CN107382966B - A kind of longinamide-ligustrazine hybrid compound, preparation method and medicinal use - Google Patents

Abstract

The invention discloses a piperlongumine-ligustrazine heterocomplex, a preparation method and medical application thereof. The compound is a compound shown as a formula (I), an isomer or a pharmaceutically acceptable salt thereof, wherein R¹、R³Or R⁴Are each independently selected from C_1～3Alkyl or C_1～3An alkoxy group; r²Selected from hydrogen or halogen. The compound has better water solubility, and the proliferation inhibition activity on 4 colon cancer cells is obviously better than that of piperlongumine. The compound, the isomer or the pharmaceutically acceptable salt thereof can be applied to the preparation of medicaments for treating or preventing tumors or medicaments related to metastatic cancers, and related diseases which can be applied include but are not limited to brain glioma, colon cancer, metastatic colon cancer, non-small cell lung cancer, leukemia and the like.

Description

A kind of longinamide-ligustrazine hybrid compound, preparation method and medicinal use

technical field

The invention belongs to the fields of medicinal chemistry and pharmaceutical therapy, and in particular relates to a class of piperonamide-ligustrazine hybrids or pharmaceutically acceptable salts thereof, their preparation methods, pharmaceutical compositions containing these compounds and their medicines use

Background technique

Piperlongumine (PL), also known as pipelartine, is an alkaloid compound. It was originally isolated from the root of Piper Piper, and also in the roots of P. sylvaticum Roxb. and P. tuberculatum Jacq.

Longinamide has a variety of pharmacological effects, which can regulate blood lipid metabolism in hyperlipidemia rats, anti-platelet aggregation, analgesia, antifungal and many other pharmacological activities. It is a chemical substance with great medicinal value. In recent years, studies have found that piperamide can inhibit the proliferation, adhesion, invasion and metastasis of tumor cells; when piperamide is used in combination with some anti-tumor drugs, it can reduce or eliminate the adverse reactions of anti-tumor drugs. Mechanistic studies have shown that longinamide regulates the homeostasis of redox and reactive oxygen species by targeting oxidative stress proteins, resulting in a decrease in the level of GSH and an increase in the level of GSSG in tumor cells, which ultimately leads to an increase in the concentration of ROS in tumor cells. apoptosis or necrosis of cells. Since there is little or no oxidative stress in normal cells, longinamide has little effect on normal cells. Although longinamide has good antitumor activity, it also has some shortcomings, such as poor water solubility, which restricts its in-depth research and clinical development.

Ligustrazine (TMP), a natural product, is one of the main active components of TMP, and its structure contains a water-soluble pyrazine skeleton. Ligustrazine has various pharmacological activities such as scavenging free radicals, anti-platelet aggregation, anti-thrombosis, vasodilator, etc. It is clinically used in the treatment of respiratory system diseases, coronary heart disease and ischemic cardiovascular and cerebrovascular diseases, and has achieved good curative effect. . Recent studies have found that ligustrazine is a ROS inducer, which can increase the level of ROS in tumor cells and kill tumor cells. In addition, ligustrazine can also inhibit the proliferation and metastasis of tumor cells. However, ligustrazine requires high doses to obtain the desired efficacy, which may lead to the accumulation of toxicity. In addition, ligustrazine generally needs to be used in combination with chemotherapeutic drugs when it is anti-tumor, so it may change the pharmacokinetic properties and increase the toxic and side effects.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a class of piperonamide-ligustrazine hybrids, isomers or pharmaceutically acceptable salts thereof, which are better in water solubility and pharmacological activity than piperamide on the basis of the prior art .

Another object of the present invention is to provide a preparation method of piperonamide-ligustrazine hybrid.

The third object of the present invention is to provide the application of a piperonamide-ligustrazine hybrid compound in medicine.

The object of the present invention can be achieved by the following measures:

A kind of longinamide-ligustrazine hybrid compound, namely the compound represented by formula (I), isomer or pharmaceutically acceptable salt thereof,

in,

R ¹ , R ³ or R ⁴ are each independently selected from C _1-3 alkyl or C _1-3 alkoxy;

R ² is selected from hydrogen or halogen.

The present invention includes isomers such as cis-trans isomers and stereoisomers. In particular, in formula (I), the straight-chain double bond connected to the pyrazine ring is in the E configuration.

