CN116102468B - Lead compound of targeted human lipocalin 2 or pharmaceutically acceptable salt thereof, and preparation method and application thereof - Google Patents

Abstract

A lead compound targeting human lipocalin 2 or a pharmaceutically acceptable salt thereof and a preparation method and application thereof, wherein the lead compound has a structure shown in the following formula (I): Wherein, m is 4 to 12. The lead compound of the present invention or a pharmaceutically acceptable salt thereof can achieve an anti-tumor immunotherapy effect by inhibiting the targeted human lipocalin 2.

Description

Lead compound targeting human lipocalin 2 or its pharmaceutically acceptable salt and its preparation method and application

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to a lead compound targeting human lipocalin 2 (LCN2, also known as neutrophil gelatinase-associated lipocalin) or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof.

technical background

Cancer is one of the major diseases that endanger human life and health. my country ranks first in the world in terms of the number of new cases and deaths from cancer each year, and the incidence rate is increasing year by year, which has put tremendous pressure on people's life and health and social economic development. At present, my country's research and development of cancer treatment drugs still lags behind major developed countries. The main methods of cancer treatment at this stage include using targeted drugs to accurately kill tumor cells or activate the body's immune system, and use the immune system to inhibit the development of tumors. Therefore, finding molecules that are specifically expressed or highly expressed by tumor cells and developing targeted drugs for such molecules are the key to cancer treatment research.

Summary of the invention

In view of this, the main purpose of the present invention is to propose a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof and a preparation method and application thereof, in order to at least partially solve at least one of the above-mentioned technical problems.

In order to achieve the above object, as one aspect of the present invention, a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof is provided, wherein the lead compound has a structure shown in the following formula (I):

Among them, m is 4 to 12.

As another aspect of the present invention, a method for preparing the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof is provided, comprising the following steps:

Compound (1) is reacted with 1,1'-(5-amino-1,3-phenyl)di(ethyl-1-one) to produce compound (2),

The compound (2) is reacted with an aminoguanidine acid addition salt to generate the lead compound,

As another aspect of the present invention, a pharmaceutical composition is provided, comprising the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

As another aspect of the present invention, there is provided a use of the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for anti-tumor immunotherapy.

As another aspect of the present invention, provided is a use of the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting LCN2.

Based on the above technical solution, it can be known that a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof and a preparation method and application thereof of the present invention have one or part of the following beneficial effects:

The lead compound or a pharmaceutically acceptable salt thereof provided by the present invention has been determined to target LCN2 and to bind to LCN2 relatively stably through in vitro affinity tests, and has been verified through in vivo model tests to show that the interaction between the two can effectively inhibit tumor growth. It can be seen that the lead compound or a pharmaceutically acceptable salt thereof of the present invention can achieve an anti-tumor immunotherapy effect by inhibiting LCN2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of the correlation between LCN2 expression and tumorigenesis and cancer survival in Example 1 of the present invention, wherein a is the increase in LCN2 content in the serum of tumor-bearing mice along with tumor development, b is the knockdown efficiency of the LCN2 gene in the LLC mouse lung cancer cell line, c is the cell growth curve of the LLC cell line with knockdown of the LCN2 gene; d is the subcutaneous tumor growth curve of tumor-bearing mice inoculated subcutaneously with the LLC cell line with knockdown of the LCN2 gene, and e to f are curves related to the expression of LCN2 and the survival of patients with pan-cancer, cholangiocarcinoma and pancreatic cancer;

Figure 2 shows the construction of the prokaryotic recombinant expression vector pET28a-LCN2 in Example 2 of the present invention, the expression, purification and determination results of the human LCN2 recombinant protein, wherein a is the construction model of pET28a-LCN2, b is the expression amount of the LCN2 protein under different induction conditions, c is the PAGE electrophoresis purity result of the purified LCN2, and d is the Western Blot identification result of the purified LCN2 protein;

FIG3 shows the post-synthesis identification results of compound KDX001 of Example 3 of the present invention, wherein a is the hydrogen nuclear magnetic resonance spectrum of compound KDX001, and b is the mass spectrum of compound KDX001;

