CN116102468A - Lead compound of targeted human lipocalin2 or pharmaceutically acceptable salt thereof, and preparation method and application thereof - Google Patents
Lead compound of targeted human lipocalin2 or pharmaceutically acceptable salt thereof, and preparation method and application thereof Download PDFInfo
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- CN116102468A CN116102468A CN202310148418.6A CN202310148418A CN116102468A CN 116102468 A CN116102468 A CN 116102468A CN 202310148418 A CN202310148418 A CN 202310148418A CN 116102468 A CN116102468 A CN 116102468A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
- C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
- C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
A lead compound of targeted human lipocalin2 or pharmaceutically acceptable salt thereof, and a preparation method and application thereof, wherein the lead compound has a structure shown in the following formula (I):
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a lead compound of targeted human lipocalin2 (LCN 2 for short, also called neutrophil gelatinase related lipocalin for short) or pharmaceutically acceptable salt thereof, and a preparation method and application thereof.
Technical Field
Cancer is one of the major diseases that endanger human life and health. The new cases and the number of cancer deaths in China are the first worldwide each year, the morbidity rises year by year, and huge pressure is caused to the life health of people and the economic development of society. At present, the research and development of tumor treatment medicines in China still fall behind the main developed countries. The main methods of tumor treatment at the present stage comprise the steps of precisely killing tumor cells or activating the immune system of an organism by using targeted drugs, and inhibiting the development of tumors by using the immune system. Thus, finding molecules that are specifically expressed or highly expressed by tumor cells, and developing targeted drugs against such molecules, is a key to the research of tumor therapy.
Disclosure of Invention
Accordingly, the present invention is directed to a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof, so as to at least partially solve at least one of the above technical problems.
In order to achieve the above object, as one aspect of the present invention, there is provided a lead compound targeting LCN2, or a pharmaceutically acceptable salt thereof, the lead compound having a structure represented by the following formula (I):
As another aspect of the present invention, there is provided a method for preparing the above LCN 2-targeting lead compound or pharmaceutically acceptable salt thereof, comprising 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,
as a further aspect of the invention there is provided a pharmaceutical composition comprising a lead compound as described above that targets LCN2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
As a further aspect of the invention, there is provided the use of a lead compound targeting LCN2 as described above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in anti-tumour immunotherapy.
As a further aspect of the invention there is provided the use of a lead compound as described above for targeting LCN2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting LCN 2.
Based on the technical scheme, the lead compound targeting LCN2 or the pharmaceutically acceptable salt thereof, the preparation method and the application thereof have one or a part of the following beneficial effects:
the lead compound or the pharmaceutically acceptable salt thereof provided by the invention can target LCN2 and can be combined to LCN2 more stably through an in vitro affinity test, and the interaction of the lead compound or the pharmaceutically acceptable salt thereof can be verified to effectively inhibit tumor growth through an in vivo model test, so that the lead compound or the pharmaceutically acceptable salt thereof can realize an anti-tumor immunotherapy effect through the inhibition effect on LCN 2.