We unexpectedly found that when the 3,5,6-positions of pyrazine have alkyl substituents at the same time, compared with mono-substituted or piperamide, the compound's proliferation inhibitory activity against some human tumor cells and in aqueous solution can be significantly improved Soluble in the compound, so that this kind of compound has more excellent application prospect.

In a preferred embodiment, R ¹ , R ³ or R ⁴ are each independently selected from methyl or ethyl.

In a more preferred embodiment, R ¹ , R ³ or R ⁴ are each independently selected from methyl.

In a preferred embodiment, R ² is selected from hydrogen, chlorine, bromine or iodine.

In a preferred embodiment, the compound of the present invention or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

(E)-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (I ₁ )

(E)-3-Chloro-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (I ₂ )

(E)-3-Bromo-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (I ₃ )

(E)-3-iodo-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (I ₄ )

(E)-3-Chloro-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one hydrochloride Salt (I ₅ )

The invention provides a kind of preparation method of the compound shown in formula (I), and its reaction scheme is as follows:

wherein R is a R ¹ , R ³ and R ⁴ group, and the R ¹ , R ³ , R ⁴ or R ² group is as defined above.

In the preparation of compound III, the solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide or dioxane species; the reaction temperature is room temperature.

When compound IV is prepared from compound III, the solvent is selected from one of acetonitrile, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide or dioxane or more; the reaction is carried out under alkaline conditions, and the base is selected from potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, pyridine, 4-methylaminopyridine, triethylamine, N,N-dicarbonate Isopropyl methylamine; the reaction temperature is room temperature.

When preparing V from IV, the solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide or dioxane. The reaction is carried out under basic conditions, the base is selected from pyridine, 4-methylaminopyridine, triethylamine, N,N-diisopropylmethylamine; the reaction temperature is -20°C.

When preparing I from V, the solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide or dioxane. The reaction is carried out under basic conditions, the base is selected from pyridine, 4-methylaminopyridine, triethylamine, N,N-diisopropylmethylamine; the reaction temperature is -70°C.

These intermediates or target compounds can be purified according to conventional separation techniques, separated into their monomers if desired, and further converted into addition salts with pharmaceutically acceptable acids as desired.

Unless otherwise specified, the following terms used in the specification and claims have the meanings discussed below:

"Alkyl" means a saturated aliphatic hydrocarbon group of 1-20 carbon atoms, including straight-chain and branched-chain groups (numerical ranges mentioned in this application, such as "1-20", refer to this group, which is an alkyl group, which may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). Alkyl groups containing 1-3 carbon atoms are called lower alkyl groups. When lower alkyl has no substituent, it is called unsubstituted lower alkyl. More preferably, the alkyl group is a medium-sized alkyl group having 1-10 carbon atoms (ie, a C _1-10 alkyl group), such as methyl, ethyl, propyl, 2-propyl, n-butyl, Isobutyl, tert-butyl, pentyl, etc. Further preferred alkyl groups are those having 1-8 or 1-6 carbon atoms, most preferably, the alkyl group is a lower alkyl group having 1-3 carbon atoms, such as methyl, ethyl, propyl, 2-propyl Base et al. Alkyl groups can be substituted or unsubstituted.

"Halogen" means fluorine, chlorine, bromine or iodine.

"Pharmaceutically acceptable salts" as referred to in the present invention refer to those salts which retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) Forming a salt with an acid, obtained by the reaction of the free base of the parent compound with an inorganic acid or an organic acid. The inorganic acid includes hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid, etc. , organic acids include acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, tartaric acid, maleic acid acid, ascorbic acid, benzoic acid, hydroxybenzoic acid, gamma-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, camphoric acid, oxalic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene- 2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, lauryl sulfate, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid or malonic acid, etc.

(2) The acid proton present in the parent compound is replaced by a metal ion or a salt formed by complexation with an organic base, such as an alkali metal ion, alkaline earth metal ion or aluminum ion, an organic base such as ethanolamine, diethanolamine, triethanolamine Ethanolamine, tromethamine, N-methylglucamine, quinine, etc.

The "pharmaceutical composition" in the present invention refers to combining one or more of the compounds of the present invention or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof with other chemical components, such as pharmaceutically acceptable carrier, mix. The purpose of a pharmaceutical composition is to facilitate the process of administration to an animal.