Figure 4 shows the detection results of the interaction between LCN2 protein and compound KDX001 in Example 4 of the present invention, wherein a is the nuclear magnetic resonance saturation transfer difference spectrum of LCN2-KDX001, b is the relationship curve between the dissolution temperature of LCN2 protein and the concentration of compound KDX001 obtained based on the thermal shift assay, and c is the isothermal calorimetric titration curve of the interaction between LCN2 protein and KDX001;

Figure 5 shows the mouse tumor model treated with compound KDX001 in Example 5 of the present invention, wherein a is the tumor growth curve of LLC cell line tumor-bearing mice, b is the tumor tissue weight of LLC cell line tumor-bearing mice, c is the tumor growth curve of MC38 cell line tumor-bearing mice, d is the tumor tissue weight of MC38 cell line tumor-bearing mice, e is the in vivo imaging of tumor progression in E0771 cell line tumor-bearing mice, and f is the tumor tissue imaging of E0771 cell line tumor-bearing mice.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the process of realizing the present invention, it was found that by focusing on the formation mechanism of tumor cachexia, it was first found that the expression of LCN2 protein was significantly upregulated in the serum of patients with tumor cachexia, which is a protein related to lipid nutrition transport. Then it was found that LCN2 protein was positively correlated with the secretion of various tumor-related inflammatory cytokines and the weight loss of tumor patients. In animal models, it was found that LCN2 protein can directly cause atrophy of mouse adipose tissue. When the molecule is missing or blocked, the progression of mouse tumors is significantly inhibited and the tumor cachexia phenotype is effectively alleviated. Therefore, the development of small molecule drugs targeting LCN2 for anti-tumor immunotherapy has important practical significance for restoring anti-tumor immune activity. On this basis, the present invention further designs drugs based on the structure of LCN2, screens and obtains the lead compound provided by the present invention, verifies that it can be more stably bound to LCN2 through in vitro and in vivo experiments, and effectively inhibits tumor growth through the interaction between the two, thereby realizing anti-tumor immunotherapy, and has good application prospects.

Specifically, according to some embodiments of the present invention, a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof is provided, wherein the lead compound has a structure as shown in the following formula (I):

Among them, m is 4 to 12.

According to an embodiment of the present invention, LCN2 is a pan-cancer marker that promotes the secretion of tumor-related inflammatory cytokines and promotes tumor growth, and the lead compound shown in the above formula (I) has a significant LCN2 inhibitory effect, thereby playing an anti-tumor immunotherapy role of inducing tumor ferroptosis and enhancing anti-tumor immune response.

According to an embodiment of the present invention, m is preferably 8, which has suitable drug solubility and affinity for targeting LCN2, but is not limited thereto. In some embodiments, m may also be 4, 5, 7, 10, etc.

According to an embodiment of the present invention, the pharmaceutically acceptable salt of the lead compound is an acid addition salt of the lead compound, and the acid addition salt of the lead compound of the present invention can be prepared by reacting the free base form of the lead compound with a pharmaceutically acceptable inorganic or organic acid. Preferably, the acid addition salt of the lead compound includes, but is not limited to, hydrochloride, carbonate, sulfate, hydrobromide or phosphate, etc.

According to an embodiment of the present invention, there is also provided a method for preparing the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof, comprising the following steps:

Step A: reacting compound 1 with 1,1'-(5-amino-1,3-phenyl)di(ethyl-1-one) 1a to generate compound 2, as shown in the following formula (A1);

Step B: Compound 2 is reacted with aminoguanidine acid addition salt 2a to generate a lead compound I, specifically represented by the following formula (B1).

According to an embodiment of the present invention, step A specifically includes: dissolving compound 1, 1,1'-(5-amino-1,3-phenyl)di(ethyl-1-one) 1a and a base in an organic solvent, and reacting at 16 to 37° C. (e.g., 20° C., 30° C., etc.) for 0.5 to 4 hours (e.g., 1 hour, 2 hours, etc.).

More specifically, the base may be, for example, an inorganic base or an organic base, preferably pyridine, which is conducive to the reaction. The organic solvent may be, for example, dichloromethane, etc., which can dissolve the reactants.

According to an embodiment of the present invention, step B specifically comprises: dissolving compound 2 and aminoguanidine acid addition salt in an organic solvent, and reacting at 60-120° C. (eg 80° C., 100° C., etc.) for 15-20 hours (eg 16 hours, 18 hours, etc.).

