A piperazine dione ring compound organic acid salt, a preparation method and application thereof

Abstract

This invention discloses an organic acid salt of a piperazine dione cyclic compound, its preparation method, and its applications, belonging to the field of medicinal chemistry. This invention provides an organic acid salt of a piperazine dione cyclic compound or its solvate, which can serve as an inhibitor of FLT3-ITD mutant cell lines and can be used in the development of tumor therapeutic drugs. In the organic acid salt of compound 44 provided by this invention, the mesylate salt of compound 44 has significantly higher water solubility than the unsalted compound 44; the high water solubility of this organic salt significantly improves its bioavailability and enhances the therapeutic efficacy of the drug, providing a novel route of administration for the development of intravenous injection, oral liquid formulations, etc. This organic salt exhibits significant advantages in many aspects, including pharmacokinetic behavior and the development of new drug formulations. The organic acid salt or its solvate provided by this invention can also serve as an inhibitor of FLT3-ITD and FLT3-ITD-F691L leukemia cells, showing broad application prospects and promotional value.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a piperazine dione cyclic compound organic acid salt, its preparation method, and its application. Background Technology

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy caused by the malignant proliferation of hematopoietic stem / progenitor cells. It is primarily characterized by excessive proliferation of abnormal primitive cells in the bone marrow, suppressing normal hematopoietic function and leading to clinical symptoms such as anemia, bleeding, and infection. The occurrence of AML is closely related to various gene mutations, among which FLT3 (Fms-like tyrosine kinase 3) gene mutation is one of the most common genetic alterations. FLT3 is a type III receptor tyrosine kinase that plays a crucial role in regulating the proliferation, differentiation, and survival of hematopoietic stem / progenitor cells.

[0003] FLT3 (FMS-like tyrosine kinase 3) is an important receptor tyrosine kinase (RTK) in cell signaling pathways, also known as fetal liver kinase 2 (FLK-2) or human stem cell kinase 1 (STK-1). It belongs to the type III receptor tyrosine kinase (RTK) family, along with FMS, KIT, and platelet growth factor receptor (PDGFR) kinases. The FLT3 gene is located on chromosome 13q12, contains 24 codons, and encodes a receptor-type tyrosine kinase consisting of 993 amino acids. It is expressed in various lymphohematopoietic cells and tissues and is crucial for the normal development of stem cells and the immune system.

[0004] In FLT3-targeted inhibitor therapy, some AML patients develop resistance mutations at FLT3 gating sites, such as the F691L mutation. This mutation occurs in the FLT3 tyrosine kinase domain, leading to the ineffectiveness of multiple FLT3 inhibitors (such as quizartinib / AC220), thus resulting in drug resistance. This resistance mutation significantly increases the difficulty of AML treatment. Type II FLT3-ITD inhibitors are small molecule kinase inhibitors that bind to the inactive conformation of FLT3. They are sensitive to FLT3-ITD cells, but have limited activity against resistance mutations such as F691L. Therefore, there is a need to develop a novel FLT3-ITD inhibitor. However, due to the large molecular weight, high structural symmetry, multiple hydrogen bond donors and acceptors, few polar groups, and a large conjugated system, these inhibitors have poor water solubility, resulting in reduced oral absorption and bioavailability, and poor drug metabolism.

[0005] In animal studies, poor water solubility often results in higher dosages, and the limited dosing volume restricts the choice of solvents or excipients. Poor water solubility also limits the development of candidate compounds when evaluating their pharmacokinetic (PK) characteristics, pharmacodynamic (PD) effects, and toxicity profiles, providing crucial data for subsequent clinical trials. With the widespread application of combinatorial chemistry and high-throughput screening technologies, candidate compounds often exhibit characteristics such as large molecular weight, high lipid solubility, and poor water solubility. Statistics show that over 70% of compounds in the R&D pipeline suffer from insufficient water solubility. Therefore, improving the water solubility of compounds, enhancing pharmacokinetic properties, and developing dosage forms suitable for different patient needs are core issues for the future development of FLT3-ITD inhibitors. Summary of the Invention

[0006] To address the problems in the prior art, the present invention aims to provide an organic acid salt of piperazine dione cyclic compounds, its preparation method, and its applications. The present invention provides an organic acid salt of piperazine dione cyclic compounds, which has good water solubility, can improve in vivo pharmacokinetic behavior, and can be formulated into various dosage forms, including injections, thereby enabling more effective use in the preparation of drugs for tumor treatment.

