Solid form of hydroxyamide compound and preparation method and application thereof

Abstract

The invention relates to a solid form of a hydroxyamide compound as well as a preparation method and application of the solid form, provides a pharmaceutically acceptable salt of the compound shown in the formula (I), the pharmaceutically acceptable salt is inorganic acid salt or organic acid salt, and further provides a preparation method of the salt form of the compound. The salt form of the compound shown in the formula (I) has excellent physical and chemical stability, is beneficial to medicine storage, and is also beneficial to effective control of the curative effect and safety of the medicine.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical chemistry, and specifically relates to a solid form of a compound of formula (I), particularly disclosing its salt form and preparation method thereof; pharmaceutical compositions comprising the solid form of the compound of formula (I), and the use of said solid form in the preparation of medicaments for the prevention or treatment of diseases mediated by LSD1 and / or HDAC. Background Technology

[0002] Histone demethylase (LSD1) is a flavin adenine dinucleotide (FAD)-dependent aminooxidase that specifically removes mono- and dimethyl groups from histone H3K4. Furthermore, LSD1 can remove methyl groups from non-histone proteins such as p53, DNMT1, STAT3, E2F1, MYPT1, ERa, and HIF-1, further regulating the stability and activity of their downstream groups. It plays a crucial regulatory role in many cellular processes, such as promoting tumor cell proliferation, inhibiting energy metabolism, promoting lipid synthesis, inhibiting lipolysis, and regulating cell differentiation. Inhibiting LSD1 function can enhance the expression of endogenous retroviral elements (ERVs) and inhibit the function of the RISC (RNA-induced silencing complex) complex, leading to overexpression of double-stranded RNA (dsRNA) and activation of type I interferon (IFN), thereby enhancing the efficacy of tumor immunotherapy (Doll, S., Kriegmair, MC, Santos, A., Wierer, M., Coscia, F., Neil, HM, et al. Molecular Oncology, 2018, 12(8), 1296–1307.).

[0003] LSD1 is widely expressed in the body, with lower secretion in the liver, pancreas, and salivary glands, higher expression in the testes, and similar expression levels in other tissues. Researchers have found an overexpression trend of LSD1 in some cancer types using public databases of human cancers. LSD1 expression levels were significantly elevated in various tumor tissues, including neuroblastoma, breast cancer (Wang, Y.; Zhang, H.; et al., Cell 2009, 138(4), 660-72.), prostate cancer (Zhao, L.-J.; Fan, Q.-Q.; et al., Pharmacol. Res. 2020, 159, 104991), pancreatic cancer (Sehrawat, A.; Gao, L.; et al., Pr℃. Nat. Acad. Sci. USA 2018, 115(18), E4179-E4188.), colon cancer, glioma, and hematologic malignancies (Hatzi, K.; Geng, H.; et al., Nature Immunology). 2019, 20(1), 86-96.), and high expression of LSD1 is often associated with poor tumor prognosis and recurrence after treatment (Lynch, J.; Harris, W.; et al., Expert Opinion on Therapeutic Targets 2012, 16(12), 1239-1249.). In addition, the survival time of patients with high LSD1 expression is significantly shortened, which also suggests that LSD1 overexpression is a poor prognostic factor. More and more reports indicate that LSD1, as an epigenetic regulator, participates in various tumor processes and embryonic development. Therefore, the development of LSD1 inhibitors is one of the hot topics in the field of tumor research.

[0004] Histone deacetylases (HDACs) are a class of epigenetic regulatory proteins responsible for removing acetyl groups from the tail of histones. They have a wide range of biological functions, including neurodegeneration, inflammation, metabolic disorders, and cancer. They are divided into four classes: Class I includes HDAC1, 2, 3, and 8; Class II includes HDAC4, 5, 6, 7, 9, and 10; Class IV is HDAC11; and Class III includes SIRT1 to SIRT7. Classes I, II, and IV are collectively referred to as HDACs, which are highly conserved zinc-dependent deacetylases. Histone acetylation is jointly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histones in transcriptionally activated regions often exhibit a highly acetylated state, while deacetylation typically results in gene silencing. The mechanism of action of HDACs is to remove the acetyl group of lysine residues of nucleosome histones, thereby reducing the space between the nucleosome and DNA. At the same time, the histones return to positive charge, enhancing the electrostatic interaction between the nucleosome and DNA, causing the DNA to wrap tightly around the nucleosome, thus hindering the binding of transcription factors to DNA and inhibiting the transcription process (Yan L., et al, Current Topic in Medicinal Chemistry, 2019, 19, 223).

[0005] In various malignant tumors such as leukemia, lymphoma, cervical cancer, colorectal cancer, and breast cancer, the expression and activity of HDAC family members are significantly upregulated. Furthermore, the poor prognosis of these malignant tumors is positively correlated with high HDAC expression. Currently, five HDAC inhibitors have been approved for the treatment of various cancers. Vorinostat, romidepsin, and belinostat have been approved for the treatment of cutaneous T-cell lymphoma, while panibinostat has been approved for the treatment of multiple myeloma. Chidamide has been approved in China for the treatment of relapsed and refractory peripheral T-cell lymphoma, and several other HDAC inhibitor candidates are in clinical trials.

[0006] As two important histone epigenetic regulatory proteins, LSD1 and HDACs are both closely related to the occurrence and development of cancer, and there is a strong correlation between them. LSD1 and HDAC1 / 2 co-exist in multiple co-repressive complexes such as NuRD, CoREST, and Sin3A, participating in the regulation of transcription of various genes. LSD1 activity is regulated by HDACs; inhibiting HDAC activity also inhibits LSD1 activity. In various cancers, including bladder cancer, breast cancer, and lung cancer, reducing LSD1 expression or inhibiting LSD1 activity can significantly enhance the sensitivity of cancer cells to HDAC inhibitors.

[0007] Studies have found that the combination of LSD1 and HDAC small molecule inhibitors has a significant synergistic effect on the inhibitory activity of various tumors. Huang et al. found that the combined use of LSD1 and HDAC inhibitors can increase the levels of H3K4me2 and AcH3K9, and show a synergistic effect in inhibiting breast cancer cell growth (Huang, Y., et al., Breast Cancer Res. Treat., 2012, 131, 777-789). Vasilatos et al. also reported the simultaneous use of LSD1 and HDAC inhibitors to treat triple-negative breast cancer (Vasilatos, SN, et al., Carcinogensis, 2013, 34, 1196-1207). However, due to the different pharmacokinetic properties of the drugs, combination therapy may pose challenges to clinical research.

