Novel compound CX-1 and its therapeutic use in inflammatory diseases and tumors

Abstract

The present invention relates to a novel compound CX-1 and its therapeutic use in inflammatory diseases and tumors. Specifically, the present invention relates to a compound CX-1 shown in formula I, a pharmaceutical composition containing the same, and its use in treating inflammatory diseases, autoimmune diseases or tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of treatment of inflammatory diseases and tumors. Specifically, the present invention relates to spermine derivatives and their therapeutic uses in inflammatory diseases and tumors. Background Art

[0002] Spermine is a polyamine substance containing two amino groups and two imino groups. It is produced in the body by putrescine (butanediamine) and S-adenosylmethionine catalyzed by various enzymes. It and spermidine are both present in bacteria and most animal cells and are important substances for promoting cell proliferation. Under acidic conditions, it exhibits the characteristics of polycationic polyamines and can bind to DNA in viruses and bacteria. It makes DNA molecules more stable and flexible, and is also one of the necessary components in cell culture medium.

[0003] Spermine plays a key role in regulating cell growth, division, differentiation and proliferation of animal tissues, and is an important cell growth and proliferation factor. Pharmacological studies have shown that spermine and spermidine also have antioxidant, anti-aging, anti-inflammatory and immunomodulatory effects.

[0004] In addition, dietary intake and intestinal flora production are also important sources of spermidine for the human body. Many foods, such as milk, soy products and cheese, are rich in spermidine. Spermidine ingested through diet will be quickly absorbed and distributed by the intestines without being degraded. The average daily nutritional intake of spermidine is about 7 to 25 mg, or even higher, which is beneficial to human health.

[0005] The therapeutic uses of spermine and spermidine have been reported in the prior art, for example, the therapeutic effect of spermine in periprosthetic osteolysis (CN111096959A); the use of spermidine in weight loss (CN 109820844A), treatment of nerve cell damage (CN107929270 A), treatment of aneurysms (CN 110548020A) and treatment of hepatitis B virus (CN111529516A).

[0006] Inflammation or inflammatory diseases are associated with inflammatory cytokines, also known as inflammatory cytokines, which are produced by lymphocytes and macrophages and are involved in inflammatory responses associated with bacterial or viral infections, tumors or tissue damage. Known inflammatory cytokines include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), type I interferons, tumor necrosis factor (TNF-α) and GM-CSF, etc.

[0007] The development of more spermine derivatives and / or analogs will help promote its therapeutic use in inflammatory diseases and tumors. Summary of the invention

[0008] After unexpectedly discovering that spermine has an immunosuppressive effect, the present invention further optimized and modified the structure of spermine analog 1,2-bis(3-aminopropylamino)ethane based on the molecular structure of spermine, and finally obtained a new compound CX-1 with a broad immunosuppressive effect. Among them, we found that compound CX-1 significantly inhibited the growth of human lymphoma and various solid tumor cells and induced their death, proving that it has a therapeutic effect on blood tumors such as lymphoma and other tumors.

[0009] Based on the above findings, in the first aspect, the present invention provides a spermine derivative or a pharmaceutically acceptable salt thereof, wherein the compound CX-1 has a structure selected from the following:

[0010]

[0011] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound CX-1 of the present invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0012] In a third aspect, the present invention provides use of the compound CX-1 according to the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the present invention, in the preparation of a medicament for treating a disease selected from an inflammatory disease, an autoimmune disease or a tumor.

[0013] In a specific embodiment, provided is the use of a compound CX-1 according to the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the present invention, in the preparation of a drug for treating a disease, wherein the disease is selected from acute inflammation, chronic inflammation, Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, neurodegenerative diseases associated with inflammation, and tumors.

[0014] In a fourth aspect, the present invention provides a method for preparing a compound CX-1 as shown in Formula I or a pharmaceutically acceptable salt thereof, the method comprising the step of reacting 2,5-bistrifluoroethoxybenzoic acid with a spermine analog 1,2-bis(3-aminopropylamino)ethane.