More specifically, the aminoguanidine acid addition salt may be, for example, aminoguanidine hydrochloride, aminoguanidine carbonate, aminoguanidine sulfate, aminoguanidine hydrobromide or aminoguanidine phosphate, etc. The organic solvent may be, for example, anhydrous ethanol, etc., which can dissolve the reactants.

According to an embodiment of the present invention, there is further provided a pharmaceutical composition, comprising the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

According to an embodiment of the present invention, the above-mentioned pharmaceutical composition can be formulated into several dosage forms according to the mode of administration, such as oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which can be used immediately by adding injection water before injection); topical preparations (such as ointments or solutions).

According to the embodiments of the present invention, the carrier used in the pharmaceutical composition of the present invention is a common type available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for local preparations. The pharmaceutical preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically).

According to an embodiment of the present invention, there is also provided a use of the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for anti-tumor immunotherapy, especially a drug for immunotherapy of subcutaneous lung cancer tumors or subcutaneous colorectal cancer tumors.

According to an embodiment of the present invention, there is further provided a lead compound or a pharmaceutically acceptable salt thereof for use in preparing a drug for inhibiting LCN2.

The above-mentioned lead compound inhibits LCN2, thereby inducing tumor ferroptosis and enhancing anti-tumor immune response in anti-tumor immunotherapy.

The technical scheme of the present invention is further described below by specific examples in combination with the accompanying drawings. It should be noted that the following specific examples are only used as examples, and the protection scope of the present invention is not limited thereto. The drugs or reagents used in the following examples are all commercially available or homemade by known preparation methods. The methods used in the following examples, such as Western blot, are all well-known methods in the art and can be carried out by description in textbooks or relevant documents, and will not be repeated here.

Example 1: LCN2 protein promotes tumor growth and is an important tumor treatment target

Serum LCN2 protein levels increase with tumor progression

In vivo, several B6 mice (Slack) were ordered and inoculated subcutaneously with LLC cell lines (5×105 cells/mouse). After tumors were formed in mice, the LCN2 content in mouse serum was measured by Elisa (R&D). It was found that the expression level of LCN2 gradually increased with the development of tumors, as shown in Figure 1a.

Knockdown of LCN2 gene inhibits tumor cell growth in vitro

Using gene interference technology, two gene interference reagents shLCN2-1 (sh1) and shLCN2-2 (sh2) were used to knock down the LCN2 gene in LLC mouse lung cancer cell lines, and the knockdown efficiency was detected by real-time quantitative PCR experiments, as shown in Figure 1b. In vitro, the cell growth curve was determined using a real-time label-free cell analysis (RTCA) system. First, 50 μL of serum-free culture medium was added to the bottom of a 96-well plate, and a baseline calibration was performed on the RTCA system. Then, three groups of LLC cell lines, shNC, shLCN2-1, and shLCN2-2, were digested and recovered, and the cell density was adjusted to 5000 cells/150 μL. 150 μL of cells were inoculated on the bottom of a 96-well plate, and the RTCA system was clicked again to run, and the growth rates of the three groups of cells were recorded, as shown in Figure 1c. It can be seen that after LCN2 deficiency, the growth of tumor cells was significantly inhibited.

Knockdown of LCN2 gene inhibits tumor cell growth in vivo

In vivo, several B6 mice (Slack) were ordered and subcutaneously inoculated with the three cell lines mentioned above (5×105 cells/each mouse). After the tumors were formed in the mice, the tumor growth curves were measured at regular intervals and statistically analyzed according to the calculation principle of length×width×width/2, as shown in Figure 1d. This proves that LCN2 deficiency in vivo can also lead to slow tumor growth, which is sufficient to prove that LCN2 is a reliable target for tumor treatment.

Correlation between high expression of LCN2 and survival of cancer patients

LCN2 is associated with poor prognosis in cancer patients. Based on the TCGA (The Cancer Genome Atlas) database, a total of 9637 cancer patients with traceable disease progression were analyzed for Kaplan-Meier survival curve estimation. According to the median TPM (Transcripts Per Million) of the LCN2 gene in the transcriptome of the patient's tumor tissue, patients with higher LCN2 gene expression showed worse survival, Figure 1e. Specifically, for cholangiocarcinoma (CHOL, data included 36 patients) Figure 1f, and pancreatic cancer (PAAD, data included 178 patients) Figure 1g, the survival difference associated with LCN2 gene levels was particularly obvious.