[0008] Studies on dual-target inhibitors of LSD1 and HDAC have shown that small molecule inhibitors simultaneously inhibiting the activity of LSD1 and HDACs have a synergistic antitumor effect. These studies also indicate that dual-target drugs have more predictable metabolic pathways compared to multidrug combinations, superior pharmacokinetic / pharmacodynamic properties, and better bioavailability (Giulia S.; et al, Current Opinion in Chemical Biology 2019, 50, 89-100). Furthermore, compared to combination therapy, dual-target drugs ensure that both pharmacophores act synchronously in the same cells at the same time (de Lera, AR; Ganesan, A., Clin Epigenetics 2016, 8:105). Additional advantages of dual-target monotherapy include improved patient compliance and reduced treatment costs (Fu, RG, Sun, Y., Sheng, WB, Liao, DF, Eur. J. Med. Chem. 2017, 136, 195-211). Corin, an LSD1 / HDAC dual-target inhibitor, acts on the CoREST complex and has shown superior activity in multiple cell lines compared to single LSD1 or HDAC inhibitors (Kalin, JH, et al, Nat. Commun., 2018, 9, 1-13). 4-SC-202, another LSD1 / HDAC dual-target inhibitor, inhibits cancer cell growth in multiple cell lines and is currently in clinical trials (Hoffman, MM, et al, Cancers, 2020, 12, 756). The recently reported LSD1 / HDAC6 dual-target compound JBI-802 has shown significant efficacy and good tolerability in leukemia models and is in clinical trials (Dhanalakshmi, S., et al, Blood, 2020, 136, 29). Cole and colleagues reported a class of LSD1 / HDAC1 dual-target inhibitors that showed good in vivo activity in a mouse model of melanoma (Kalin, JH; et al; Nat. Commun., 2018, 9, 53). Other academic institutions, such as Xinxiang Medical University, have also recently published several patents for LSD1 / HDAC dual-target inhibitors (CN111592487; CN113444038; CN113527195). It is evident that the advantages of dual-target inhibitors are increasingly valued by scientists.

[0009] PCT application (PCT / CN2023 / 095304) discloses a hydroxyamide compound of formula (I) that exhibits excellent inhibitory activity against both LSD1 and HDAC, and also shows good inhibitory activity against drug-resistant cells. The entire contents of that application are incorporated herein by reference.

[0010]

[0011] Different solid forms of the same drug may possess different properties, such as better processability, favorable powder properties, better solubility and stability, and higher bioavailability, thus affecting drug formulation preparation and further influencing the efficacy and safety of the drug. Therefore, studying the solid form of compounds, discovering and obtaining superior salt or crystal forms to facilitate drug processing, and providing more information for efficacy studies of solid drugs is of great significance. Summary of the Invention

[0012] Salt and its preparation method

[0013] This application provides a pharmaceutically acceptable salt of the compound shown in formula (I), wherein the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

[0014]

[0015] The chemical name of the compound shown in formula (I) provided in this application is: (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidine-4-yl)amino)methyl)-phenyl)-N-hydroxyacrylamide.

[0016] In some embodiments, the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application is selected from p-toluenesulfonate, sulfate, methanesulfonate, benzenesulfonate and hydrochloride.

[0017] In some embodiments, in the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application, the molar ratio of the compound represented by formula (I) to the acid molecule is selected from 1:1 to 1:2; preferably, the molar ratio of the compound represented by formula (I) to the acid molecule is 1:1.

[0018] In a first aspect, the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application is a p-toluenesulfonate, wherein the molar ratio of the compound represented by formula (I) to p-toluenesulfonic acid molecules is 1:1.

[0019] Furthermore, the p-toluenesulfonate of the compound shown in formula (I) provided in this application has the following structure:

[0020]

[0021] Secondly, the pharmaceutically acceptable salt of the compound shown in formula (I) provided in this application is a sulfate, wherein the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1.

[0022] Furthermore, the sulfate of the compound shown in formula (I) provided in this application has the following structure:

[0023]

[0024] Thirdly, the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application is a mesylate, wherein the molar ratio of the compound represented by formula (I) to the mesylate molecule is 1:1.

[0025] Furthermore, the methanesulfonate salt of the compound shown in formula (I) provided in this application has the following structure:

[0026]

[0027] Fourthly, the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application is a benzenesulfonate, wherein the molar ratio of the compound represented by formula (I) to the benzenesulfonic acid molecule is 1:1.

[0028] Furthermore, the benzenesulfonate salt of the compound shown in formula (I) provided in this application has the following structure:

[0029]

[0030] Fifthly, the pharmaceutically acceptable salt of the compound represented by formula (I) provided in this application is a hydrochloride salt, wherein the molar ratio of the compound represented by formula (I) to the hydrochloric acid molecule is 1:1.

[0031] Furthermore, the hydrochloride salt of the compound shown in formula (I) provided in this application has the following structure:

[0032]

[0033] Sixthly, this application provides a method for preparing a pharmaceutically acceptable salt of the compound shown in formula (I), the method comprising the following steps:

[0034] The compound shown in formula (I) is dispersed in an organic solvent, wherein the mass-to-volume ratio of the compound shown in formula (I) to the organic solvent is 0.3 g: (2-20) mL, and then an acid is added, wherein the molar ratio of the compound shown in formula (I) to the corresponding acid is 1: (2-20); nitrogen gas is introduced for protection, and the reaction is stirred at 0-50 °C for 0.5-24 h; the mixture is filtered, the filter cake is collected, and the filter cake is dried under vacuum to obtain the salt of the compound shown in formula (I).

[0035] In some embodiments, the organic solvent is an alcohol solvent; preferably, the organic solvent is selected from isopropanol and ethanol.

[0036] In some embodiments, the mass-to-volume ratio of the compound of formula (I) to the organic solvent is 0.3 g: (3-6) mL.

[0037] In some embodiments, the acid is selected from p-toluenesulfonic acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, and hydrochloric acid.