[0015] In a fifth aspect, the present invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of the compound CX-1 described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the present invention to a subject in need thereof; the disease is selected from an inflammatory disease, an autoimmune disease or a tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A The results showed that CX-1 (10 μM) inhibited the IL-2 production of immature CD4+ T cells under the stimulation of Phorbol 12-myristate 13-acetate (PMA) and ionomycin. Figure 1B Shown are the levels of IL-2 production by naive CD4+ T cells upon TCR stimulation.

[0017] Figure 2A Flow cytometry was used to detect the inhibitory effect of CX-1 (10 μM) on CD4+ T cell proliferation under TCR activation conditions; Figure 2B It is a bar chart; Figure 2C It showed that CX-1 had no effect on CD4+ T cell activity.

[0018] Figure 3A is the result of flow cytometry, which shows that CX-1 (10 μM) induces cell apoptosis; Figure 3B It is a bar chart.

[0019] Figure 4A-4C CX-1 (10 μM) induced the proliferation of mouse breast cancer cell line 4T1 ( Figure 4A ); Human breast cancer cell line BT549 ( Figure 4B ) and mouse melanoma cell line B16 ( Figure 4C ) apoptosis.

[0020] Figure 5 Displays the results of H NMR spectrum detection.

[0021] In the figure, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001. Unpaired two-tailed T test was used for data analysis of the two groups. DETAILED DESCRIPTION

[0022] In the first aspect of the present invention, there is provided a compound CX-1 or a pharmaceutically acceptable salt thereof represented by Formula I:

[0023]

[0024] In a specific embodiment, the compound represented by formula I of the present invention is obtained by the following synthesis steps:

[0025]

[0026] In a specific embodiment, the compound of formula I of the present invention is obtained by reacting 2,5-bistrifluoroethoxybenzoic acid with spermine analog 1,2-bis(3-aminopropylamino)ethane. It should be understood that the pharmaceutically acceptable salt forms are within the scope of the present invention, and reference to compound CX-1 includes its salt forms.

[0027] Salts of the present invention can be synthesized by conventional chemical methods. Acid addition salts (mono or di-salts) can be formed with a wide variety of inorganic and organic acids. Examples of acid addition salts include mono or di-salts formed with acids selected from acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphor, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid).

[0028] A particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One specific salt is the hydrochloride. Another specific salt is the acetate. Another specific salt is the phosphate.

[0029] If the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then it can form a salt with an organic or inorganic base to generate a suitable cation. Examples of suitable inorganic cations include, but are not limited to: alkali metal ions (such as Li + 、Na + and K + ), alkaline earth metal cations (such as Ca 2+ and Mg 2+ ) and other cations (such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R + NH2R2 + 、NHR3 + NR4 + ). Some examples of suitable substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + .

[0030] In a specific embodiment of the present invention, the compound CX-1 or a pharmaceutically acceptable salt thereof can be formulated into a pharmaceutical composition with one or more conventional carriers and / or excipients according to techniques well known in the art. Generally, the pharmaceutical composition of the present invention is suitable for oral or parenteral administration, for example, by intradermal, subcutaneous, intraperitoneal or intravenous injection. Therefore, suitable pharmaceutical dosage forms include plain tablets or coated tablets, capsules, suspensions and solutions, containing active ingredients and optionally containing one or more conventional inert carriers and / or diluents, such as corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinyl pyrrolidone, citric acid, tartaric acid, water, water / ethanol, water / glycerol, water / sorbitol, water / polyethylene glycol, propylene glycol, octadecyl alcohol, carboxymethyl cellulose or fatty substances (such as stearin) or any suitable mixture of these substances.

[0031] In a specific embodiment, the pharmaceutical composition is formulated as tablets, capsules, granules, oral liquids, granules, dropping pills or micropills.