Drawings
FIG. 1 shows the correlation results of LCN2 expression with tumorigenesis and cancer survival in example 1 of the present invention, wherein a is the increase of LCN2 content in serum of tumor-bearing mice with tumor development, b is the LCN2 gene knockdown efficiency of LLC mouse lung cancer cell line, and c is the LLC cell line cell growth curve of knockdown LCN2 gene; d, subcutaneously inoculating tumor-bearing mice of LLC cell lines with the knocked-down LCN2 genes, wherein e to f are curves related to survival of patients with the flood cancer, cholangiocarcinoma and pancreatic cancer of LCN2 expression;
FIG. 2 shows the construction, purification and measurement results of prokaryotic recombinant expression vector pET28a-LCN2 of the embodiment 2, wherein a is a construction model of pET28a-LCN2, b is the expression level of LCN2 protein under different induction conditions, c is the result of PAGE electrophoresis purity of purified LCN2, and d is the result of Western Blot identification of purified LCN2 protein;
FIG. 3 shows the post-synthesis identification of compound KDX001 of example 3 of the present invention, wherein a is the nuclear magnetic resonance hydrogen spectrum of compound KDX001 and b is the mass spectrum of compound KDX 001;
FIG. 4 shows the results of the detection of the interaction between LCN2 protein and compound KDX001 according to example 4 of the present invention, wherein a is the nuclear magnetic resonance saturation transfer spectrum of LCN2-KDX001, b is the relationship between the dissolution temperature of LCN2 protein and the concentration of compound KDX001 based on the thermal shift assay, and c is the isothermal calorimetric titration curve of the interaction of LCN2 protein-KDX 001;
fig. 5 shows a tumor model of a compound KDX001 of example 5 of the invention, wherein a is the tumor growth curve of a tumor-bearing mouse of the LLC cell line, b is the tumor tissue weight of a tumor-bearing mouse of the LLC cell line, c is the tumor growth curve of a tumor-bearing mouse of the MC38 cell line, d is the tumor tissue weight of a tumor-bearing mouse of the MC38 cell line, E is in vivo imaging of tumor progression of a tumor-bearing mouse of the E0771 cell line, and f is tumor tissue imaging of a tumor-bearing mouse of the E0771 cell line.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
In the process of realizing the invention, the protein LCN2 is obviously up-regulated in the serum of a patient suffering from tumor cachexia by focusing a mechanism of forming tumor cachexia, which is a protein related to lipid nutrition transport. The LCN2 protein was then found to be positively correlated with the secretion of various tumor-associated inflammatory cytokines and weight loss in tumor patients. The LCN2 protein can directly cause atrophy of adipose tissues of mice, and when the molecule is deleted or blocked, the tumor progress of the mice is obviously inhibited and the malignant tumor cytoplasmic phenotype is effectively relieved. Therefore, development of the small molecule drug targeting LCN2 for anti-tumor immunotherapy has important practical significance for recovering anti-tumor immunity. On the basis, the invention further carries out drug design based on the structure of LCN2, and the lead compound provided by the invention is obtained through screening, and the lead compound can be relatively stably combined with LCN2 through in vitro and in vivo experiments, and effectively inhibit tumor growth through interaction between the lead compound and the LCN2, thereby realizing antitumor immunotherapy and having better application prospect.
Specifically, according to some embodiments of the present invention, there is provided a lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof, the lead compound having a structure represented by the following formula (I):
According to the embodiment of the invention, LCN2 is a flood cancer marker and has the effects of promoting the secretion of inflammatory cytokines related to tumors and promoting the growth of the tumors, and the lead compound shown in the formula (I) has a remarkable LCN2 inhibition effect, so that the effect of anti-tumor immunotherapy for inducing death of tumor iron and enhancing anti-tumor immune response is achieved.
According to embodiments of the present invention, m is preferably 8, with suitable drug solubility and affinity for targeting LCN2, but is not limited thereto, and in some embodiments, m may be, for example, 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 salts of the lead compounds described above include, but are not limited to: hydrochloride, carbonate, sulfate, hydrobromide, phosphate, or the like.
According to an embodiment of the present invention, there is also provided a method for preparing the above LCN 2-targeting lead compound or a pharmaceutically acceptable salt thereof, comprising the steps of:
step A: reacting compound 1 with 1,1' - (5-amino-1, 3-phenyl) bis (ethyl-1-one) 1a to produce compound 2, specifically represented by formula (A1) below;
and (B) step (B): compound 2 is reacted with aminoguanidine acid addition salt 2a to produce lead compound I, specifically, formula (B1) below.
According to an embodiment of the present invention, the step a specifically includes: compound 1,1' - (5-amino-1, 3-phenyl) bis (ethyl-1-one) 1a and a base are dissolved in an organic solvent and reacted at 16 to 37 ℃ (e.g., 20 ℃, 30 ℃ and the like) for 0.5 to 4 hours (e.g., 1 hour, 2 hours and the like).