From the above results, it can be seen that blocking LCN2 protein is beneficial to inhibiting tumor growth, and LCN2 is an important target for tumor treatment.

Example 2: Construction of prokaryotic recombinant expression vector pET28a-LCN2, expression, purification and determination of human LCN2 recombinant protein

The full-length LCN2 gene was artificially synthesized, and 6×His-Tag was introduced at the N-terminus. First, the pET28a(+) plasmid was linearized using restriction endonucleases NotⅠ and XhoⅠ, and the PCR product of the LCN2 gene was treated with these two enzymes to make its two ends sticky. Finally, the LCN2 gene was connected to the pET28a(+) vector using T4 ligase to successfully construct the prokaryotic expression plasmid of LCN2. The plasmid was transferred into DH5α competent cells, and positive clones were screened for DNA sequencing identification. The sequencing results showed that the recombinant expression vector pET28a-LCN2 was successfully constructed. The construction process of the prokaryotic recombinant expression vector pET28a-LCN2 is shown in Figure 2a.

In order to obtain the LCN2 protein (uniprot: P80188) of the sequence shown in SEQ ID NO.1, different concentrations of isopropyl β-D-Thiogalactoside (IPTG) (0mM, 0.1mM, 0.2mM, 0.4mM) were used to induce the expression strain containing the synthetic LCN2 gene amplified to the logarithmic phase, and samples were taken before induction, 2h induction, and 4h induction. Through sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) and Coomassie brilliant blue staining of the whole bacteria, it was found that within a certain range, as the IPTG concentration and induction time increased, the expression of the corresponding size protein also increased and was significantly different from the impurity protein. At the same time, it was also found that the background expression of the target protein existed in the expression bacteria in the control group, as shown in Figure 2b.

The induced expression bacterial solution was further ultrasonically lysed and centrifuged at high speed to obtain a supernatant and a precipitate containing inclusion bodies. The supernatant was passed through a nickel column and gradient eluted with eluents containing different concentrations of imidazole. After obtaining the purified protein solution, the recombinant LCN2 protein solution was concentrated by an ultrafiltration tube and further purified using a molecular sieve. Finally, an SDS-PAGE experiment was performed to detect the purity of the protein, and it was found that the purity of the purified protein was high, as shown in Figure 2c.

In order to verify whether the protein is recombinant human LCN2 protein, the sample was tested by Western Blot, and the primary antibody used was Anti-His tag. The result showed clear bands, as shown in Figure 2d. This proves that the recombinant LCN2 protein was successfully obtained.

Example 3: Synthesis and identification of KDX001

Compound 1-1 sebacic acid dichloride, CAS: 111-19-3, (100 mg, 418 umol, 1.00 eq), compound 1a1, 1'-(5-amino-1,3-phenyl) di(ethyl-1-one), CAS: 87533-49-1, (146 mg, 828 umol, 2.00 eq) and pyridine (109 mg, 1.38 mmol, 111 uL, 3.33 eq) were dissolved in dichloromethane (2.60 mL). The reaction solution was stirred at 20 ° C for 2 hours. TLC (developing solvent ratio: petroleum ether/ethyl acetate = 0/1, compound 1a Rf = 0.50, compound 2 Rf = 0.30) showed that compound 1a was completely consumed. The reaction solution was poured into water (20.0 mL), the dichloro organic phase was separated from the water, the dichloro organic phase was first washed with a saturated aqueous sodium bicarbonate solution (20.0 mL), then washed with brine (20.0 mL), and finally dried over anhydrous sodium sulfate. The dichloro organic phase was filtered and concentrated in vacuo to obtain compound 2-1 (470 mg, crude) as a yellow solid.

Compound 2-1 (270 mg, 518 umol, 1.00 eq) and 2a (286 mg, 2.59 mmol, 5.00 eq, HCl) were dissolved in anhydrous ethanol (8.10 mL) and stirred at 80 ° C for 16 hours. TLC (petroleum ether/ethyl acetate = 0/1, compound 2 Rf = 0.30, compound Target Rf = 0) showed that compound 2 was completely consumed. The reaction solution was cooled to 20 ° C, the solid was filtered, and rinsed with ethanol (10.0 mL). The crude product was dissolved in ethyl acetate and slurried at 20 ° C for 30 minutes to obtain compound KDX001 (95.0 mg, 121 umol, 23.3% yield, 95.3% purity) as a yellow solid.