[0007] To achieve the above objectives, the present invention is implemented through the following solution:

[0008] The first aspect of this invention provides a piperazine dione cyclic compound organic acid salt or a solvate thereof, the structure of which is shown in Formula I. , Formula I, Wherein: n is 1, 2, 3, 4 or 5, and the organic acid is any one of methanesulfonic acid, fumaric acid, maleic acid, acetic acid, oxalic acid, formic acid, and D-pyroglutamic acid.

[0009] Furthermore, the piperazine dione cyclic compound organic acid salt is: 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide fumarate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide maleate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide acetate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide oxalate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide carboxylate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide pyroglutamate.

[0010] This invention also provides a method for preparing the organic acid salt of the piperazine dione cyclic compound, specifically comprising the following steps:

[0011] , After adding 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide to methanol, various organic acid salts of this compound were prepared by adding the corresponding organic acids.

[0012] Furthermore, the specific steps of the preparation method of the general formula are as follows:

[0013] Dissolve 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide in methanol, add organic acid and stir at room temperature for 10 h; concentrate the mixture under reduced pressure, add methanol and methyl tert-butyl ether, stir at room temperature for 1-2 h, filter, wash 2-3 times with methyl tert-butyl ether, collect the filter cake, concentrate under reduced pressure to obtain the product.

[0014] The molar concentration of the 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide in methanol is 0.1 M.

[0015] The present invention also provides a pharmaceutical composition comprising: a therapeutically effective dose of the said piperazine dione cyclic compound organic acid salt or solvate thereof and a pharmaceutically acceptable carrier.

[0016] Furthermore, the pharmaceutical composition includes an oral formulation, an injectable formulation, or a transdermal formulation.

[0017] The present invention also provides the use of the described piperazine dione cyclic compound organic acid salt or solvate thereof or the pharmaceutical composition in the preparation of tyrosine kinase inhibitors.

[0018] Furthermore, the tyrosine kinase inhibitors include FLT3 and FLT3-ITD leukemia cell inhibitors.

[0019] The present invention also provides the use of piperazine dione cyclic compound organic acid salts or solvates thereof or the pharmaceutical compositions thereof in the preparation of medicaments for the prevention and treatment of tumors.

[0020] Furthermore, the tumor cells include acute myeloid leukemia (AML) cells.

[0021] Furthermore, this invention focuses on the use of 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate, fumarate, maleate, acetic acid, oxalic acid, formic acid, D-pyroglutamic acid or solvates thereof, and pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of tumor diseases.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Among the organic acid salts disclosed in this invention, the water solubility of 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate is 2.62 mg / mL, while the water solubility of 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene) The solubility of (methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide is less than 0.0001 mg / mL, meaning that the solubility of the methanesulfonate of compound 44 is more than 20,000 times higher than that of compound 44. After improving water solubility, the solubility of the methanesulfonate of compound 44 in water is 2.58 mg / mL, which significantly improves bioavailability, enhances the therapeutic efficacy of the drug, and can simplify formulation, reduce the risk of toxic side effects, and reduce dosage in future development. The good water solubility of this compound provides a new route of administration for the development of intravenous injection, oral liquid formulations, and other dosage forms.

[0023] 2. The 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide organic acid salts provided by this invention retain their original antitumor activity and can be used in the development of drugs for the treatment of malignant tumors. Simultaneously, the organic acid salts provided by this invention exhibit significant advantages in many aspects, such as pharmacokinetic behavior and the development of new drug formulations. They are practically applicable for improving formulation processes and developing new dosage forms. In experiments inhibiting cell proliferation, the mesylate salt compound showed an IC50 of 5.3 nM for inhibiting the growth of FLT3-ITD mutant cells, demonstrating outstanding and significant technical effects. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] This invention comprises a pharmaceutical composition containing a therapeutic amount of the compound of this invention, and one or more pharmaceutically acceptable carriers and / or excipients. Carriers include, for example, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof, discussed in more detail below. If desired, the composition may also contain smaller amounts of a wetting agent or emulsifier, or a pH buffer. The composition may be a liquid, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate, etc. Depending on the desired formulation, formulation may be designed to mix, granulate, and compress or dissolve the components. In another approach, the composition may be formulated into nanoparticles and enteric-coated microcapsules.