"Pharmaceutically acceptable carrier" in the present invention refers to an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, phosphoric acid Calcium, various sugars (eg lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin, water, polyethylene glycol , propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxy hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

The present invention provides a pharmaceutical composition, which uses the compound, isomer or a pharmaceutically acceptable salt thereof described in the present invention as an active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable salt. The active ingredient of the pharmaceutical composition can be only the compound of the present invention, or can be used in combination with other existing medicines.

In the present invention, the compounds of formula I and their pharmaceutically acceptable salts, as well as solvates of these compounds (collectively referred to herein as "therapeutic drugs"), can be used alone, or preferably in accordance with standard pharmaceuticals, when administered to mammals Methods It is used in combination with a pharmaceutically acceptable carrier or diluent. Administration can be by various routes, including oral, parenteral, or topical. Parenteral administration as referred to herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

The compound provided by the present invention has better water solubility, which is about 9-26 times that of piperamide. Pharmacological tests have shown that the compounds have significantly better inhibitory activity on the proliferation of four types of colon cancer cells than piperamide. The selectivity of _I2 for colon cancer cells (the ratio of the _IC50 of normal colon cell CCD-841 to the _IC50 of colon cancer cells) was 43-172-fold, while that of piperamide was only 5-17-fold. I ₂ can selectively increase the level of ROS in tumor cells, and has excellent anti-proliferation and metastasis activities of colon cancer cells in vitro and in vivo, and the effect is better than that of piperamide.

The compounds, isomers or pharmaceutically acceptable salts thereof of the present invention can be used in the preparation of medicaments for treating or preventing tumors. On the other hand, the compounds, isomers or pharmaceutically acceptable salts thereof of the present invention can be used in medicines related to metastatic cancer, especially in medicines for the treatment or prevention of cancer, specifically including but not limited to brain colloids stromal tumor, colon cancer, metastatic colon cancer, non-small cell lung cancer, leukemia, etc.

Description of drawings

Fig. 1 The effect of the representative compound I ₂ of the present invention on the growth of mouse colon cancer xenografts;

Figure 2 The effect of the representative compound _I2 of the present invention on lung metastasis of colon cancer in mice.

Detailed ways

The content of the present invention is specifically described below by means of examples. In the present invention, the examples described below are for better illustrating the present invention, and are not intended to limit the scope of the present invention.

Example 1: (E)-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (I ₁ )

Intermediate V (0.5 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, triethylamine (0.6 mL) was added dropwise thereto, and pivaloyl chloride (0.6 mL) was added dropwise to the reaction solution at -20°C, followed by reaction for 45 minutes At the same time, the intermediate VI (0.5 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, n-butyllithium (2.9 mL) was added at -70 °C, and then stirred for about 45 minutes, the reaction solution of the previous step was directly added to In this reaction, the reaction was monitored by TLC after 1 hour of reaction. 10 mL of saturated aqueous ammonium chloride solution was added to the reaction solution to quench the n-butyllithium in the reaction, extracted, the aqueous phase was washed with ethyl acetate (20 mL × 3), the organic phases were combined and washed with saturated brine (30 mL × 3 ), dried over anhydrous sodium sulfate, and concentrated to obtain crude product, which was purified by column (PE/EA=3:1) to obtain 236 mg of pure pale yellow solid (I ₁ ), pale yellow solid.

Yield 53%; mp 115-118°C. ¹ H NMR (300 MHz, CDCl ₃ ), δ (ppm): 2.41-2.45 (m, 2H, CH ₂ ), 2.48 (s, 6H, 2×CH ₃ ), 2.59 (s, 3H, CH ₃ ), 4.01 (t, J=6.0Hz, 2H, NCH ₂ ), 6.03 (d, J=4.5Hz, 1H, CH =CH), 6.90～6.96 (m, 1H, CH= CH ), 7.80～7.91 (m , 2H, CH=CH); ¹³ C NMR (75MHz, CDCl ₃ ): δ 20.9, 21.9, 22.2, 26.5, 42.3, 126.2, 138.4, 143.2, 149.3, 150.1, 152.6, 161.4, 169.0; ESI-MS: 294.1[M+Na] ⁺ ; HRMS calculated for C ₁₅ H ₁₇ N ₃ O ₂ Na[M+Na] ⁺ 294.1218, found 294.1219, ppm error 0.3.