A small amount of the raw material was directly taken for thin layer chromatography monitoring (developer polarity: petroleum ether/ethyl acetate = 0/1; detection wavelength: 254 nm). Subsequently, the product was characterized by hydrogen nuclear magnetic resonance (1HNMR, parameters: ^1H NMR: ET62357-4-P1A1 400MHzDMSO-d6; δ11.38 (s, 4H), 10.22 (s, 2H), 8.15 (s, 4H), 8.02 (s, 2H), 7.86 (s, 14H), 2.32-2.37 (m, 16H), 1.58-1.60 (m, 4H), 1.30 (s, 8H).)). The results showed that the peak shape was specific, indicating that the purity of the final product was high, as shown in Figure 3a. Liquid chromatography-mass spectrometry (LC/MS, parameters: ET62357-4-P1A1, product RT＝4.647min, [M+H] ⁺ ＝745.5) mass spectrometry was further used for identification. The test results showed that the peak shape was specific, and the purity of the final product was 95% according to quantitative analysis, as shown in Figure 3b.

Example 4: In vitro affinity assay to determine the interaction between protein and KDX001

The interaction between LCN2 and KDX001 was identified by the nuclear magnetic resonance saturation transfer difference spectroscopy (STD-NNR) technique. For the STD spectra, all protein-ligand samples were prepared at a ligand/protein ratio of 50:1. Typically, the final concentration of the ligand in the sample was 0.4 mM, and the final concentration of the LCN2 recombinant protein was 20 μM in 20 mM deuterated phosphate buffer. The final volume of the analyzed sample was 200 μL. STD-NMR experiments were performed on a 700 MHz spectrometer. In the STD experiment, selective pre-saturation of the protein was achieved by a series of Gaussian pulses. A blank experiment was performed in the absence of protein to avoid artifacts. After the protein was added, the protein was stimulated by a saturation pulse again. Observing the nuclear magnetic spectrum peaks, the appearance of the characteristic peaks of binding proves that there is an interaction relationship between the protein and the small molecule, as shown in Figure 4a.

Thermoshift assay (TSA) was used to determine the interaction between proteins and KDX001. The TSA was performed using a melting curve program of a real-time fluorescence quantitative PCR system (Roche LightCycler 480) with a temperature increment of 0.01°C and a temperature range of 25-95°C. All reactions were incubated in a final volume of 20 μL and assayed in 384-well plates using a 1:5000 dilution of 5000× SYPRO Orange stock solution (Sigma-Aldrich) and the indicated concentration (10 μM) of recombinant protein diluted in a buffer containing 20 mM Tris. Different concentrations of KDX001 (5 μm-100 μm) were added to the reaction to evaluate the small molecule binding-dependent thermal stability of the protein. The real-time fluorescence quantitative PCR system software (Roche LightCycler480) was used to construct the positive derivative (d(F)/dT) curve and estimate the melting temperature of the protein unfolding transition (Tm), as shown in Figure 4b. The results showed that KDX001 can enhance the thermal stability of LCN2 protein, proving that there is a direct and strong interaction between the two.

Isothermal calorimetric titration (ITC) was used to determine the interaction between protein and KDX001. The dialyzed protein and small molecule solvent entered the 20mM hepes buffer system and the pH was adjusted to be consistent. The PEAQ-ITC isothermal titration microcalorimeter (MicroCal) was turned on to rinse the sample needle and the bottom pool with buffer. 20μM protein buffer was added to the bottom pool, the bubbles were discharged, and 400μM small molecule inhibitors were added drop by drop. The reaction exotherm was detected, and the titration curve was fitted. Based on the slope at the midpoint of the jump in the titration curve, the binding constant Kd between LCN2 and KDX001 was determined to be 1.56μM, as shown in Figure 4c, proving that KDX001 has a direct and stable interaction with the LCN2 protein, and the interaction ability is strong.