[0038] In some embodiments, the compound of formula (I) is in a molar ratio of 1:4 with p-toluenesulfonic acid, sulfuric acid or methanesulfonic acid, and in a molar ratio of 1:10 with hydrochloric acid or benzenesulfonic acid.

[0039] In some implementation schemes, the reaction is carried out under nitrogen protection at 20–45°C with stirring for 2–16 hours.

[0040] In some embodiments, the vacuum drying temperature is 30–50°C, preferably 45°C; the vacuum drying time is 10–24 h, preferably 16 h.

[0041] The salt form of the compound shown in formula (I) provided in this application has excellent physical and chemical stability, good solubility, and low hygroscopicity, which is beneficial for the development, storage and transportation of pharmaceutical formulations, as well as for the effective control of drug efficacy and safety.

[0042] Pharmaceutical Compositions and Uses

[0043] This application provides a pharmaceutical composition or pharmaceutical preparation, characterized in that the pharmaceutical composition or pharmaceutical preparation comprises a salt of the compound shown in formula (I) above, and one or more pharmaceutically acceptable carriers.

[0044] The term "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium that is administered co-administered with the therapeutic agent and is suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0045] The pharmaceutical formulations of this application can act systemically and / or locally. For this purpose, they can be administered via suitable routes, such as by injection, intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or transdermal administration; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulations, or inhalation administration.

[0046] For these routes of administration, the formulations of the present invention can be administered in suitable dosage forms.

[0047] The dosage form may be a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form, including but not limited to tablets, capsules, powders, granules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, suspensions, elixirs, and syrups.

[0048] This application provides the use of salts of the compounds represented by formula (I) above, or pharmaceutical compositions or pharmaceutical preparations of this application, in the preparation of medicaments for the prevention and / or treatment of related diseases mediated by LSD1 and / or HDAC, respectively or synergistically.

[0049] This application provides a method for preventing and / or treating related diseases mediated by LSD1 and / or HDAC, individually or synergistically, comprising administering to a patient in need a therapeutically effective amount of a salt of the compound of formula (I) described above or a pharmaceutical composition or pharmaceutical preparation of this application.

[0050] This application provides the use of salts of the compounds of formula (I) described above in the preparation of medicaments for treating diseases mediated by one or more of LSD1 and HDAC.

[0051] In some embodiments, the HDAC enzyme includes, but is not limited to, subtypes such as HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC8, with HDAC1 and HDAC8 subtypes being preferred, and HDAC1 subtype being even more preferred.

[0052] This application provides the use of salts of the compounds shown in formula (I) above, or pharmaceutical compositions or pharmaceutical preparations of this application, in the preparation of medicaments for the prevention and / or treatment of related diseases mediated by LSD1 protein and / or HDAC1 protein, LSD1 protein and / or HDAC8 protein, respectively or synergistically.

[0053] In some implementations, the disease is a disease mediated by abnormal protein activity as described above.

[0054] In some implementations, the disease is cancer or an autoimmune disease.

[0055] In some implementation schemes, the cancer is selected from: non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, chronic myeloid leukemia, colorectal cancer, brain cancer, liver cancer, kidney cancer, stomach cancer, breast cancer, triple-negative breast cancer, skin cancer, melanoma, head and neck cancer, bone cancer, cervical cancer, pelvic cancer, vaginal cancer, oral cancer, lymphoma, leukemia, esophageal cancer, urethral cancer, and nasal cancer.

[0056] The salts of the compounds shown in formula (I) provided in this application not only have excellent effects in the prevention or treatment of LSD1 and / or HDAC-mediated related diseases, but also exhibit excellent chemical stability, physical stability, good solubility, and low hygroscopicity, which are beneficial for drug formulation development, storage and transportation, as well as for the effective control of drug efficacy and safety. Detailed Implementation

[0057] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain this application.

[0058] I. Definition

[0059] The terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other elements or method steps not listed. Those skilled in the art will understand that the foregoing term “comprising” encompasses the meaning of “consisting of.”

[0060] The term "one or more" or similar expression "at least one" can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0061] The term "solid form" includes all solid forms of the compound represented by formula (I), such as crystalline or amorphous forms, salt forms, and crystalline or amorphous forms of salts.

[0062] The term "therapeutic effective dose" refers to a sufficient amount of a non-toxic drug or agent that achieves the desired effect. In embodiments of this disclosure, when treating a patient according to this disclosure, the amount of a given drug depends on many factors, such as the specific dosing regimen, the type and severity of the disease or condition, and the unique characteristics of the patient or host requiring treatment (e.g., weight). However, depending on the specific surrounding circumstances, including, for example, the specific drug used, the route of administration, the condition being treated, and the patient or host being treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. This required dose can conveniently be expressed as a single dose, or concurrent (or over a short period of time) or fractions at appropriate intervals, such as two, three, four, or more doses per day. Those skilled in the art will understand that although the above dosage ranges are given, the specific effective dose can be appropriately adjusted based on the patient's condition and in conjunction with the physician's diagnosis.

[0063] The term "prevention" includes suppressing and delaying the onset of disease, encompassing not only prevention before the disease develops but also prevention of recurrence after treatment.

[0064] The term "treatment" includes curing symptoms, improving symptoms, and suppressing the progression of symptoms.

[0065] The starting material used in the salt preparation method of this application can be any form of the compound shown in formula (I), including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0066] II. Examples

[0067] The following specific embodiments illustrate the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0068] The structures of the compounds in this application were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR measurements were performed using an AVANCE NEO 400MHz Bruker instrument with deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and tetramethylsilane (TMS) as the internal standard. NMR chemical shifts (δ) are given in parts per million (ppm). MS measurements were performed using an ISQ-EC Thermo Fisher LC-MS instrument.

[0069] Unless otherwise specified, the solvents used in this application are those that are commercially available.

[0070] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0071] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0072] The method for detecting anion content in this application is as follows:

[0073] Anion content detection instrument: Thermo ICS-6000 ion chromatograph

[0074] Chromatographic conditions for anion content detection:

[0075] Column: Dionex Ionpac™ AS11-HC, 4 x 250 mm

[0076] Flow rate: 1 mL / min

[0077] Column temperature: 30℃

[0078] Equilibrium temperature: 30℃

[0079] Suppressor model: Anion suppressor, Dinnex ASRS 300, 4mm (ERS-4mm)

[0080] Suppressor current: 174mA

[0081] Detector: Conductivity detector

[0082] Injection volume: 25uL

[0083] Runtime: 25min

[0084] Eluent A: Water; Phase B: 100 mmol / L NaOH solution

[0085] Gradient procedure:

[0086]

[0087] Solution preparation:

[0088] 100 mmol / L NaOH solution: Transfer 5.2 mL of 50% sodium hydroxide solution to 1000 mL of purified water, mix well and sonicate.