[0032] In a specific embodiment, the pharmaceutical composition comprises compound CX-1 or a pharmaceutically acceptable salt thereof as an active ingredient and one or more pharmaceutically acceptable carriers selected from the following: for example, diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes and combinations thereof.

[0033] In a specific embodiment, the pharmaceutical composition comprises a tablet or gelatin capsule of compound CX-1 or a pharmaceutically acceptable salt thereof as an active ingredient and one or more of the following pharmaceutically acceptable carriers:

[0034] a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0035] b) lubricants, such as silicon dioxide, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; in the case of tablets, also

[0036] c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if necessary

[0037] d) disintegrating agents, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and

[0038] e) Absorbents, colorants, flavorings and sweeteners.

[0039] The compound CX-1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same according to the present invention can treat inflammatory diseases or autoimmune diseases by inhibiting the immune inflammatory response in a subject.

[0040] As used herein, the term "treating" includes treating a disease or condition described herein in a subject, such as a human, and includes: (i) inhibiting the disease or condition, i.e., preventing its occurrence; (ii) relieving the disease or condition, i.e., causing regression of the condition; (iii) slowing the progression of the disease; and / or (iv) inhibiting, relieving or slowing the progression of one or more symptoms of the disease or condition.

[0041] The terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any animal or its cells suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0042] As used herein, "inflammatory diseases" or "autoimmune diseases" include, but are not limited to, acute and chronic inflammatory diseases, such as inflammatory bowel disease (Crohn's disease, ulcerative colitis), rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis and / or neurodegenerative diseases associated with inflammation.

[0043] The compound CX-1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same according to the present invention can treat tumors by inducing apoptosis of tumor cells.

[0044] The tumors described herein include, but are not limited to, liver cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, medulloblastoma, skin cancer (e.g., melanoma), non-small cell lung cancer, small cell lung cancer, cervical cancer, ovarian cancer, bladder cancer, thyroid cancer, head and neck cancer, prostate cancer, fibroblastoma, leukemia, myeloma, and lymphoma.

[0045] In a specific embodiment, the compound CX-1 or its pharmaceutically acceptable salt as described herein, or a pharmaceutical composition comprising it, for treating a disease, can be used for a specific route of administration, such as oral administration, parenteral administration (e.g., by injection, instillation, transdermal or topical administration) and rectal administration. Topical administration also includes inhalation or intranasal administration. The compound CX-1 of the present invention or its pharmaceutically acceptable salt or a pharmaceutical composition comprising it can be made into a solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories), or a liquid form (including but not limited to solutions, suspensions or emulsions). Tablets can be added with film coatings or enteric coatings according to methods known in the art.

[0046] The present invention is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention. Experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or commodity manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0047] Example 1. Preparation and confirmation of CX-1

[0048] Preparation of CX-1 compound:

[0049] 2,5-bis(trifluoroethoxybenzoic acid) (1.1 g, 3.5 mM), CDI (648 mg, 4 mM), and DCM (10 mL) were added to a 25 mL reaction bottle. After 30 min of reaction under an ice bath, the spermine analogue 1,2-bis(3-aminopropylamino)ethane (274 uL, 1.5 mM) was slowly added and reacted at room temperature overnight. TLC monitored the reaction to be complete, and saturated 20 mL NaHCO3 and DCM (20 mL x 3) were added for extraction three times. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude product, which was purified by a 200-300 mesh alkaline silica gel column to obtain compound JA-6-12-B (371 mg), with a yield of 48%, MS: [M+H] 775.35; 1H NMR (400MHz, CD3OD) δ7.38 (t, J = 1.8 Hz, 2H), 7.15 (d, J = 1.8 Hz, 4H), 4.65 (q, J = 8.4 Hz, 4H), 4.53 (q, J = 8.4 Hz, 4H), 3.46 (t, J = 7.0 Hz, 4H), 2.78-2.73 (m, 4H), 2.72-2.63 (m, 4H), 1.84-1.71 (m, 4H).