[0026] The drug carrier used can be solid or liquid.

[0027] Typical solid carriers include lactose, gypsum powder, sucrose, talc, gel, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc. A solid carrier may include one or more substances that can simultaneously function as a flavoring agent, lubricant, solubilizer, suspending agent, filler, gliding agent, compression aid, binder, or tablet-disintegrant; it can also be an encapsulating material. In powders, the carrier is a finely pulverized solid mixed with a finely pulverized active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compressibility properties in a suitable proportion and compressed to the desired shape and size. Powders and tablets preferably contain up to 99% active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gel, cellulose, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins.

[0028] Typical liquid carriers include syrups, peanut oil, olive oil, water, and so on. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, tinctures, and sealed compositions. Active ingredients can be dissolved or suspended in pharmaceutically acceptable liquid carriers such as water, organic solvents, mixtures of both, or pharmaceutically acceptable oils or fats. Liquid carriers may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, pigments, viscosity modifiers, stabilizers, or osmotic pressure regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). Carriers for parenteral administration can also be oils such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used for sterile liquid compositions for parenteral administration. The liquid carrier used for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Sterile solutions or suspensions of the liquid pharmaceutical compositions can be used, for example, for intravenous, intramuscular, intraperitoneal, or subcutaneous injection. Injection can be a single bolus or gradual infusion, such as a 30-minute intravenous infusion. The compound can also be administered orally in liquid or solid composition form.

[0029] The carrier or excipient may include time-retarding materials known in the art, such as glyceryl monostearate or glyceryl distearate, and may also include waxes, ethyl cellulose, hydroxypropyl methylcellulose, methyl isobutyl acrylate, etc. When the formulation is intended for oral administration, 0.01% Tween 80 in PHOSALPG-50 (phospholipids and 1,2-propanediol concentrate, A. Nattermann & Cie. GmbH) is recognized for the formulation of acceptable oral formulations of other compounds and can be adapted to the formulation of various compounds of this invention.

[0030] Various pharmaceutical forms can be used when administering the compounds of the present invention. If a solid carrier is used, the formulation can be an enteric-coated tablet, enteric-coated microcapsule, tablet, or sugar tablet encapsulated in a hard capsule. The amount of solid carrier varies considerably, but is preferably from about 25 mg to about 1.0 g. If a liquid carrier is used, the formulation can be a syrup, emulsion, soft capsule, sterile injectable solution or suspension in an ampoule or vial or a non-aqueous liquid suspension.

[0031] Various release systems are known and can be used for the administration of compounds or other various formulations, including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, etc. Methods of introduction include, but are not limited to, skin, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and (generally preferred) oral routes. Compounds can be administered via any convenient or other suitable route, such as by injection or rapid concentration, absorption through epithelial or mucosal pathways (e.g., oral mucosa, rectal and intestinal mucosa, etc.), or through a drug-loaded stent, and may be administered co-administered with other bioactive agents. Administration can be systemic or local.

[0032] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0033] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0034] Compound 44 mentioned in this invention is compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide.

[0035] Example 1

[0036] This embodiment provides the preparation of compound 1, specifically including the following: 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide maleate (compound 44-1)

[0037] 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide (10 mg, 0.014 mmol, 1 eq) and maleic acid (8 mg, 0.07 mmol, 5 eq) were used as reactants. 1 ml of methanol was added to the mixture, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The mixture was evaporated to dryness, and then 1 ml of ethanol was added to the flask. After stirring for 3 h, the mixture was filtered and evaporated to dryness to obtain 10 mg of a yellow solid, which was the compound 44-1.