[0089] Solvent: Measure 100 mL of acetonitrile into 400 mL of purified water, mix well and sonicate.

[0090] Chloride ion control solution: Take an appropriate amount of sodium chloride, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing approximately 0.5 mg per 1 mL, and then inject it for analysis.

[0091] Sulfate ion control solution: Take an appropriate amount of sodium sulfate, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing approximately 0.5 mg per 1 mL, and then inject it for analysis.

[0092] Test solution: Take an appropriate amount of each salt form of the compound shown in formula (I) prepared in the example, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 0.5 mg of the salt of the compound shown in formula (I) per 1 mL, inject it for analysis, and calculate the external standard method.

[0093] Example 1: Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)-phenyl)-N-hydroxyacrylamide (compound shown in formula (I))

[0094]

[0095] Step 1): Preparation of tert-butyl (1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)carbamate

[0096] The following ingredients were added to a 1000 mL reaction flask: (10.0 g, 19.8 mmol) 1-(5'-bromo-3'-cyano-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)carbamate tert-butyl (10.0 g, 19.8 mmol), 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenol (5.5 g, 21.8 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (0.8 g, 0.99 mmol), cesium carbonate (12.9 g, 39.6 mmol), 1,4-dioxane (100 mL), and water (25 mL). The mixture was purged with nitrogen for protection. The reaction was then carried out at 80–90 °C for 6.0–8.0 h, after which the reaction was stopped. The reaction solution was cooled to 40°C, and ethyl acetate (100 mL) and water (30 mL) were added. The mixture was separated. The organic phase was concentrated to obtain a crude product. Methanol (50 mL) was added to the crude product, and the mixture was stirred at 50°C for 2 h. Then, the temperature was lowered to 20–25°C, and the mixture was stirred for 16 h. The mixture was then filtered. The filter cake was washed twice with methanol (10 mL), and then dried under vacuum at 50°C to obtain tert-butyl (1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)carbamate. Yield: 45.0%;

[0097] Step 2): Preparation of 2'-(4-aminopiperidin-1-yl)-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-3'-nitrile

[0098] (4.8 g, 8.8 mmol) of tert-butyl (1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)carbamate was added to a 100 mL reaction flask, followed by dichloromethane (48 mL), and then trifluoroacetic acid (10.0 g, 88 mmol). The reaction was carried out at 20–25 °C for 16.0–20.0 h, after which the reaction was stopped. The reaction solution was concentrated to obtain a crude product. Acetonitrile (15 mL) and water (50 mL) were added to the crude product, and a saturated sodium bicarbonate aqueous solution was slowly added dropwise under stirring to adjust the pH of the system to 7–8. The filter cake was filtered, washed with water (15 mL), and dried by forced air at 55 °C to give 2'-(4-aminopiperidin-1-yl)-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-3'-nitrile. Yield: 80.0%;

[0099] Step 3): Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridin]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide

[0100] 2'-(4-aminopiperidin-1-yl)-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-3'-nitrile (3.1 g, 7.0 mmol) was added to a 100 mL reaction flask, followed by dichloromethane (60 mL), methanol (6 mL), (E)-3-(4-formylphenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (2.1 g, 7.7 mmol), and acetic acid (0.4 g, 7.0 mmol). The mixture was then purged with nitrogen for protection. Sodium triacetoxyborohydride (2.9 g, 14.0 mmol) was slowly added in portions at 0–5 °C. The reaction was then stopped at 20–25 °C for 16.0–20.0 h. The reaction solution was slowly poured into a 600 mL solution of sodium bicarbonate (5.9 g, 70.0 mmol) in water, stirred for 20 min, filtered, and the filter cake was dried at 45 °C to obtain the crude product. The crude product was added to methanol (60 mL), stirred at 55 °C for 1 h, then cooled to 20–25 °C and stirred for 16 h, and filtered. The filter cake was washed with methanol (8 mL), and dried under vacuum at 50 °C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide. Yield 80.0%;

[0101] Step 4): Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide

[0102] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.30 g, 0.42 mmol) was added to a 10 mL reaction flask, followed by the addition of ethanol (3.0 mL) and hydrochloric acid-ethanol solution (4N, 1.05 mL, 4.20 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 20–25 °C for 2.0–6.0 h, and the reaction was stopped. Saturated sodium bicarbonate solution was added dropwise to adjust the pH of the reaction system to 8–9. The mixture was filtered, and the filter cake was washed three times with purified water (3.0 mL). The filter cake was transferred to water (5.0 mL) and acetonitrile (3.0 mL) and lyophilized to give (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide. Yield 75.0%;

[0103] 1 H NMR(400MHz,DMSO-d6)δppm 10.71(br,1H),9.01(s,1H),8.59(d,J=1.8Hz,1H),8.45(s,1H),8.36(d,J=8.4Hz ,1H),8.21(dd,J=8.4Hz,2.1Hz,1H),7.56-7.36(m,5H),6.79(d,J=8.1Hz,1H),6.5 0-6.38(m,3H),4.23-4.15(m,2H),3.79(s,2H),3.70(s,3H),3.23-3.15(m,2H),2 .76-2.65(m,1H),2.03-1.93(m,2H),1.50-1.38(m,2H); ESI-MS(m / z): 622.0[M+H] + .

[0104] Example 2 Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide-4-methylbenzenesulfonate

[0105]

[0106] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.30 g, 0.42 mmol) was added to a 25 mL reaction flask, followed by the addition of isopropanol (6.0 mL) and p-toluenesulfonic acid (0.29 g, 1.70 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 40–45 °C for 10.0–16.0 h, and the reaction was stopped. The mixture was filtered, and the filter cake was washed three times with isopropanol (1 mL). The filter cake was vacuum dried at 45°C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide-4-methylbenzenesulfonate.