Example 2: (E)-3-Chloro-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridine-2(1H) - Ketone (I ₂ )

Referring to the synthesis method of compound I ₁ , 236 mg of pure light yellow solid (I ₂ ) was obtained. Yield 56%; mp 111-114°C. ¹ H NMR (300MHz, CDCl ₃ ), δ (ppm): 2.49 (s, 6H, 2×CH ₃ ), 2.54～2.56 (m, 2H, NCH ₂ CH ₂ ), 2.59 (s, 3H, CH ₃ ) ,4.06(t,J＝6.0Hz,2H,N _CH2CH2 ),7.06(t,J＝4.5Hz,1H,COClC＝ _CH ),7.82～7.95(m,2H,CH＝ CH ); ^13C NMR (75MHz, _CDCl3 ): δ 21.0, 21.9, 22.2, 25.5, 42.0, 126.2, 128.3, 138.4, 141.3, 143.2, 149.3, 150.1, 152.6, 161.4, 168.8; ESI-MS: 328.1 [M+Na] ⁺ ;HRMScalculated for C ₁₅ H ₁₆ N ₃ O ₂ ClNa[M+Na] ⁺ 328.0829,found 328.0835,ppm error 1.8.

Example 3: (E)-3-Bromo-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridine-2(1H) - Ketone (I ₃ )

Referring to the synthesis method of compound I ₁ , pure pale yellow solid (I ₃ ) was obtained. Yield 43%; mp 109-113°C. ¹ HNMR (300MHz, CDCl ₃ ), δ(ppm): 2.44～2.59 (m, 11H, NCH ₂ CH ₂ and 3×CH ₃ ), 4.06 (t, J=7.5Hz, 2H, N CH ₂ CH ₂ ) , 7.06 (t, J=4.5Hz, 1H, COClC= CH ), 7.83～7.93 (m, 2H, CH=CH); ¹³ C NMR (75MHz, CDCl ₃ ): δ 21.0, 21.9, 22.2, 26.9, 42.1, 113.2, 126.3, 138.3, 143.3, 147.3, 149.3, 150.1, 152.6, 161.3, 169.0; ESI-MS: 372.0[M+Na] ⁺ ; HRMS calculated for C ₁₅ H ₁₆ N ₃ O ₂ BrNa[M+Na] ] ⁺ 372.0368,found372.0371,ppm error 0.8.

Example 4: (E)-3-iodo-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridine-2(1H) - Ketone (I ₄ )

Iodine (104 mg) was added to a mixture of I ₁ (50 mg) of pyridine and tetrahydrofuran (V(Py)/V(THF)=1:1), stirred at room temperature overnight, and 16 mL of saturated ammonium chloride was added to the reaction The aqueous solution, after adding an appropriate amount of sodium sulfite, was extracted, the aqueous phase was washed with ethyl acetate (20 mL×3), the organic phases were combined and dried over magnesium sulfate, filtered, and the filtrate was concentrated to obtain a crude product, which was purified by column (PE/EA=3: 1) 23 mg of yellow solid (I ₇ ) was obtained, with a yield of 31.4%.

mp 108-113°C. ¹ H NMR (300MHz, CDCl ₃ ), δ (ppm): 2.45-2.51 (m, 8H, NCH ₂ CH ₂ and 2×CH ₃ ), 2.59 (s, 3H, CH ₃ ), 4.07 (t, J= 7.5Hz, 2H, NCH ₂ ), 7.65 (t, J=4.5Hz, 1H, C=CH), 7.84 (s, 2H, CH=CH); ¹³ C NMR (75MHz, CDCl ₃ ): δ 21.0, 21.9, 22.2, 28.7, 42.3, 96.6, 126.4, 138.2, 143.3, 149.3, 150.1, 152.6, 154.7, 161.3, 169.1; ESI-MS: 420.0[M+Na] ⁺ ; HRMS calculated for C ₁₅ H ₁₆ N ₃ O ₂ INa[M+Na] ⁺ 420.0185, found 420.0191, ppm error 1.4.

Example 5: (E)-3-Chloro-1-{3-[2-(3,5,6-trimethylpyrazine)]acryloyl}-5,6-dihydropyridine-2(1H) - Keto hydrochloride (I ₅ )

Compound I ₂ (50 mg, 0.16 mmol) was dissolved in ethyl acetate (5 mL), saturated HCl in ethyl acetate (15 mL) was added, and the mixture was stirred at room temperature overnight. _The product I5 was obtained by filtration as a pale yellow powder, yield: 49%, mp: 95-97°C. ¹ H NMR (300MHz, DMSO), δ (ppm): 2.49 (s, 6H, 2×CH ₃ ), 2.54～2.56 (m, 2H, NCH ₂ CH ₂ ), 2.59 (s, 3H, CH ₃ ), 3.97 (t, J=6.3 Hz, 2H, NCH ₂ CH ₂ ), 7.36 (t, J=4.5 Hz, 1H, COClC= CH ), 7.77-7.67 (m, 2H, CH=CH).

