阅读说明：本技术 甲酰胺类化合物在制备抗脊索肿瘤药物中的应用及试剂盒 (Application of formamide compound in preparation of spinal cord tumor resisting medicine and kit ) 是由 赵曜 张启麟 韩瑞 郭国骥 费丽江 马增翼 叶钊 姚博远 于 2021-01-27 设计创作，主要内容包括：本发明公开了甲酰胺类化合物在制备抗脊索肿瘤药物中的应用及试剂盒,药理实验表明,所述的化合物或其盐和晶体具有较强的抑制脊索瘤TGF-β信号通路的活性,与阳性对照药Vactosertib活性相当,脊索瘤TGF-β信号通路的抑制活性高于另一阳性化合物LY3200882,对人颅底脊索瘤细胞系的侵袭性有较强的抑制活性,预示了所述的化合物可以制备治疗抗脊索肿瘤药物。本发明对脊索瘤具有较好的抑制活性,安全性优于现有技术报道的化合物,由此可见,所述化合物：毒性更低、更具开发前景的新型脊索瘤侵袭性抑制剂,具有作为临床治疗剂使用前景。甲酰胺类化合物的结构式如下：(Pharmacological experiments show that the compound or salt and crystal thereof have stronger activity of inhibiting a chordoma TGF-beta signal path, the activity of inhibiting the chordoma TGF-beta signal path is equivalent to that of a positive control drug Vactorib, the inhibitory activity of the chordoma TGF-beta signal path is higher than that of another positive compound LY3200882, and the compound has stronger inhibitory activity on the invasiveness of a human skull base chordoma cell line, so that the compound can be used for preparing the anti-chordoma drug. The compound has better inhibitory activity to chordoma and better safety than the compounds reported in the prior art, so that the compound: the novel chordoma invasive inhibitor has lower toxicity and better development prospect, and has application prospect as a clinical therapeutic agent. CarboxamidesThe structural formula of the compound is as follows:)

1. The application of the formamide compound in the preparation of the anti-spinal cord tumor medicament is that the formamide compound is a compound shown in the formula I or salt and crystal thereof,

2. the use according to claim 1, wherein the salt is a pharmaceutically acceptable salt of an anion.

3. Use according to claim 1, wherein the salt is a hydrochloride, hydrobromide, sulphate, acetate, trifluoroacetate, citrate, tartrate, maleate, fumarate, methanesulphonate, p-toluenesulphonate or oxalate salt.

4. The use according to any one of claims 1 to 3, wherein said medicament comprises a therapeutically effective amount of said carboxamide compound or salts and crystals thereof and a pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the medicament is in the form of a tablet, capsule, aqueous suspension, oily suspension, dispersible powder, granule, lozenge, emulsion, syrup, cream, suppository or injection.

6. A kit for treating an anti-chordoma drug, which comprises the carboxamide compound as claimed in any of claims 1 to 3.

Technical Field

The invention relates to a new application of formamide compounds in pharmacy.

Background

Chordoma is a relatively rare primary tumor, originating from residual spinal cord tissue of the embryo, preferably around the age of 40 to 60 years. Among them, the skull base chordoma is a rare disease, only accounts for one thousandth of the intracranial tumor, occurs in the slope and saddle area of the skull base, and grows slowly and invasively. Epidemiological surveys have shown that the annual incidence of craniofacial chordoma is 0.2-0.8 per million people. The skull base chordoma, due to its special location, can cause serious consequences and even death in deep brain where surrounding tissues are involved. The current treatment means for patients mainly comprise:

1. and (3) operation: radical surgical resection is the only effective treatment at present. However, complete tumor resection is not achieved in nearly 70% of patients.

2. Radiotherapy: among various radiotherapy methods, only proton knives have been proven to have certain efficacy on chordoma at present. However, the proton knife may damage the tissue around the tumor, especially the brain stem and optic nerve. There is still a considerable risk with this approach. In addition, proton knife therapy requires multiple objective factors, such as high cost, making proton knife therapy unavailable to many patients.

3. Medicine preparation: at present, no drug which is really effective on the chordoma is available, including common chemotherapy and targeted therapy;

therefore, the urgent need in the field is to explore new means for treating chordoma, especially to discover a specific drug target, which can effectively treat chordoma patients and meet the needs of clinical application.

Disclosure of Invention

The invention aims to disclose application of a formamide compound in preparation of a spinal cord tumor resisting medicine and a kit, so as to overcome the defects in the prior art and meet the requirement of clinical application.