[0107] Yield: 91.5%; 1 H NMR(400MHz,DMSO-d6)δppm 10.82(br,1H),9.05(br,1H),8.94(br,2H),8.62(d,J=1.9Hz,1H),8.51(s,1H),8.39(d,J=8.4Hz,1H),8.24(dd,J= 8.4Hz,2.1Hz,1H),7.66(d,J=8.0Hz,2H),7.58(d,J=8.0Hz,2H),7.53-7.45(m,3H),7.12(d,J=8.0Hz,2H),6.82(d, J=8.9Hz,1H),6.56(d,J=15.8Hz,1H),6.51-6.47(m,2H),4.40-4.32(m,2H),4.31-4.25(m,2H),3.71(s,3H),3.51- 3.41(m,1H),3.16(t,J=12.4Hz,2H),2.28(s,3H),2.27-2.24(m,2H),1.83-1.70(m,2H); ESI-MS(m / z): 622.0[M+H] + .

[0108] One molecule of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide forms a salt with one molecule of p-toluenesulfonic acid.

[0109] Example 3: Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide sulfate

[0110]

[0111] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.30 g, 0.42 mmol) was added to a 25 mL reaction flask, followed by the addition of isopropanol (6.0 mL) and sulfuric acid (0.16 g, 1.68 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 40–45 °C for 6.0–10.0 h, and the reaction was stopped. The mixture was filtered, and the filter cake was washed three times with isopropanol (1 mL). The filter cake was vacuum dried at 45°C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide sulfate.

[0112] Yield: 88.0%; 1 H NMR(400MHz,DMSO-d6)δppm 8.91(br,2H),8.61(d,J=1.9Hz,1H),8.50(s,1H),8.40(d,J=8.4Hz,1H),8.26(dd,J =8.4Hz,2.4Hz,1H),7.67(d,J=8.0Hz,2H),7.60(d,J=8.0Hz,2H),7.50(d,J=15.8Hz 1H),6.82(d,J=8.7Hz,1H),6.60(d,J=15.8Hz,1H),6.50-6.47(m,2H),4.39-4.32(m,2H),4.31-4.25(m,2H),3.7 1(s,3H),3.50-3.40(m,1H),3.21-3.11(m,2H),2.31-2.24(m,2H),1.83-1.71(m,2H); ESI-MS(m / z): 622.0[M+H] + .

[0113] Sulfate anion content: 13.3%, 1 molecule of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide forms a salt with 1 molecule of sulfuric acid.

[0114] Example 4: Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide methanesulfonate

[0115]

[0116] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.30 g, 0.42 mmol) was added to a 25 mL reaction flask, followed by the addition of isopropanol (6.0 mL) and methanesulfonic acid (0.16 g, 1.68 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 40–45 °C for 10.0–16.0 h, and the reaction was stopped. The mixture was filtered, and the filter cake was washed three times with isopropanol (1 mL). The filter cake was vacuum dried at 45°C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide methanesulfonate.

[0117] Yield: 87.5%; 1 H NMR(400MHz,DMSO-d6)δppm 9.33(br,2H),8.61(s,1H),8.50(s,1H),8.39(d,J=8.3Hz,1H),8.24(d,J=8.1Hz,1H),7.6 8-7.40(m,4H),7.48(d,J=15.8Hz,1H),6.81(d,J=8.5Hz,1H),6.56(d,J=15.8Hz,1H),6.51 -6.46(m,2H),4.41-4.31(m,2H),4.29-4.21(m,2H),3.71(s,3H),3.46-3.35(m,1H),3.21- 3.09(m,2H),2.38(s,3H),2.34-2.26(m,2H),1.90-1.74(m,2H); ESI-MS(m / z): 622.0[M+H]+ .

[0118] One molecule of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide forms a salt with one molecule of methanesulfonic acid.

[0119] Example 5: Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide benzenesulfonate

[0120]

[0121] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.30 g, 0.42 mmol) was added to a 25 mL reaction flask, followed by the addition of isopropanol (6.0 mL) and benzenesulfonic acid (0.66 g, 4.20 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 40–45 °C for 10.0–16.0 h, and the reaction was stopped. The mixture was filtered, and the filter cake was washed three times with isopropanol (1 mL). The filter cake was vacuum dried at 45 °C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide benzenesulfonate.

[0122] Yield: 85.0%; 1H NMR(400MHz,DMSO-d6)δppm 8.97(br,2H),8.62(d,J=2.0Hz,1H),8.50(s,1H),8.39(d,J=8.4Hz,1H),8.26(dd,J=8.4Hz,2.0Hz,1H ),7.67-7.61(m,3H),7.58(d,J=8.0Hz,2H),7.50(d,J=15.8Hz,1H),7.35-7.33(m,4H),6.82(d,J=8.8H z,1H),6.61(d,J=15.8Hz,1H),6.51-6.46(m,2H),4.39-4.32(m,2H),4.31-4.24(m,2H),3.70(s,3H), 3.49-3.44(m,1H),3.18-3.11(m,2H),2.32-2.23(m,2H),1.85-1.71(m,2H); ESI-MS(m / z): 622.0[M+H] + .

[0123] One molecule of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide forms a salt with one molecule of benzenesulfonic acid.

[0124] Example 6: Preparation of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide hydrochloride

[0125]

[0126] (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (0.3 g, 0.42 mmol) was added to a 25 mL reaction flask, followed by the addition of ethanol (3.0 mL) and hydrochloric acid-ethanol solution (4N, 1.00 g, 4.20 mmol). The mixture was then purged with nitrogen for protection. The mixture was stirred at 20–25 °C for 2–4 h, and the reaction was stopped. The mixture was filtered, and the filter cake was washed twice with ethanol (1 mL). The filter cake was vacuum dried at 45°C to obtain (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide hydrochloride.

[0127] Yield: 80.0%; 1 H NMR(400MHz,DMSO-d6)δppm 10.83(br,1H),9.32(s,1H),9.06(br,1H),8.62(d,J=1.9Hz,1H),8.51(s,1H),8.39(d,J=8.4Hz,1H ),8.23(dd,J=8.4Hz,2.2Hz,1H),7.68-7.61(m,4H),7.48(d,J=15.8Hz,1H),6.82(d,J=9.0Hz,1H),6 .53(d,J=15.8Hz,1H),6.50-6.47(m,2H),4.36(d,J=13.1Hz,2H),4.29-4.22(m,2H),3.71(s,3H),3. 39-3.38(m,1H),3.19-3.12(m,2H),2.35-2.25(m,2H),1.88-1.76(m,2H); ESI-MS(m / z): 622.0[M+H] + .