[0038] 1 H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 12.15 (s, 1H), 10.59 (s,1H), 8.51 (d, J = 3.2 Hz, 1H), 8.19 (d, J = 2.8 Hz, 1H), 7.95 (s, 1H), 7.88(dd, J = 9.2, 2.8 Hz, 1H), 7.68 – 7.60 (m, 2H), 7.53 (dd, J = 8.4, 2.8 Hz,1H), 7.01 (s, 1H), 6.64 (s, 1H), 5.98 (s, 2H), 4.89 (s, 2H), 4.25 (t, J = 6.4Hz, 2H), 3.55 (t, J = 4.4 Hz, 5H), 2.61 (t, J = 6.4 Hz, 2H), 2.42 (t, J = 4.4Hz, 4H), 1.44 (s, 9H).

[0039] Example 2

[0040] This embodiment provides the preparation of compound 2, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide fumarate (compound 44-2)

[0041] Using 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide (10 mg, 0.014 mmol, 1 eq) and fumaric acid (8 mg, 0.07 mmol, 5 eq) as reactants, 1 ml of methanol was added to a 10 ml round-bottom flask, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The mixture was then evaporated to dryness, and 1 ml of ethanol was added to the flask. After stirring for 3 hours, the mixture was filtered and evaporated to dryness to obtain 10 mg of a pale yellow solid, which was compound 44-2.

[0042] 1 H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 12.16 (s, 1H), 10.57 (s,1H), 8.52 (d, J = 2.8 Hz, 1H), 8.19 (d, J = 2.8 Hz, 1H), 7.96 (s, 1H), 7.88(dd, J = 8.8, 2.4 Hz, 1H), 7.67-7.61 (m, 2H), 7.53 (dd, J = 8.8, 3.2 Hz, 1H), 7.01 (s, 1H), 6.64 (s, 1H), 6.13 (s, 2H), 4.90 (s, 2H), 4.35 (t, J = 6.0 Hz,2H), 3.64 (br s, 4H), 2.90 (br s, 2H), 2.71 (br s, 4H), 1.45 (s, 9H).

[0043] Example 3

[0044] This embodiment provides the preparation of compound 3, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate (compound 44-3)

[0045] Dissolve 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide (20 mg, 0.028 mmol, 1 eq) in 1 mL of methanol, add methanesulfonic acid (13 mg, 0.14 mmol, 5 eq), and stir at room temperature for 10 h. Concentrate the mixture under reduced pressure, add methanol and methyl tert-butyl ether, stir at room temperature for 1-2 h, filter, wash 2-3 times with methyl tert-butyl ether, collect the filter cake, concentrate under reduced pressure, and obtain 20 mg of yellow solid product, which is compound 44-3.

[0046] 1 H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.61 (s, 1H), 8.52 (d, J =2.8 Hz, 1H), 8.19 (d, J = 2.4 Hz, 1H), 8.15 (s, 1H), 7.87 (dd, J = 8.4, 2.8Hz, 1H), 7.70 – 7.60 (m, 2H), 7.54 (dd, J = 9.2, 3.2 Hz, 1H), 6.96 (s, 1H), 6.66 (s, 1H), 4.91 (s, 2H), 4.61 (t, J = 8.8 Hz, 2H), 3.99 (s, 2H), 3.19 (s, 2H), 2.33 (s, 9H), 1.45 (s, 9H) LCMS: m / z 701.95 [M+H] + .

[0047] Example 4

[0048] This embodiment provides the preparation of compound 4, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide acetate (compound 44-4)

[0049] Using 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide (10 mg, 0.014 mmol, 1 eq) and acetic acid (4 mg, 0.07 mmol, 5 eq) as reactants, 1 ml of methanol was added to a 10 ml round-bottom flask, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The mixture was then evaporated to dryness, and 1 ml of ethanol was added to the flask. After stirring for 3 hours, the mixture was filtered and evaporated to dryness to obtain 10 mg of a yellow solid, which was compound 44-4.