Example 5: In vivo functional assay to verify the biological activity of KDX001

KDX001 treats LLC lung cancer subcutaneous tumor model in mice

Several B6 mice (Slack) were ordered, and LLC lung cancer cell lines (2×10 ⁵ cells/each mouse) were subcutaneously inoculated in the mice. After the tumors were formed in the mice, the tumors were randomly divided into a control group (200 μL/mouse normal saline) and a treatment group (10 mg/kg/200 μL/mouse KDX001). The mice were intraperitoneally injected with drugs once every two days until the measurement endpoint. The tumor size of the mice was monitored at all times and a tumor growth curve was drawn as shown in Figure 5a. At the measurement endpoint, the tumor tissues were collected and weighed as shown in Figure 5b; this indicates that KDX001 has the ability to significantly inhibit the growth of LLC tumor cell lines in mice.

KDX001 treats MC38 subcutaneous colorectal cancer model in mice

Several B6 mice (Slack) were ordered, and the mice were subcutaneously inoculated with MC38 colorectal cancer cell line (2×10 ⁵ cells/each mouse). After the tumors were formed in the mice, the tumors were randomly divided into a control group (200 μL/mouse normal saline) and a treatment group (10 mg/kg/200 μL/mouse KDX001). The mice were intraperitoneally injected with drugs once every two days until the measurement endpoint. The tumor size of the mice was monitored at all times and the tumor growth curve was drawn as shown in Figure 5c. At the measurement endpoint, the tumor tissues were collected and weighed, as shown in Figure 5d; this shows that KDX001 has the ability to significantly inhibit the growth of MC38 tumor cell line in mice.

KDX001 treats E0771 breast cancer tail vein lung metastasis model in mice

Several B6 mice (Slack) were ordered, and the MC38 colorectal cancer cell line (2×10 ⁵ cells/each mouse) was inoculated through the tail vein of the mice. After the tumors were formed in the mice, the tumors were randomly divided into a control group (200 μL/mouse normal saline) and a treatment group (10 mg/kg/200 μL/mouse KDX001). The mice were intraperitoneally injected with drugs once every three days until the measurement endpoint. The tumor progression was monitored at all times using small animal imaging in vivo as shown in Figure 5e. At the measurement endpoint, the tumor tissues were collected and photographed for imaging as shown in Figure 5f; this indicates that KDX001 has the ability to significantly inhibit the growth of the E0771 tumor cell line in mice.

It can be seen that through mouse model experiments, it was found that KDX001 can inhibit the growth of subcutaneous lung cancer tumors, subcutaneous colorectal cancer tumors and lung metastases of breast cancer, indicating that the combination of KDX001 and LCN2 can inhibit LCN2, which is beneficial to the realization of anti-tumor immunotherapy.

The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (7)

1. Use of a lead compound targeting human lipocalin 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for anti-tumor immunotherapy by inhibiting human lipocalin 2, said tumor being a colorectal subcutaneous tumor, said lead compound having the structure according to formula (I):

Wherein m is 4-12.

2. The use according to claim 1, wherein m is 8.

3. The use according to claim 1, wherein the pharmaceutically acceptable salt of the lead compound is selected from the group consisting of hydrochloride, carbonate, sulfate, hydrobromide or phosphate.

4. The use according to claim 1, wherein the method of preparing the lead compound comprises the steps of:

Reacting the compound (1) with 1,1' - (5-amino-1, 3-phenyl) di (ethyl-1-one) to produce a compound (2),

Reacting said compound (2) with an aminoguanidine acid addition salt to form said lead compound,

5. The use according to claim 4, wherein said reacting compound (1) with 1,1' - (5-amino-1, 3-phenyl) bis (ethyl-1-one) comprises:

The compound (1), 1' - (5-amino-1, 3-phenyl) di (ethyl-1-ketone) and alkali are dissolved in an organic solvent and reacted for 0.5 to 4 hours at the temperature of 16 to 37 ℃.

6. The use according to claim 4, wherein said reacting compound (2) with an aminoguanidine acid addition salt comprises:

and (2) dissolving the compound (2) and the amino guanidine acid addition salt in an organic solvent, and reacting for 15-20 hours at 60-120 ℃.

7. The use according to claim 4, wherein the amino guanidine acid addition salt is amino guanidine hydrochloride, amino guanidine carbonate, amino guanidine sulfate, amino guanidine hydrobromide or amino guanidine phosphate.