[0128] Chloride ion content: 5.4%, 1 molecule of (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide forms a salt with 1 molecule of hydrochloric acid.

[0129] Experimental Example 1: Solubility Test

[0130] The solubility of different salt forms of compound (I) in pure water was determined by high performance liquid chromatography with external standard method.

[0131] 1.1 Preparation of external standard reference material

[0132] The solvent is a mixture of acetonitrile and water in a ratio of 3:7.

[0133] Accurately weigh appropriate amounts of p-toluenesulfonate, sulfate, methanesulfonate, benzenesulfonate, and hydrochloride reference standards of compound (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridine]-2'-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide, dissolve them in solvent, and quantitatively prepare a solution containing approximately 0.2 mg of the salt of compound (I) per 1 mL. This solution serves as the external standard reference solution for the salt type of compound (I).

[0134] 1.2 Preparation of Solubility Test Samples

[0135] Each salt form was investigated independently: an appropriate amount of the salt form compound prepared in each example was accurately weighed (as much as possible the solution was supersaturated during the test) into a 100 mL volumetric flask, 20 mL of purified water was precisely added, and the sample was placed in a constant temperature air bath shaker at 25 °C and 100 rpm for 24 hours. Samples were taken at 3 h, 7 h and 24 h.

[0136] At each time point, 100 μL of sample was taken and filtered through a 0.45 μm filter membrane. The filtrate was then diluted appropriately according to the sample concentration to serve as the test solution for each compound at each time point.

[0137] 1.3 Sample Detection

[0138] The content of each sample was determined by high performance liquid chromatography (HPLC) and calculated using the external standard method.

[0139] 1.4 High Performance Liquid Chromatography Method

[0140] Column: Wates Xbridge C18 4.6*250mm 5μm

[0141] Mobile phase: 50 mmol / L ammonium formate solution (adjusted to pH 3.0 with formic acid): methanol: acetonitrile = 50:25:25

[0142] Flow rate: 1.0 mL / min

[0143] Detection wavelength: 280nm

[0144] Column temperature: 30℃

[0145] Injection volume: 10 μL

[0146] Table 1 Results of saturated solubility determination in purified water

[0147] Salt type Saturated solubility in purified water (mg / mL) sulfates 0.43 hydrochloride 2.40 p-Toluenesulfonate 0.28 benzenesulfonate 0.36 Methanesulfonates 0.86

[0148] The salt forms of the compound shown in formula (I) (especially the hydrochloride salt) all have certain solubility in purified water, which is beneficial to the requirements of subsequent drug development.

[0149] Experiment Example 2 Stability Test

[0150] Test protocol:

[0151] High humidity: Take an appropriate amount of each example sample, spread them evenly in a dry and clean vial, and take samples for testing at different times under high humidity conditions (25°C, RH 92.5%, saturated NaCl solution);

[0152] Light exposure: Take an appropriate amount of each example sample, spread them evenly in a dry and clean transparent vial, and take samples for testing at different times in a light exposure test chamber (5000lx & 84μw / cm2);

[0153] Humid heat: Take an appropriate amount of each example sample, spread them evenly in a dry and clean vial, and take samples for testing at different times under the conditions of 40℃±2℃ / RH75%±5%.

[0154] Detection method:

[0155] Solvent: Water-acetonitrile (50:50, v / v)

[0156] Test solution: Take an appropriate amount of the sample prepared in each example, weigh it accurately, dissolve it in solvent and prepare a solution containing about 0.5 mg of the salt of the compound shown in formula (I) per 1 mL.

[0157] Chromatographic conditions:

[0158] Column: SVEA TM C18 Opal 4.6*250mm, 5μm

[0159] Mobile phase A: 10 mmol / L potassium dihydrogen phosphate solution (adjusted to pH 2.1 with phosphoric acid)

[0160] Mobile phase B: Methanol: Acetonitrile = 80:20

[0161] Flow rate: 1.0 mL / min

[0162] Detection wavelength: 280nm

[0163] Column temperature: 30℃

[0164] Injection volume: 10 μL

[0165] Gradient elution:

[0166] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 2 75 25 45 25 75 55 25 75 56 75 25 65 75 25

[0167] Table 2 Results of stability test

[0168]

[0169] Data shows that, compared with the free state and other salt forms, the stability of p-toluenesulfonate and hydrochloride of the compound shown in formula (I) is significantly improved, which is beneficial to meeting the requirements of subsequent formulation research.

[0170] Experimental Example 3 Hygroscopicity Test

[0171] Hygroscopicity test method: The test was conducted according to the General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia (9103 Guidelines for Hygroscopicity Testing of Drugs), specifically as follows:

[0172] (1) Take a dry, stoppered glass weighing bottle and place it in a suitable constant temperature desiccator at 25℃±1℃ (with a saturated ammonium chloride solution at the bottom and a relative humidity of 80%±2%) one day before the test, and weigh it accurately.

[0173] (2) Take an appropriate amount of the sample prepared in each embodiment, spread it evenly in the weighing bottle above, and accurately weigh it.

[0174] (3) Leave the weighing bottle open and place it under the constant temperature and humidity conditions described above for 24 hours, then close the weighing bottle cap and accurately weigh the mass.

[0175] (4) Calculate the moisture gain of each sample according to the pharmacopoeia requirements.

[0176] Table 3. Description of hygroscopic characteristics and definition of hygroscopic weight gain in the Chinese Pharmacopoeia

[0177] Hygroscopic characteristics Moisture absorption increases weight deliquescence Absorbs sufficient water to form a liquid Extremely hygroscopic No more than 15% Hygroscopic Less than 15% but not less than 2% Slightly hygroscopic Less than 2% but not less than 0.2% None or almost none hygroscopic Less than 0.2%

[0178] Table 4. Hygroscopicity Test Results

[0179]

[0180]

[0181] Data shows that the hydrochloride, benzenesulfonate, and p-toluenesulfonate salts of the compound shown in formula (I) have low hygroscopicity.