More specifically, the base may be, for example, an inorganic base or an organic base, preferably pyridine, which is advantageous in the progress of the reaction. The organic solvent may be, for example, methylene chloride, and the reactant may be dissolved.
According to an embodiment of the present invention, the step B specifically includes: the compound 2 and the aminoguanidine acid addition salt are dissolved in an organic solvent and reacted at 60 to 120 ℃ (e.g., 80 ℃, 100 ℃ and the like) for 15 to 20 hours (e.g., 16 hours, 18 hours and the like).
More specifically, the aminoguanidine acid addition salt may be, for example, aminoguanidine hydrochloride, aminoguanidine carbonate, aminoguanidine sulfate, aminoguanidine hydrobromide, aminoguanidine phosphate, or the like. The organic solvent may be, for example, absolute ethanol or the like, and may dissolve the reactant.
According to an embodiment of the present invention, there is also provided a pharmaceutical composition comprising the above-described LCN 2-targeting lead compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
According to embodiments of the present invention, the above pharmaceutical composition may be formulated into several dosage forms, such as oral formulations (e.g., tablets, capsules, solutions or suspensions) according to the administration mode; injectable formulations (e.g., injectable solutions or suspensions, or injectable dry powders, ready for use by addition of water for injection prior to injection); topical formulations (e.g. ointments or solutions).
According to an embodiment of the present invention, the carriers for the pharmaceutical compositions of the present invention are of a common type available in the pharmaceutical arts, including: binders, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, pigment-free agents, flavoring agents and the like for oral preparations; preservatives, solubilizing agents, stabilizers and the like for injectable formulations; matrix for topical formulations, diluents, lubricants, preservatives and the like. The pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically).
According to an embodiment of the invention, there is also provided an application of the above-mentioned lead compound targeting LCN2 or a pharmaceutically acceptable salt thereof in preparing a medicament for anti-tumor immunotherapy. In particular to the application of the medicine for resisting lung cancer subcutaneous tumor or colorectal cancer subcutaneous tumor immunotherapy.
According to an embodiment of the present invention, there is additionally provided a lead compound as described above or a pharmaceutically acceptable salt thereof, which can be used for the preparation of a medicament for inhibiting LCN 2.
The lead compound has the effects of inducing death of tumor iron and enhancing anti-tumor immune response by inhibiting LCN 2.
The technical scheme of the invention is further described below by means of specific embodiments and with reference to the accompanying drawings. It should be noted that the following specific examples are given by way of illustration only and the scope of the present invention is not limited thereto. The drugs or reagents used in the examples below were obtained commercially or were prepared by a known preparation method. Methods such as Western blot and the like used in the following examples are all known in the art and may be described in textbooks or related documents, and are not described in detail.
Example 1: LCN2 protein promotes tumor growth and is an important tumor treatment target point
Serum LCN2 protein content increases with tumor progression
In vivo, a number of ordered B6 mice (schliek), which were subcutaneously vaccinated with LLC cell lines (5×105 cells/each) and, after tumor formation in the mice, were subjected to Elisa (R & D) mice serum LCN2 levels, which were found to gradually increase as the tumor progressed, as shown in fig. 1a.
Knocking down LCN2 gene to inhibit in vitro tumor cell growth
The gene interference technology is utilized, the two gene interference reagents shLCN2-1 (sh 1) and shLCN2-2 (sh 2) are utilized to knock down the LCN2 gene on the LLC mouse lung cancer cell line, and the real-time quantitative PCR experiment is utilized to detect the knock down efficiency, as shown in figure 1b. In vitro, the determination of the cell growth curve was performed using a real-time label-free cell analysis (RTCA) system. Firstly, 50 mu L of serum-free culture medium is added to the bottom of a 96-well plate, baseline calibration is carried out on an RTCA system, then three groups of LLC cell lines of shNC, shLCN2-1 and shLCN2-2 are digested and recovered, the cell density is adjusted to 5000 cells/150 mu L, 150 mu L of cells are inoculated to the bottom of the 96-well plate, the RTCA system is clicked again to run, and the growth rates of the three groups of cells are recorded, and the growth of tumor cells is obviously inhibited after the defect LCN2 is seen in the figure 1 c.
