystalline group of the present invention (20 mg / kg, ig); morphine (2 mg / kg, ip); and gabapentin (100 mg / kg, ig). The new crystalline and non-crystalline forms of the AC1 inhibitor NB001 were prepared with sterile saline. Morphine and gabapentin were used as positive control drugs. All drugs were administered twice daily for 3 days. The pain threshold was measured within 2 hours after the last administration on the 4th day.

[0118] 7. Rat pain behavior assessment:

[0119] Heat-induced paw shrinkage latency (PWL): Measured using a PL-200 thermal pain stimulation device (8V, 50W, 100% intensity). The time from irradiation of the mid-stomach of the affected side of the rat to the rat raising its leg to avoid the stimulation was defined as TWL. To prevent tissue damage, a cutoff time of 80 seconds was set. Measurements were taken three times per rat, with a 5-minute interval between each measurement, and the average value was recorded.

[0120] The 2-hour post-administration latency (PWL) of heat-induced foot contraction was determined after the 7th administration (21 days): Compared with the model group, the non-crystalline group of NB001 (20 mg / kg, ig); the crystalline group of the present invention (20 mg / kg, ig); morphine (2 mg / kg, ip); and gabapentin (100 mg / kg, ig) all showed better analgesic effects. Figure 7 As shown, the analgesic effect of the crystalline group (20 mg / kg, ig) of the present invention is extremely significant (P < 0.01).

[0121] Example 6: The effect of NB001 crystals in inhibiting chronic pain and anxiety

[0122] Chronic visceral pain model

[0123] Visceral pain was induced in mice by intracolonic injection of yeast polysaccharide derived from *Saccharomyces cerevisiae* (Sigma-Aldrich), in which a glucan is attached to the yeast cell wall and identified as a protein-carbohydrate complex. Specifically, mice were anesthetized by inhalation of 1-3% isoflurane, then fed a 24mm long, size 22 plastic feeding tube. A 0.1ml suspension of yeast polysaccharide (prepared to a 30mg / ml solution with physiological saline) was injected into the colon over 2 minutes. The control group received 0.1ml of physiological saline. Yeast polysaccharide or physiological saline was injected daily for 3 consecutive days. Visceral pain behavioral tests were performed according to Laird's method. The number of times the abdomen was licked within 10 minutes was recorded, including behaviors such as grooming, full-body stretching, pressing the abdomen to the floor, and arching postures for 1-2 seconds. Open field tests were performed on days 1, 7, and 14 after intracolonic injection, from 9:00 AM to 12:00 PM. The entire test was double-blind. Mice were acclimatized in an observation room for 30 minutes before the behavioral tests. The mouse was placed in a new open area (43.2×43.2×30.5cm). 3 In the middle of the room, there is a dim light (<50 lux) and a fan. The animal's movement distance, vertical count, movement count, stereotyped behavior count, and jumping count are recorded over 30 minutes using an activity monitoring system equipped with multiple pairs of beams.

[0124] Behavioral anxiety test

[0125] Anxiety was modeled using an existing animal model—the elevated plusmaze test (EPM). Mice were acclimatized indoors for 30 minutes prior to behavioral observation. The EPM consisted of two open arms (250 lux) and two closed arms (350 lux) set up facing each other. For each test, a single animal was placed in the center of the test position and allowed free movement for 5 minutes. The number of times the animal entered each arm and the time taken were recorded.

[0126] Light / Dark Box Test: The test method is performed according to a modified method in the prior art. The apparatus consists of a rectangular plexiglass box (44×8.5×25cm). 3 The plexiglass enclosure is divided into equal-sized bright and dark compartments, separated by doors. A 60-watt (400 lux) lamp is lit in the bright compartment, positioned 30 cm above the enclosure. Each animal is placed in the dark compartment for 20 seconds before the door to the bright compartment is opened. The time each animal spends in the dark compartment and the time it enters the bright compartment are recorded over 10 minutes.

[0127] result

[0128] Mice treated with yeast polysaccharides were administered physiological saline, non-crystalline NB001 (3 mg / kg), crystalline form of the present invention (3 mg / kg), and gabapentin (30 mg / kg) as a positive control via intraperitoneal injection (IP). Mouse behavior was observed 28 days after yeast polysaccharide treatment. The results are shown in Table 6. Both non-crystalline NB001 (3 mg / kg) and crystalline form of the present invention (3 mg / kg) showed significant therapeutic effects compared to the positive control group. Furthermore, the therapeutic effect of the crystalline form of the present invention was extremely significant.

[0129] Table 6. The therapeutic effects of the crystals of this invention on chronic pain.

[0130] physiological saline 8 27.5±2.4 Old NB001 7 19.6±1.2 New NB001 8 11.0±0.7 Gabapentin 8 20.7±1.9

[0131] The other two sets of experiments were to test the inhibitory effect of the crystals of this invention on anxiety caused by chronic pain and neuropathic pain.

[0132] The EPM test examined the anxiety behavior of mice after experimental treatment. Mice treated with yeast polysaccharides were administered saline, non-crystalline NB001 (3 mg / kg), the crystalline form of this invention (3 mg / kg), and gabapentin (30 mg / kg) as a positive control via intraperitoneal injection (IP). Mouse behavior was observed 28 days after yeast polysaccharide treatment. The results are shown in Table 7. Both non-crystalline NB001 (3 mg / kg) and the crystalline form of this invention (3 mg / kg) showed significant therapeutic effects compared to the positive control group. Furthermore, the therapeutic effect in the crystalline group of this invention was extremely significant.

[0133] Table 7 EPM Test Results

[0134]

[0135] To confirm the effect of the crystals of this invention on anxiety suppression, a light / dark box test was conducted on mice, and the results are shown in Table 8. Mice treated with yeast polysaccharides were administered physiological saline, non-crystalline NB001 (3 mg / kg), the crystals of this invention (3 mg / kg), and gabapentin (30 mg / kg) as a positive control via intraperitoneal injection (IP). Mouse behavior was observed 28 days after yeast polysaccharide treatment. The results, as shown in Table 8, indicate that both non-crystalline NB001 (3 mg / kg) and the crystals of this invention (3 mg / kg) showed significant therapeutic effects compared to the positive control group. Furthermore, the therapeutic effect of the crystal group was extremely significant.

[0136] Table 8 Results of Open / Dark Box Tests

[0137]

Claims

The use of crystals of 1,5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-pentanol in the preparation of a medicament for treating anxiety caused by neuropathic pain, characterized in that... The crystallization was measured using Cu-Ka, and the interplanar spacing d was 13.8 mm according to powder X-ray diffraction. o ±0.2 o 16.0 o ±0.2 o 17.1 o ±0.2 o 21.6 o ±0.2 o 22.1 o ±0.2 o 22.5 o ±0.2 o 23.7 o ±0.2 o 24.7 o ±0.2 o 31.7 o ±0.2 o Characteristic peaks are displayed at this location; The crystal has the following powder X-ray data: 。

Images