[0182] Experiment 4: LSD1 enzyme activity assay

[0183] The effect of compounds on LSD1 enzyme activity was assessed using HTRF technology to evaluate their inhibitory levels. First, a 90 μM stock solution of the test compound (dissolved in DMSO) was serially diluted 5-fold with DMSO to obtain nine working solutions (90×). Then, each of the nine working solutions 1 was serially diluted 30-fold, i.e., 2 μL of working solution 1 was added to 58 μL of buffer and thoroughly mixed using a vortex mixer to obtain nine screening compound working solutions (3×). In a 384-well shallow-well white plate, add 2 μL of compound working solution 2 (3×) to each well, then add 2 μL of a 1:1 premixed solution of 6×LSD1 (Activemotif, 31426) and 6×FAD (Sigma, F8384), mix well, and incubate at room temperature for 15 min; add 2 μL of 3×H3K4me1 (Anaspec, AS-64355-025) substrate solution to each well, mix well, and incubate at room temperature for 60 min; add 2 μL of stop solution (containing 5.4 mM 2-PCPA) to each well, mix well, and incubate at room temperature for 15 min; add 4 μL of a 1:1 premixed antibody solution of Eu-anti H3K4 (PerkinElmer, TRF0404-D) and Prozyme (PJ27S) to each well, mix well, and incubate at room temperature for 60 min. The 384-well plate was placed on a multi-mode microplate reader for reading. The excitation wavelength was set to 337 nm, and readings at 620 nm and 665 nm were recorded. The data results are presented as the ratio of the 665 nm signal value to the 620 nm signal value per well, i.e., Ratio = 10. 4 ×665nm signal value / 620nm signal value. The suppression rate is calculated using the following formula:

[0184] %Suppression = [(Ratio 阴性 -Ratio 化合物 ) / (Ratio 阴性 -Ratio Blank )]×100

[0185] Note: Negative indicates the group without inhibitors; Blank indicates the group without enzymes.

[0186] IC50 was calculated using GraphPad Prism software with the inhibition rate fitted by selecting log(inhibitor) vs. response—Variable Slope (four parameters).

[0187] Experimental Example 5: HDAC Enzyme Activity Detection

[0188] The effects of compounds on the activities of pan-HDAC and two HDAC isoforms (HDAC1 and HDAC8) were assessed using the HDAC-Glo I / II Assay and Screening System, thereby evaluating their inhibitory levels on HDAC protease activity and their selectivity in each isoform. The proteins and assay reagents used were analyzed using the HDAC-Glo I / II Assay and Screening System. TM For the I / II Assay and Screening System (Promage), the 1 mM compound was serially diluted 5-fold with DMSO to prepare 8 concentrations. A certain volume was then diluted 25-fold with HDAC Buffer to obtain a 4× working solution. In a 384-well plate, 5 μL of 4× HDAC enzyme solution and 5 μL of the test compound (4×) were added to each well, mixed, and incubated at room temperature for 30 min. Next, 10 μL of 2× Developer regent was added to each well, mixed, and incubated at room temperature for 15-45 min. The 384-well plate was then placed on a multi-plate reader to read the values. The Luminescence channel was selected, and the Luminescence value (RLU) was recorded. The inhibition rate was calculated using the following formula:

[0189]

[0190] Note: Negative indicates the group without inhibitors; Blank indicates the group without enzymes.

[0191] IC50 was calculated using GraphPad Prism software with the inhibition rate fitted by selecting log(inhibitor) vs. response—Variable Slope (four parameters).

[0192] Table 5. Data on the Enzymatic Activity Tests of Compounds

[0193]

[0194] Note: "NT" indicates that it has not been tested.

[0195] Data shows that the salt of the compound shown in formula (I) provided in this application has a strong inhibitory effect on both LSD1 and HDAC enzymes.

[0196] Experiment 6: In vitro detection of the inhibitory activity of the compound on the proliferation of H1417 cells.

[0197] pass The number of viable cells was measured using reagents to assess the inhibitory effect of the compound on cell proliferation. NCI-H1417 cells in the logarithmic growth phase were collected and seeded into 96-well plates with white walls and clear bottoms, 100 μL per well, at a density of 7 × 10⁶ cells / well. 3Cells / well were incubated overnight at 37°C and 5% CO2. The compound was serially diluted 5-fold with DMSO to obtain 8 concentration gradient dilutions, which were then diluted with BEGM (10% FBS) cell culture medium to obtain the working solution (2×). 100 μL of the working solution was added to the cell supernatant in each well, and the cells were incubated for another 7 days at 37°C and 5% CO2. The plates were then removed and allowed to thaw at room temperature (25°C). Mix the reagents and allow them to equilibrate for approximately 10-30 minutes; after centrifugation, carefully discard 100 μL of culture medium, then add 85 μL of the culture medium. Reagents. Use a microporous shaker to mix the cells with... Mix the reagents thoroughly for 2 minutes, then incubate at room temperature for 10 minutes. Place the 96-well plate on a multi-plate reader and record the Luminescence (RLU) value.

[0198] The inhibition rate is calculated using the following formula:

[0199]

[0200] Note: Negative indicates the group without inhibitors; Blank indicates the group without cells.

[0201] IC 50 The inhibition rate was calculated using GraphPad Prism software.

[0202] Experiment 7: In vitro detection of the inhibitory activity of the compound on the proliferation of NCI-H69 cells.

[0203] The LSD1 / HDAC dual inhibitor compounds in this invention typically exhibit excellent proliferative inhibitory activity against LSD1 inhibitor-resistant tumor cells, such as small cell lung cancer cells NCI-H69. The reagents were used to detect the number of viable cells, thereby assessing the inhibitory effect of the compound on cell proliferation. NCI-H69 cells in the logarithmic growth phase were collected and seeded into clear-bottomed 96-well plates at a density of 1×10⁶ cells / well (100 μL per well). 3 Cells / well were incubated overnight at 37°C and 5% CO2. The compound was serially diluted 5-fold with DMSO to obtain 8 concentration gradient dilutions, which were then diluted with RPMI-640 (20% FBS) cell culture medium to obtain the working solution (2×). 100 μL of each solution was added to the cell supernatant and incubated for another 7 days at 37°C and 5% CO2. The plate was then removed and allowed to thaw at room temperature (25°C). Mix the reagents and allow them to equilibrate for approximately 10-30 minutes; centrifuge the plate at 1200 rpm for 5 minutes, carefully discard 120 μL of culture medium, and then add 60 μL of culture medium. Reagents. Use a microporous shaker to mix the cells with... Mix the reagents thoroughly for 2 minutes, then incubate at room temperature for 10 minutes. Place the 96-well plate on a multi-plate reader and record the Luminescence (RLU) value.