Knocking down LCN2 gene to inhibit in vivo tumor cell growth
In vivo, a plurality of ordered B6 mice (Schlenk) are inoculated with the three groups of cell lines (5×105 cells/each mouse) subcutaneously respectively, after the tumors of the mice are formed, tumor growth curves of the mice are measured regularly and counted according to the calculation principle of length×width×width/2, as shown in fig. 1d, the fact that LCN2 defects can cause slow tumor growth in vivo is proved, and the LCN2 is a reliable tumor treatment target point is proved.
Correlation of the height of LCN2 expression with the survival of cancer patients
LCN2 is associated with poor prognosis in tumor patients. For the TCGA (The Cancer Genome Atlas, cancer genome map) database, a total of 9637 cancer patients with survival statistics that can track disease progression were analyzed by Kaplan-Meier survival curve estimation, and patients with higher LCN2 gene expression showed worse survival according to TPM (Transcripts Per Million) median groupings of LCN2 genes in their tumor tissue transcriptome, fig. 1e. In particular, for cholangiocarcinoma (CHOL, data containing 36 patients total), fig. 1f, and pancreatic cancer (PAAD, data containing 178 patients total), fig. 1g, the survival differences associated with LCN2 gene levels are particularly pronounced.
From the above results, it can be seen that blocking LCN2 protein is beneficial to inhibit tumor growth, and LCN2 is an important tumor therapeutic target.
Example 2: construction of prokaryotic recombinant expression vector pET28a-LCN2, expression, purification and determination of human LCN2 recombinant protein
The LCN2 full-length gene is synthesized artificially, and 6 XHis-Tag is introduced at the N end. The pET28a (+) plasmid was first linearized using restriction enzymes NotI and XhoI, and the PCR products of the LCN2 gene were treated simultaneously with these two enzymes to make both ends sticky, and finally the LCN2 gene was ligated into the pET28a (+) vector using T4 ligase to construct a successful LCN2 prokaryotic expression plasmid. Transferring into DH5 alpha competent cells, screening positive clones for DNA sequencing identification, and the sequencing result shows that the recombinant expression vector pET28a-LCN2 is successfully constructed. The construction flow of the prokaryotic recombinant expression vector pET28a-LCN2 is shown in FIG. 2 a.
In order to obtain LCN2 protein (uniprot: P80188) having the sequence shown in SEQ ID No.1, isopropyl Thiogalactoside (IPTG) was used at various concentrations (0 mM, 0.1mM, 0.2mM, 0.4 mM) to induce expression strains containing LCN2 gene of synthetic origin amplified to logarithmic phase, and sampled before induction, 2 hours and 4 hours of induction, respectively. Through sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, abbreviated as SDS-PAGE electrophoresis) and coomassie brilliant blue staining of the whole strain, the expression level of the corresponding protein is increased along with the increase of the IPTG concentration and the induction time within a certain range and is obviously different from that of the hybrid protein, and meanwhile, the background expression of the target protein of the expressed strain is found in a control group, as shown in figure 2b.
Further carrying out ultrasonic pyrolysis on the induced expression bacterial liquid, and then carrying out high-speed centrifugation to obtain supernatant and sediment containing inclusion bodies. Passing the supernatant through nickel column, and gradient eluting with eluent containing imidazole with different concentrations. After the purified protein solution was obtained, the recombinant LCN2 protein solution was concentrated by ultrafiltration tubing and further purified using molecular sieves. Finally, SDS-PAGE experiments are carried out to detect the purity of the protein, and the purity of the purified protein is higher as shown in figure 2c.
In order to verify whether the protein is recombinant human LCN2 protein, western Blot detection is performed on the sample, the primary antibody used is Anti-His tag, and clear bands are visible as a result, as shown in FIG. 2d. This demonstrates the success of recombinant LCN2 proteins.