The formamide compound is a compound shown as a formula I, or salt and crystal thereof,

the salt of the compound is a chemically acceptable anion salt containing a medicament, and preferably hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, citrate, tartrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate and oxalate.

Such crystals are disclosed in WO 2019/114792 a1, such as formula I;

the preparation method of the formamide compound or the salt and the crystal thereof is well known and can be referred to the report of the WO 2019/114792A 1 patent, and the invention is not repeated.

Pharmacological experiments show that the compound or the salt and the crystal thereof have stronger activity of inhibiting the TGF-beta signal pathway of the chordoma, and the activity is equivalent to that of a positive control drug Vactosertib (CAS No.:1352608-82-2), and the inhibition activity of the TGF-beta signal pathway of the chordoma is higher than that of another positive compound LY3200882(CAS No.: 1898283-02-7;

in addition, the compound disclosed by the application has strong inhibition activity on the invasiveness of the human skull base chordoma cell line (UM-Chor1), is equivalent to the positive compound LY-364947(CAS No.:396129-53-6) and Vactosertib, and indicates the potential application value of the compound in the aspect of resisting skull base chords;

on the basis, the inventor verifies that the compound can be used for preparing the anti-spinal cord tumor medicament in vitro and in vivo.

In combination with the above description, the compound of the present invention has a better inhibitory activity on chordoma and is safer than the compounds reported in the prior art, so that the compound has an obvious therapeutic effect on chordoma tumors and can be used for preparing anti-chordoma drugs;

the compounds of the present invention can be administered to a patient in need of treatment by intravenous injection, oral administration, etc., and the dosage can be determined by a physician according to the specific condition of the patient.

The invention also relates to a pharmaceutical composition of formamide compounds, which comprises a therapeutically effective amount of the formamide compounds or salts thereof and a pharmaceutically acceptable carrier, wherein the carrier comprises excipients such as water, an antioxidant such as sodium sulfite, a lubricant such as magnesium stearate;

the pharmaceutical composition is tablets, capsules, aqueous suspensions, oily suspensions, dispersible powders, granules, pastilles, emulsions, syrups, creams, suppositories or injections;

the invention also relates to a kit for treating the spinal cord tumor resisting medicine, and the kit comprises the formamide compound.

The invention has the beneficial effects that:

the compound has better inhibitory activity to chordoma and better safety than the compounds reported in the prior art, so that the compound: the novel chordoma invasive inhibitor has lower toxicity and better development prospect, and has application prospect as a clinical therapeutic agent.

Drawings

FIG. 1 is a graph showing the results of an invasion test.

FIG. 2 is a graph showing the in vivo tumor suppression results of nude mice

Detailed Description

The present invention is described in further detail with reference to the following examples, but the embodiments of the present invention are not limited thereto, and the technical scope of the present invention is defined by the claims.

Example 1

The compounds were tested for their inhibitory activity against the normal cell lines 293-T (human renal epithelial cells) and HL7702 (human hepatocytes) using the CELL TITER-GLO method. The initial concentration of the compound was 100 micromolar, 2-fold dilution, and 3 concentration duplicate wells were assayed, with specific experimental procedures being performed according to the kit instructions. The half maximal inhibitory concentration (IC50) values for the compounds of the present application are shown in table 1 below.

TABLE 1 half inhibitory concentrations of the compounds of the invention against two normal cell lines

As can be seen from Table 1, compared with positive compounds LY364947 and Vactosertib, the compound GFH-018 disclosed by the application has weak inhibitory activity on human normal renal epithelial cells (293-T) and human normal hepatic cells (HL7702) and has lower toxic and side effects.

Example 2

Compounds were tested for inhibitory activity against UM-Chor1 (human skull base chordoma), MCF7 (human breast cancer cells), U87MG (human glioblastoma cells) using the CELL TITER-GLO method. The initial concentration of the compound was 100 micromolar, 2-fold dilution, and 3 concentration duplicate wells were assayed, with specific experimental procedures being performed according to the kit instructions. The specific results are shown in Table 2 below. TABLE 1 half inhibitory concentrations of the compounds of the invention against two normal cell lines

TABLE 2 half inhibitory concentration of the compounds of the invention against sensitive tumor cell lines

As can be seen from Table 2, the GFH-018 compound expressed in the present application has higher inhibitory activity against UM-Chor1 (human skull base chordoma), MCF7 (human breast cancer cell) and U87MG (human glioblastoma cell) than the positive compounds LY364947 and Vactosertib.