Example 6: Solubility Test Results of Representative Compounds of the Invention

The solubility of ligustrazine-piperamide hybrid I _1-4 in pure water and PBS solution (50 mM, pH 7.4) was determined by HPLC. It can be seen from Table 1 that the water solubility of these compounds is better than that of piperamide, wherein the solubility of I ₂ (22.84 μg/mL) and I ₁ (42.63 μg/mL) in pure water are respectively piperamide (1.63 μg/mL). μg/mL) about 14 times and 26 times. The results show that the compounds of the present invention can significantly improve the water solubility of the overall molecule.

Table 1.I _1-4 Determination of water solubility

Example 7: Partial pharmacological tests and results of representative compounds of the present invention

1. In vitro anti-tumor screening

experimental method:

(1) Cells were digested, counted, and prepared into a cell suspension with a concentration of 3 to 5×10 ⁴ cells/mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate (3 to 5×10 ³ cells per well). );

(2) The 96-well cell culture plate was placed in a 37°C, 5% _CO2 incubator for 24 hours;

(3) Dilute the drug to the required concentration with complete medium, add 100 μL of the corresponding drug-containing medium to each well, and set up a negative control group and a positive control group at the same time;

(4) The 96-well cell culture plate was placed in a 37°C, 5% _CO2 incubator for 72 hours;

(5) MTT staining was performed on the 96-well plate, λ=490 nm, and the OD value was determined;

a. Add 20 μL MTT (5 mg/mL) to each well, and continue to culture in the incubator for 4 hours;

b. Discard the medium, add 150 μL DMSO to each well to dissolve, shake gently for 10 minutes;

c. λ=490nm, the microplate reader reads the OD value of each well, and calculates the inhibition rate.

(6) Calculate the inhibition rate of each group.

Test results: Table 2 lists the activity data of some compounds of the present invention in inhibiting the proliferation of various tumor cells, and the positive control drug is PL.

In addition, our research group has disclosed a group of nitrogen-containing heterocycle-derived PL analogs and their anti-tumor activities (European Journal of Medicinal Chemistry, 2017, 138, 313-319), among which three compounds contain pyrazine structures, which are (E) -1-{3-[2-(pyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (Compound A), (E)-1-{3-[2-( 3-Methylpyrazine)]acryloyl}-5,6-dihydropyridin-2(1H)-one (Compound B) and (E)-1-{3-[2-(5-methylpyrazine) )]acryloyl}-5,6-dihydropyridin-2(1H)-one (compound C). We compared the antitumor activity of compounds I _1-4 with AC. It can be seen from Table 1 that the antitumor activity of compounds I _1-4 is significantly better than that of PL, among which compound I ₂ has the strongest activity. Notably, the proliferation inhibitory activity of _I2 on HCT-116 cells was 10–30 times higher than that of AC. The results show that the hybridization of ligustrazine and piperamide can effectively enhance the in vitro antitumor activity of the derivatives.

Table 2 IC ₅₀ values of the anti-proliferation activity of the compounds on some human tumor cells

Table 3 The proliferation inhibitory activity of compounds on different human colon cancer cells and normal colon epithelial cells IC ₅₀ (μM)

It can be seen from Table 3 that the proliferation inhibitory activity of I ₂ on four kinds of colon cancer cells is obviously better than that of piperamide, and the effect on HCT-116 is the best. The selectivity of _I2 for colon cancer cells (the ratio of the _IC50 of normal colon cell CCD-841 to the _IC50 of colon cancer cells) was 43-172-fold, while that of PL was only 5-17-fold, suggesting that _I2 was effective for colon cancer Cells are more selective than PL.