Example 3: synthesis and identification of KDX001
Compound 1-1 sebacoyl dichloride, CAS:111-19-3, (100 mg,418umol,1.00 eq), compound 1a1, 1' - (5-amino-1, 3-phenyl) bis (ethyl-1-one), CAS:87533-49-1, (146 mg, 238 umol,2.00 eq) and pyridine (109 mg,1.38mmol,111uL,3.33 eq) were dissolved in dichloromethane (2.60 mL). The reaction solution was stirred at 20℃for 2 hours. TLC (developing solvent ratio: petroleum ether/ethyl acetate=0/1, compound 1arf=0.50, compound 2rf=0.30) showed complete consumption of compound 1a. The reaction solution was poured into water (20.0 mL), the dichloro organic phase was separated from the water, the dichloro organic phase was washed with saturated aqueous sodium bicarbonate (20.0 mL), then brine (20.0 mL) and finally dried over anhydrous sodium sulfate, and the dichloro organic phase was filtered and concentrated in vacuo to give compound 2-1 (470 mg, trude) as a yellow solid.
Compounds 2-1 (270 mg,518umol,1.00 eq) and 2a (284 mg,2.59mmol,5.00eq, HCl) were dissolved in absolute ethanol (8.10 mL) and stirred at 80℃for 16 hours. TLC (petroleum ether/ethyl acetate=0/1, compound 2rf=0.30, compound targetrf=0) showed complete consumption of compound 2. The reaction solution was cooled to 20 ℃, the solid was filtered and washed with ethanol (10.0 mL). The crude product was dissolved in ethyl acetate and slurried at 20℃for 30 minutes to give compound KDX001 (95.0 mg,121umol,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). Subsequent hydrogen nuclear magnetic resonance spectroscopy (1 HNMR, parameters: 1 h NMR ET62357-4-P1A1 400MHz DMSO-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 detection result shows peak specificity, indicating higher purity of the final product, as shown in fig. 3a; further using liquid chromatography-mass spectrometry (LC/MS, parameters: ET62357-4-P1A1, product RT=4.647 min, [ M+H ]] + = 745.5), the detection result shows that the peak type is specific, and the purity of the quantitative analysis end product is 95%, as shown in fig. 3b.
Example 4: in vitro affinity assay to determine protein interaction with KDX001
Nuclear magnetic resonance saturation transfer differential spectroscopy (STD-NNR) techniques identified interactions between LCN2 and KDX 001. For the STD spectrum, all protein-ligand samples were taken at 50:1 in the ligand/protein ratio. Typically, the final concentration of ligand in the sample is 0.4mM, and the final concentration of LCN2 recombinant protein is 20. Mu.M in a solution consisting of 20mM deuterated phosphate buffer. The final volume of the sample analyzed was 200. Mu.L. STD-NMR experiments were performed on a 700MHz spectrometer. In STD experiments, selective pre-saturation of proteins was achieved by a series of gaussian pulses. Blank experiments were performed without protein to avoid artifacts. After the addition of the protein, the protein was again subjected to saturation pulse stimulation. The nuclear magnetic spectrum peaks were observed and characteristic peaks of binding appeared to demonstrate the interaction relationship between protein and small molecule as shown in fig. 4a.
Thermal displacement assay (Thermal shift assay, TSA for short) measures protein interactions with KDX 001. Thermal displacement assays were performed using a real-time fluorescent quantitative PCR system (roses LightCycler 480) melting curve program with temperature increments of 0.01 ℃ and temperature ranges of 25-95 ℃. All reactions were incubated in a final volume of 20 μl and 1 of 5000×sypro orange stock solution (Sigma-Aldrich) was used: 5000 dilutions were assayed in 384 well plates and diluted in buffer containing 20mM Tris at the indicated concentration (10. Mu.M) of recombinant protein. Different concentrations of KDX001 (5 μm-100 μm) were added to the reaction to assess the small molecule binding dependent thermostability of the protein. The use of real-time fluorescent quantitative PCR system software (roses LightCycler 480) to construct a positive derivative (d (F)/dT) curve and estimate the melting temperature of the protein unfolding transition (Tm) as shown in fig. 4b, results show KDX001 can enhance the thermal stability of LCN2 protein, demonstrating the direct powerful interaction of the two.