[0050] Confirmation: After the compound is synthesized, the product is verified by H NMR. The results are shown in Figure 5 .

[0051] Example 2. CX-1 inhibits the activation of mouse naive CD4+ T cells and IL-2 production.

[0052] (A) CX-1 pretreated mouse naive CD4+ T cells

[0053] Mouse naive CD4+ T cells were isolated from the spleen of female mice aged 6-8 weeks. After obtaining single cells from mouse spleen, MojoSort TM Mouse CD4 T Cell Isolation Kit (Biolegend), strictly follow the instructions to obtain relatively pure naive CD4+T cells. The cells were cultured in RPMI1640 containing 10% FBS, non-essential amino acids and β-mercaptoethanol, and pretreated with 10μM CX-1 for 3 hours. The above CX-1 needs to be prepared into a fresh concentrate with a concentration of 20mM with DMSO before each use, and diluted as needed. For the cells in the control group (dose 0), an equal volume of DMSO was added to the culture medium.

[0054] (B) Activation of naive mouse CD4+ T cells

[0055] PMA and ionomycin activation of naive CD4+T cells: cells treated with CX-1 or DMSO were inoculated in culture plates, and PMA and ionomycin at a dose of 100 ng / ml were used to continuously activate the cells for 24 hours. The cell culture supernatant was obtained, and the IL-2 level was detected by ELISA ( Figure 1A ).

[0056] In the case of TCR-activated naive CD4+T cells, 96-well cell culture plates were coated with anti-mouse CD3 antibodies (α-CD3) at a concentration of 5 μg / ml. Specifically, the antibody stock solution was diluted to 5 μg / ml using sterile PBS (-), added to the 96-well plate at 200 μl / well, and placed in a 37-degree water-saturated constant temperature cell culture incubator for coating for more than 2 hours. The coated culture plate was washed 3 times with sterile PBS (-) before use. Cells treated with CX-1 or DMSO were inoculated in the culture plate, and anti-mouse CD28 antibodies (α-CD28) were added at a final concentration of 1 μg / ml to continuously activate the cells for 48 hours, and the cell culture supernatant was obtained. The ELISA method was used to detect the level of IL-2 ( Figure 1B ).

[0057] Experimental results and discussion:

[0058] The experimental results showed that CX-1 can effectively inhibit ion channel activation (PMA / ionomycin combined stimulation, Figure 1A ) and TCR activation (α-CD3 / α-CD28 combined stimulation Figure 1B ) showed that CX-1 has an immunosuppressive effect, which indicates that CX-1 has clinical application value in the treatment of inflammatory diseases.

[0059] Example 3. CX-1 inhibits proliferation of mouse naive CD4+ T cells.

[0060] The naive CD4+ T cells were obtained in the same manner as in Example 2. The obtained cells were purified by CellTrace TMCFSE cell proliferation kit (Thermo Company) staining. After staining, naive CD4+T cells were cultured with RPMI1640 containing 10% FBS, non-essential amino acids and β-mercaptoethanol, and the cells were pretreated with 10μM CX-1 for 3 hours. The cells in the control group (dose of 0) were added with an equal volume of DMSO to the culture medium. Subsequently, the treated cells were stimulated by α-CD3 / α-CD28 and continuously activated for 48 hours (the method is the same as in Example 2). After the activation, the cells were collected and washed twice with PBS (-), and the cells were stained with 7-aminoactinomycin D (7-AAD) to determine the cell activity. The cell proliferation level was determined by detecting the green fluorescence level of the cells by flow cytometry, and the cell activity was determined by the 7AAD negative ratio.

[0061] Experimental results and discussion:

[0062] The results showed that in naive mouse CD4+ T cells, CX-1 pretreatment significantly reduced the proportion of cells with low-level CFSE staining after TCR activation ( Figure 2A ), the bar graph shows the statistical value of the proportion of cells with low CFSE levels ( Figure 2B In addition, cell viability assays showed that CX-1 pretreatment did not significantly change the cell survival rate ( Figure 2C The above results indicate that CX-1 can inhibit the proliferation of mouse naive CD4+T cells without affecting the cell activity, proving its immunosuppressive effect.