[0050] 1 H NMR (500 MHz, DMSO-d6) δ 12.40 (s, 1H), 12.17 (s, 1H), 11.95 (s,1H), 10.60 (s, 1H), 8.54 (d, J = 3.0 Hz, 1H), 8.21 (d, J = 3.0 Hz, 1H), 7.98(s, 1H), 7.90 (dd, J = 9.0, 3.0 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.64 (d, J= 9.5 Hz, 1H), 7.55 (dd, J = 9.0, 2.5 Hz,1H), 7.03 (s, 1H), 6.66 (s, 1H),4.92 (s, 3H), 4.34 (t, J = 5.5 Hz, 2H), 4.27 (t, J = 6.5 Hz, 2H), 3.57 (t, J =4.5 Hz, 4H), 2.64-2.60 (m, 2H), 2.55-2.53 (m, 2H), 1.89 (s, 3H), 1.46 (s,9H).

[0051] Example 5

[0052] This embodiment provides the preparation of compound 5, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide oxalate (compounds 44-5)

[0053] Using 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide (10 mg, 0.014 mmol, 1 eq) and oxalic acid (6 mg, 0.07 mmol, 5 eq) as reactants, 1 ml of methanol was added to a 10 ml round-bottom flask, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The mixture was then evaporated to dryness, and 1 ml of ethanol was added to the flask. After stirring for 3 hours, the mixture was filtered and evaporated to dryness to obtain 10 mg of a yellow solid, which was compound 44-5.

[0054] 1 H NMR (500 MHz, DMSO-d6) δ 12.37 (s, 1H), 12.17 (s, 1H), 10.60 (s,1H), 8.53 (d, J = 3.0 Hz, 1H), 8.21 (d, J = 2.5 Hz, 1H), 7.98 (s, 1H), 7.90(dd, J = 8.5, 2.5 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 9.0 Hz,1H), 7.55 (dd, J = 8.5, 3.0 Hz, 1H), 7.03 (s, 1H), 6.66 (s, 1H), 4.91 (s,2H), 4.30 (t, J = 6.0 Hz, 2H), 3.60 (t, J = 4.5 Hz, 4H), 2.73 (t, J = 6.5 Hz, 2H), 2.53 (s, 4H), 1.46 (s, 9H).

[0055] Example 6

[0056] This embodiment provides the preparation of compound 6, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide carboxylate (compounds 44-6)

[0057] Dissolve 10 mg (0.028 mmol, 1 eq) of 2-((6-(((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide in 1 mL of methanol, add formic acid (3 mg, 0.07 mmol, 5 eq), and stir at room temperature for 10 h. Concentrate the mixture under reduced pressure, add methanol and methyl tert-butyl ether, stir at room temperature for 1-2 h, filter, wash 2-3 times with methyl tert-butyl ether, collect the filter cake, concentrate under reduced pressure, and give 10 mg of yellow solid product, which is compound 44-6.

[0058] Example 7

[0059] This embodiment provides the preparation of compound 7, specifically including the following: Preparation of compound 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide pyroglutamate salt (compounds 44-7)

[0060] Dissolve 10 mg (1 eq) of 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazin-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide in 1 mL of methanol, add D-pyroglutamic acid (9 mg, 0.07 mmol, 5 eq), and stir at room temperature for 10 h. Concentrate the mixture under reduced pressure, add methanol and methyl tert-butyl ether, stir at room temperature for 1-2 h, filter, wash 2-3 times with methyl tert-butyl ether, collect the filter cake, concentrate under reduced pressure, and obtain 10 mg of yellow solid product, which is compound 44-7.

[0061] Example 8

[0062] This embodiment provides the determination of the water solubility of the target compounds prepared in Examples 1-3, specifically including the following: An excess of the analyte was dissolved in water, and the solution was equilibrated by shaking at 37°C. After centrifugation, the solution was filtered to remove undissolved compounds, resulting in a saturated aqueous solution of the analyte. The peak area of ​​the saturated solution was determined using a Waters e2695 high-performance liquid chromatograph. The concentration of the analyte was calculated by establishing a standard curve of concentration versus peak area.