Isothermal calorimetric titration (ITC) determines protein interactions with KDX 001. The protein was dialyzed against small molecule solvents into a 20mM hepes buffer system and the pH was adjusted to unity. The PEAQ-ITC isothermal titration microcalorimeter (MicroCal) was turned on to buffer the loading needle and bottom well. To the bottom well, 20 μm protein buffer was added, air bubbles were removed, and 400 μm small molecule inhibitors were added dropwise. The exothermic reaction was detected, a titration curve was fitted, and the binding constant Kd between LCN2 and KDX001 was determined to be 1.56 μm based on the slope at the jump midpoint in the titration curve, as shown in fig. 4c, demonstrating the direct stable interaction of KDX001 with LCN2 protein, with strong interaction capacity.
Example 5: in vivo functional assays verify KDX001 biological Activity
KDX001 model for treating mouse LLC lung cancer subcutaneous tumor
Multiple B6 mice (Schlemk) were ordered and subcutaneously vaccinated with LLC lung cancer cell line (2X 10) 5 Individual cells/each mouse), after the mice tumor formation, tumors were randomly grouped into control group (200 μl/physiological saline) and treatment group (10 mg/kg/200 μl/KDX 001), administered by intraperitoneal injection every two days, until the measurement endpoint, the mice tumor size was monitored at the moment and the tumor growth curve was plotted as in fig. 5a, and the measurement endpoint collected tumor tissue and weighed as in fig. 5b; KDX001 was demonstrated to have the ability to significantly inhibit the growth of LLC tumor cell lines in mice.
KDX001 treatment of mouse MC38 colorectal carcinoma subcutaneous tumor model
Multiple B6 mice (Schlemk) were ordered and subcutaneously vaccinated with MC38 colorectal cancer cell line (2X 10) 5 Individual cellsAfter mice tumor formation, tumors were randomly grouped into control group (200 μl/saline) and treatment group (10 mg/kg/200 μl/KDX 001), administered intraperitoneally once every two days until the endpoint was measured, the mice tumor size was monitored at the moment and tumor growth curves were plotted as in fig. 5c, and tumor tissues were collected and weighed at the endpoint as in fig. 5d; KDX001 was demonstrated to have the ability to significantly inhibit the growth of MC38 tumor cell lines in mice.
KDX001 treatment mouse E0771 mammary cancer tail vein lung metastasis model
Multiple B6 mice (Schlemk) were ordered and the tail vein of the mice was inoculated with MC38 colorectal cancer cell line (2X 10) 5 Individual cells/each mouse), after the mice tumor formation, tumors were randomly grouped into control group (200 μl/saline) and treatment group (10 mg/kg/200 μl/KDX 001), administered by intraperitoneal injection every three days until the measurement endpoint, tumor progression was monitored with the in vivo imaging moment of small animals as in fig. 5e, tumor tissue was collected and imaged by photographing at the measurement endpoint as in fig. 5f; KDX001 was demonstrated to have the ability to significantly inhibit the growth of the E0771 tumour cell line in mice.
Therefore, the mouse model test shows that KDX001 can inhibit the growth of lung cancer subcutaneous tumor, colorectal cancer subcutaneous tumor and breast cancer lung metastasis, and the combination of KDX001 and LCN2 can inhibit LCN2, thereby being beneficial to realizing anti-tumor immunotherapy.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. A lead compound that targets human lipocalin2, or a pharmaceutically acceptable salt thereof, the lead compound having a structure represented by the following formula (I):
2. The lead compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 8.
3. The lead compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt of the lead compound comprises a hydrochloride, carbonate, sulfate, hydrobromide, or phosphate salt.