2. In vivo anti-colon cancer activity study

Experimental method: Collect cultured cells, prepare 2×10 ⁷ /mL cell suspension under sterile conditions, and inoculate 0.1 mL subcutaneously in the right armpit of nude mice. The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. After the tumor grew to 100 mm ³ , the animals were randomly divided into 4 groups with 8 mice in each group. Using the method of measuring tumor diameter, the anti-tumor effect of the test substance was dynamically observed. The model group was given no or equal dose of vehicle; the drug group: intraperitoneal injection, once a day, for 3 weeks. After the administration, the mice were sacrificed, and the tumor mass was surgically removed and weighed. The formula for calculating tumor volume (TV) is:

TV=1/2×a×b ²

where a and b represent the length and width, respectively.

The relative tumor volume (RTV) was calculated according to the measurement results, and the calculation formula was: RTV=V _t /V ₀ . Wherein V ₀ is the tumor volume measured in separate cages (ie d ₀ ), and V _t is the tumor volume at each measurement. The evaluation index of anti-tumor activity is the relative tumor proliferation rate T/C (%), and the calculation formula is as follows:

T _RTV : RTV of the treatment group; C _RTV : RTV of the negative control group.

Test results: As shown in Figure 1, two doses of I ₂ at 2.5 and 5 mg/kg were intraperitoneally injected to investigate its anti-tumor activity in vivo. PL was selected as the positive control, and the dose was 5 mg/kg. Dosing once a day for three weeks. The results showed that I ₂ could effectively inhibit the growth of transplanted tumors, and the tumor inhibition rate was 61% at a dose of 5 mg/kg, which was significantly better than that of PL (53%).

3. In vivo anti-colon cancer lung metastasis test

Experimental method: 40 nude mice, 20-22 g, male. Raised in SPF environment, one cage per 3 animals, indoor relative humidity 50%-70%, room temperature 18-29°C. The standard diet, drinking water and bedding of mice were sterilized by high temperature and high pressure and changed twice a week. Mice need to be reared for 1 week to adapt to the new environment, and can be used for experimental modeling after 1 week. HCT-116 cells in logarithmic growth phase were taken and prepared into 5×10 ⁶ /mL cell suspension with serum-free medium under aseptic conditions, and 0.2 mL was injected into the tail vein of nude mice to establish human colon cancer BALB/c nude mouse lung metastasis model of cell line HCT-116. Afterwards, compound I was administered in three doses of 1, ₂ and 4 mg/kg by intraperitoneal injection. PL and Oxaliplatin were selected as positive controls, and the dose was 4 mg/kg, which was also administered by intraperitoneal injection. Changes in mouse body weight were recorded every 3 days after injection. The mice were administered 3 times a week, and all nude mice were sacrificed after 6 weeks, and their lungs were removed and sliced for HE staining to observe the presence or absence of metastases. If metastases were found, observe the number of tumor metastases and calculate the area of tumor metastases , and perform statistical analysis.

Test results: As shown in Figure 2, I ₂ can effectively inhibit the lung metastasis of HCT-116 cells. Compared with the control group, I ₂ could dose-dependently improve the weight loss of mice in the nude mouse lung metastases model, and its effect was better than that of the control group PL and Oxaliplatin (Fig. 2A). During the modeling process, the mice in the control group started to die from the 4th week. At the end of the experiment, the final survival rate was 66.7%, while I ₂ , PL and Oxaliplatin could significantly improve the survival rate of mice (Fig. 2B). ), intraperitoneal injection of 1 mg/kg I ₂ , the survival rate of mice increased to 83.3%, while intraperitoneal injection of 2 mg/kg, 4 mg/kg of I ₂ , 4 mg/kg of PL and Oxaliplatin increased the survival rate of mice to 83.3%. 100%. More importantly, I ₂ can dose-dependently reduce the number of tumor lung metastases in mice (Figure 2D) and reduce the tumor volume of metastases (Figure 2C), which is significantly better than the control group PL and Oxaliplatin. At 4 mg/kg, the mean numbers of colon cancer tumor lung metastases in the I ₂ and PL and Oxaliplatin groups were 22, 39 and 36, respectively.

Claims (5)

1. A compound, isomer, or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

2. a pharmaceutical composition comprising the compound, isomer or pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient or a main active ingredient, in combination with a pharmaceutically acceptable salt.

3. Use of a compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the treatment or prevention of tumors.

4. Use of a compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the treatment or prevention of a disease associated with metastatic cancer.

5. Use of a compound, isomer, or pharmaceutically acceptable salt thereof of claim 1 for the manufacture of a medicament for the treatment or prevention of cancer, wherein the cancer is selected from the group consisting of brain glioma, metastatic colon cancer, non-small cell lung cancer, or leukemia.

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