Example 3

A human skull base chordoma cell line (UM-Chor1) is paved into a six-hole plate, each hole is about 3 x 10^5 cells, the plate is 24 hours, after the cells are completely attached, a Transwell invasion experiment is carried out after the cells are added for 72 hours according to a concentration gradient, the influence of the drug on the invasiveness of the cells is verified, namely 100ul of serum-free cell suspension with the concentration of 2 x 10^ 6/ml is added into an upper chamber of a Transwell chamber, and matrigel matrix glue is added according to the proportion of 1: 50 into the upper chamber and 600ul of complete medium containing serum into the lower chamber. The cell used was a Falcon Permeable Support for 24-well Plate with 8.0 μm Transparent PET Membrane, Sterile.

The results are shown in FIG. 1.

The terms: methods of invasion assay are described in detail in Shann K, Liu C, Liu B H, et al, circular numbering RNA HIPK3 media regenerative molecular dynamics in Diabetes mellitis [ J ]. Circulation,2017.

Wherein:

GFH-018 represents the drug GFH-018 as described herein;

LY364947 represents a diheteroaryl-substituted pyrazole compound useful as TGF-beta receptor I kinase; the selective ATP competitive inhibitor of (A) is described in He Fang, Li Ling, Li Pei-Pei et al. Cyclooxogene-2/systematic media TGF-beta 1-induced catalysis in vacuum smooth muscle cells and rates underlying failure [ J ]. aging (Albany NY),2020,12: 21220-;

vactoritib represents a potent, selective, orally bioavailable TGF-beta receptor ALK4/ALK5 inhibitor, specifically acting as a Jung Su Young, Park Ju-Hwan, Baek Min-Jun et al, development of an organic better-than modified-release-driven powder of human activity in a human body model of an organic activity [ J ] Int J Pharm,2020,578:119103.

In FIG. 1, the concentrations of 0uM, 50uM and 100uM were administered from left to right.

As can be seen from fig. 1: results cells stained in black in the figure represent cells that cross the matrigel-covered membrane, with a density that is positively correlated with cell invasiveness. As can be seen from the experimental results, the cell density gradually decreases with the increase of the administration concentration, which represents that the medicine GFH-018 can effectively inhibit the invasion of the chordoma cells, and the effect is slightly better than that of the other two positive compounds LY364947 and Vactosertib.

Example 4

This example is the evaluation of the efficacy of example 12 on MCF7 nude mouse xenograft model

The UM-Chor1 cell line was injected subcutaneously into nude mice by subcutaneous tumorigenesis, the injection amount was 100ml of cell suspension and 100ml of matrigel, and the number of cells was 5 x 10 x 6/mouse. A total of 15 female nude mice were injected subcutaneously with tumor cell lines. And at the time of subcutaneous neoplasia, i.e., 21 days after subcutaneous injection of the cell line, the nude mice were weighed and given intragastric administration of GFH-018, 15 nude mice were divided into 5 in the high dose group, 5 in the low dose group and 5 in the blank control group. The high dose group was administered at a dose of 50mg/kg, the low dose group was administered at a dose of 25mg/kg, and the blank control group was administered daily for 7 consecutive days with equal drug volume of solvent. The tumor volume was recorded starting from 14 days after the subcutaneous tumorigenesis, and the results are shown in FIG. 2, the body weight of the nude mice is shown in Table 3, and it can be seen that GFH-018 can inhibit chordoma in vivo.

The UM-Chor1 cell line refers to a human skull base chordoma cell line;

GFH-018 is a carboxamide drug as described herein

As shown in FIG. 2, the high dose was administered at 50mg/kg, the low dose was administered at 25mg/kg, and NC was a blank control given an equal volume of drug solvent. The abscissa is the number of days of subcutaneous injection of the tumor cell line.

TABLE 3 weight of nude mice

Example 5

Composition tablet preparation

The preparation method comprises the following steps: mixing the compound of any of examples 1-26 with sucrose and corn starch, moistening with water, stirring, drying, pulverizing, sieving, adding calcium stearate, mixing, and tabletting. Each tablet weighs 100mg, and the content of active ingredients is 10 mg.

Example 6

Preparation of injection composition

Example 10mg of the Compound in any of examples 1-26

70mg of water for injection

The preparation method comprises the following steps: dissolving active ingredient in water for injection, mixing, filtering, and packaging the obtained solution under aseptic condition into ampoule bottles with the active ingredient content of 10 mg/bottle at 80 mg/bottle.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.