4. A method of preparing the lead compound of targeted human lipocalin2 or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, comprising 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 process of 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 process according to claim 4, wherein the reacting the compound (2) with an aminoguanidine acid addition salt comprises:
dissolving the compound (2) and the amino guanidine acid addition salt in an organic solvent, and reacting for 15-20 hours at 60-120 ℃;
wherein the amino guanidine acid addition salt is preferably an amino guanidine hydrochloride, an amino guanidine carbonate, an amino guanidine sulfate, an amino guanidine hydrobromide or an amino guanidine phosphate.
7. A pharmaceutical composition comprising the lead compound of any one of claims 1 to 3 that targets human lipocalin2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
8. Use of a lead compound targeting human lipocalin2 as defined in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for anti-tumour immunotherapy.
9. The use of claim 8, wherein the tumor is a lung cancer subcutaneous tumor or a colorectal cancer subcutaneous tumor.
10. Use of a lead compound of any one of claims 1 to 3 that targets human lipocalin2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting human lipocalin 2.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144480A (en) * | 1994-01-21 | 1997-03-05 | 皮考尔医学研究院 | Guanylhydrazones for treating inflammatory conditions |
| WO2003006426A1 (en) * | 2001-07-13 | 2003-01-23 | Axxima Pharmaceuticals Ag | Aromatic guanylhydrazones as effective compounds against neurodiseases |
| US20030203969A1 (en) * | 2000-02-02 | 2003-10-30 | Dorian Bevec | Pharmaceutically active aromatic guanylhydrazones |
| EP1389480A1 (en) * | 2002-08-14 | 2004-02-18 | Mondobiotech Interferon SA | Therapeutical use of guanylhydrazones for the inhibition of CD83 dependent processes and dendritic cell maturation |
| DE102005025906A1 (en) * | 2005-06-06 | 2006-12-07 | Dorian Bevec | Use of a guanylhydrazone compound in the manufacture of medicament for the treatment and/or prevention of e.g. acute and chronic hepatitis, liver cirrhosis, liver cell carcinoma, myocarditis, pharyngitis, pneumonia and pericarditis |
| CN101010094A (en) * | 2004-08-17 | 2007-08-01 | 塞托金药品公司 | Guanylhydrazone salts, compositions, processes of making and methods of using |
-
2023
- 2023-02-22 CN CN202310148418.6A patent/CN116102468A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144480A (en) * | 1994-01-21 | 1997-03-05 | 皮考尔医学研究院 | Guanylhydrazones for treating inflammatory conditions |
| US20030203969A1 (en) * | 2000-02-02 | 2003-10-30 | Dorian Bevec | Pharmaceutically active aromatic guanylhydrazones |
| WO2003006426A1 (en) * | 2001-07-13 | 2003-01-23 | Axxima Pharmaceuticals Ag | Aromatic guanylhydrazones as effective compounds against neurodiseases |
| EP1389480A1 (en) * | 2002-08-14 | 2004-02-18 | Mondobiotech Interferon SA | Therapeutical use of guanylhydrazones for the inhibition of CD83 dependent processes and dendritic cell maturation |
| CN101010094A (en) * | 2004-08-17 | 2007-08-01 | 塞托金药品公司 | Guanylhydrazone salts, compositions, processes of making and methods of using |
| DE102005025906A1 (en) * | 2005-06-06 | 2006-12-07 | Dorian Bevec | Use of a guanylhydrazone compound in the manufacture of medicament for the treatment and/or prevention of e.g. acute and chronic hepatitis, liver cirrhosis, liver cell carcinoma, myocarditis, pharyngitis, pneumonia and pericarditis |
Non-Patent Citations (2)
| Title |
|---|
| C. CERAMI ET AL: "High-performance liquid chromatographic method for guanylhydrazone compounds", 《J. CHROMATOGR. B》, vol. 675, pages 71 - 75, XP004043441, DOI: 10.1016/0378-4347(95)00346-0 * |
| 无: "CAS号1857365-34-4", 《STN REGISTRY数据库》, pages 1 - 4 * |
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