[0063] Example 4. CX-1 induces apoptosis in Jurkat cells.

[0064] Human T lymphocyte leukemia Jurkat cells were cultured in RPMI1640 medium containing 10% FBS. Cells were treated with CX-1 at a dose of 5 μM for 6 hours, and an equal volume of DMSO was added to the culture medium for cells in the control group (dose 0). Subsequently, the cells were stained using PE Annexin V Apoptosis Detection Kit (BD Company) in strict accordance with the instructions. The stained cells were detected by flow cytometry to confirm the proportion of cell apoptosis (Figure 3).

[0065] Experimental results and discussion:

[0066] The experimental results showed that CX-1-treated human T lymphocyte leukemia cells Jurkat underwent significant apoptosis ( Figure 3A , 3B), indicating that CX-1 has the potential to be a chemotherapeutic agent.

[0067] Example 5. CX-1 (10 μM) induces apoptosis of solid tumor cells in a caspase-dependent manner.

[0068] Mouse breast cancer cell line 4T1 was cultured in RPMI1640 containing 10% FBS; human breast ductal carcinoma cell line BT549 was cultured in RPMI1640 containing 10% FBS and 0.01 mg / ml insulin; mouse melanoma cell line B16 was cultured in DMEM containing 10% FBS. Cells were routinely passaged and treated with 100 μM pan-caspase inhibitor (Zvad) for 2 hours before use. Subsequently, cells were treated with 10 μM CX-1 for 24 hours. The cells of the control group (dose 0) were added with an equal volume of DMSO to the culture medium. Cell activity was measured using 2.0Assay reagent (Promega) was used for detection. Specifically, the treated cell culture plate was placed at room temperature for 30 minutes, an equal volume of detection reagent was added to the plate, and the plate was placed on a shaker for 10 minutes for mixing, and then transferred to a special ELISA plate for detection. The instrument used was the Thermo VARIOSKAN FLASH ELISA reader ( FIG. 4A to FIG. 4C ).

[0069] Experimental results and discussion:

[0070] The experimental results showed that CX-1 can effectively reduce the viability of a variety of solid tumor cells. However, this effect can be significantly reversed by the pan-caspase inhibitor (Zvad), which fully demonstrates that CX-1 causes the reduction of solid tumor cell viability mainly through the apoptosis pathway. The above results illustrate that CX-1 has the potential to be used as a chemotherapeutic drug.

Claims

1. Compound CX-1 or a pharmaceutically acceptable salt thereof, wherein the compound CX-1 has a structure shown in Formula I:

2. The compound CX-1 or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is selected from acetate, hydrochloride, phosphate, sulfate, citrate, lactate, succinate, maleate, malate, isethionate, fumarate, benzenesulfonate and toluenesulfonate.

3. A pharmaceutical composition comprising the compound CX-1 or a pharmaceutically acceptable salt thereof according to claim 1 or 2, and one or more pharmaceutically acceptable carriers.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is formulated for oral or parenteral administration.

5. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is formulated into tablets, capsules, granules, oral liquids, dropping pills or micropills for oral administration; or The pharmaceutical composition is formulated into a solution or a lyophilized preparation for intradermal, subcutaneous, intratumoral or intramuscular injection administration.

6. Use of the compound CX-1 according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 3 to 5, in the preparation of a medicament for treating a disease, wherein the disease is selected from breast cancer, melanoma and leukemia.

7. A method for preparing a compound CX-1 of formula I, comprising: 1) reacting 2,5-bis(trifluoroethoxy)benzoic acid, CDI and DCM; and 2) Add spermine analog 1,2-bis(3-aminopropylamino)ethane and react at room temperature overnight;

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