[0204] The inhibition rate is calculated using the following formula:

[0205]

[0206] Note: Negative indicates the group without inhibitors; Blank indicates the group without cells.

[0207] IC 50 The inhibition rate was calculated using GraphPad Prism software.

[0208] Table 6. Data on the cell-inhibiting activity of the compounds.

[0209]

[0210] Data shows that the hydrochloride salt of the compound shown in formula (I) provided in this application has strong inhibitory activity against both H1417 and H69 cells.

[0211] in,

[0212] The SAHA structural formula is: Purchased from Selleck;

[0213] The structural formula of CC-90011 is: Synthesized according to the method described in the literature (Kanouni, T., et al, J. Med. Chem., 2020, 63, 14522-14529).

[0214] Experimental Example 8: Rat PK Test

[0215] Three healthy adult male SD rats (weighing 200–250 g, 6–8 weeks old) were intravenously administered the test compound. The plasma concentrations of the test compound in the rats were determined by LC-MS / MS, and the main pharmacokinetic parameters were calculated to evaluate its pharmacokinetic behavior. During the experiment, the test compound was prepared as a 20% SBE-β-CD solution at pH 6.5–7.0. Whole blood was collected before IV administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. The blood was placed in K2-EDTA anticoagulant tubes, centrifuged (4 °C) for 10 min to separate the plasma, and stored at -80 °C for analysis. Protein precipitation was performed using acetonitrile containing an internal standard, followed by thorough vortexing and centrifugation. The supernatant was then analyzed by LC-MS / MS. Pharmacokinetic parameters were calculated using a WinNonLin 8.3 non-compartmental model.

[0216] Table 8. Rat PK test data of the compounds

[0217]

[0218] Data shows that the compound has a high exposure level in the body, which is beneficial to the drug's efficacy.

[0219] The solid form of the compound shown in formula (I) and its preparation method provided in this application can be implemented by those skilled in the art by appropriately modifying the raw materials, process parameters, and other aspects, based on the content of this document. The methods and products of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and products described herein without departing from the content, spirit, and scope of this application to achieve the technology of this application. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the spirit, scope, and content of this application.

Claims

1. A pharmaceutically acceptable salt of the compound represented by formula (I), the chemical name of which is (E)-3-(4-(((1-(3'-cyano-5'-(3-hydroxy-4-methoxyphenyl)-6-nitro-[3,4'-bipyridin]-2'-yl)piperidin-4-yl)amino)methyl)-phenyl)-N-hydroxyacrylamide, wherein the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

2. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, selected from p-toluenesulfonate, sulfate, methanesulfonate, benzenesulfonate and hydrochloride.

3. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein, The molar ratio of the compound to the acid molecule shown in formula (I) is selected from 1:1 to 1:

2.

4. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein, The molar ratio of the compound shown in formula (I) to p-toluenesulfonic acid molecules is 1:1; Preferably, the p-toluenesulfonate salt of the compound shown in formula (I) has the following structure:

5. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein the molar ratio of the compound of formula (I) to sulfuric acid molecules is 1:1; Preferably, the sulfate of the compound shown in formula (I) has the following structure:

6. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein the molar ratio of the compound of formula (I) to the methanesulfonic acid molecule is 1:1; Preferably, the methanesulfonate salt of the compound represented by formula (I) has the following structure:

7. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein the molar ratio of the compound of formula (I) to the benzenesulfonic acid molecule is 1:1; Preferably, the benzenesulfonate of the compound shown in formula (I) has the following structure:

8. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or 2, wherein the molar ratio of the compound of formula (I) to the hydrochloric acid molecule is 1:1; Preferably, the hydrochloride salt of the compound shown in formula (I) has the following structure:

9. A method for preparing a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1 to 8, comprising the following steps: The compound shown in formula (I) is dispersed in an organic solvent, wherein, The mass-to-volume ratio of the compound shown in formula (I) to the organic solvent is 0.3 g: (2-20) mL. Then, acid is added, wherein the molar ratio of the compound shown in formula (I) to the corresponding acid is 1: (2-20). Nitrogen gas is introduced for protection, and the reaction is stirred at 0-50 °C for 0.5-24 h. The mixture is then filtered, and the filter cake is collected and dried under vacuum to obtain the final product. Preferably, the mass-to-volume ratio of the compound represented by formula (I) to the organic solvent is 0.3 g: (3-6) mL; Preferably, the acid is selected from p-toluenesulfonic acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, and hydrochloric acid; More preferably, the molar ratio of the compound represented by formula (I) to p-toluenesulfonic acid, sulfuric acid or methanesulfonic acid is 1:4, and the molar ratio to hydrochloric acid or benzenesulfonic acid is 1:

10.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a pharmaceutically acceptable salt of the compound of formula (I) as described in any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers.

11. Use of a salt of the compound of formula (I) according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for the prevention and / or treatment of related diseases mediated by LSD1 and / or HDAC, respectively or synergistically; Furthermore, the HDAC enzyme includes, but is not limited to, subtypes such as HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC8, with HDAC1 or HDAC8 subtypes being preferred, and HDAC1 subtype being even more preferred.

12. Use of a salt of the compound of formula (I) according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for the prevention and / or treatment of related diseases mediated by LSD1 protein and / or HDAC1 protein, LSD1 protein and / or HDAC8 protein, respectively or synergistically.

13. The use as described in claim 11 or 12, wherein the disease is cancer or an autoimmune disease; Preferably, the cancer is selected from: non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, chronic myeloid leukemia, colorectal cancer, brain cancer, liver cancer, kidney cancer, stomach cancer, breast cancer, triple-negative breast cancer, skin cancer, melanoma, head and neck cancer, bone cancer, cervical cancer, pelvic cancer, vaginal cancer, oral cancer, lymphoma, leukemia, esophageal cancer, urethral cancer, and nasal cancer.

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