[0063] The liquid chromatography detection conditions were as follows: HPLCONE 5C18A column (5μm, 250x4.6mm); detection wavelength: 254nm; mobile phase: acetonitrile / water (containing 0.05% trifluoroacetic acid) = 10 / 90 (0-20min), acetonitrile / water (containing 0.05% trifluoroacetic acid) = 90 / 10 (20-25min); injection volume: 10μL; column temperature: 25℃; flow rate: 1mL / min; detection time: 25min.

[0064] Table 1. Solubility of Compound 44 and some organic acid salts in water

[0065] a The data in the table are from three independent experiments, and the results are presented as "mean ± standard deviation".

[0066] The above results indicate that 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate has better water solubility than maleate, fumarate, acetate, oxalate, formate, and D-pyroglutamate, with a water solubility of 2.58 mg / mL, which is more than 20,000 times higher than that of compound 44. This overcomes the poor water solubility of compound 44 and provides possibilities for developing other formulations such as injectable formulations to improve bioavailability and enhance the therapeutic efficacy of the drug.

[0067] Example 9

[0068] Experimental methods: BaF3-FLT3-ITD and BaF3-FLT3-WT cells were seeded in 96-well plates, respectively. Different concentrations of 44 and 44-3 were co-incubated with both cell lines for 48 hours. Cell viability was detected using the resazurite assay. The IC50 values ​​of the compounds were obtained by plotting dose-response curves and performing nonlinear regression fitting.

[0069] Experimental results: As shown in Table 1, the IC50 values ​​of 44 and 44-3 against FLT3-ITD mutant protein overexpressing cells were 5.158 and 5.300 nM, respectively, indicating comparable inhibitory activity against cell proliferation. Neither compound showed significant inhibitory effect on the proliferation of FLT3-WT protein overexpressing cells. These results indicate that the solubility of compound 44-3 was significantly improved, and its anti-proliferative activity against FLT3-ITD mutant protein overexpressing cells was maintained.

[0070] Table 2. IC50 values ​​(nM) of compounds against the proliferation of FLT3-ITD and FLT3-WT overexpressing cells.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A piperazine dione cyclic compound organic acid salt or a solvate thereof, characterized in that, The structure of the piperazine dione cyclic compound organic acid salt is shown in Formula I: ， Formula I, Wherein: n is 1, 2 or 3, and the organic acid is selected from any one of methanesulfonic acid, fumaric acid, maleic acid, acetic acid, oxalic acid, formic acid and D-pyroglutamic acid.

2. The piperazine dione cyclic compound organic acid salt or its solvate according to claim 1, characterized in that, The piperazine dione cyclic compound organic acid salt is: 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide methanesulfonate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide fumarate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide maleate Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide acetate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide oxalate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide carboxylate, Or 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-yl)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide pyroglutamate.

3. The method for preparing the piperazine dione cyclic compound organic acid salt according to claim 1 or 2, characterized in that, The preparation method includes the following steps: ， Dissolve 2-((6-((Z)-((Z)-5-((5-tert-butyl-1-(2-morpholin-4-ylethyl)-1H-imidazol-4-yl)methylene)-3,6-dioxopiperazine-2-ylidene)methyl)pyridin-3-yl)oxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)acetamide in methanol; concentrate the mixture under reduced pressure, add methanol and methyl tert-butyl ether, stir at room temperature, filter, wash with methyl tert-butyl ether, collect the filter cake, concentrate under reduced pressure, and obtain the product.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: a therapeutically effective dose of an organic acid salt of the piperazine dione cyclic compound of claim 1 or a solvate thereof, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition includes oral, injectable, or transdermal formulations.

6. The use of the piperazine dione cyclic compound organic acid salt or solvate thereof as described in claim 1 or 2, or the pharmaceutical composition as described in claim 4, in the preparation of tyrosine kinase inhibitors.

7. The application according to claim 6, characterized in that, The tyrosine kinase inhibitors include FLT3 inhibitors and FLT3-ITD inhibitors.

8. The use of the piperazine dione cyclic compound organic acid salt or solvate thereof as described in claim 1 or 2, or the pharmaceutical composition as described in claim 4, in the preparation of a medicament for the prevention and treatment of tumors.

9. The application according to claim 4, characterized in that, The tumors include acute myeloid leukemia.

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