CN107663202B - 3-(ureido-methyl)-4-aryl-pyridine derivative and preparation method thereof and application as anti-cancer drug - Google Patents

Abstract

3-(Urea-methyl)-4-aryl-pyridine derivatives and preparation methods thereof and use as anti-liver cancer drugs. The present invention discloses a compound shown in formula I or a pharmaceutically acceptable salt, crystal form, or solvate thereof: wherein X is O or S; n is an integer of 0 to 3; R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are independently selected from H, cyano, COOR ₁₁ , C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, trifluoromethyl, amino, nitro, hydroxyl, thiol or halogen, and R ₁₁ is selected from H or C ₁ to C ₄ alkyl. At the same time, the present invention also discloses a preparation method of the compound shown in formula I. The half inhibitory concentration (IC ₅₀ ) of the compound of the present invention is small, and it has a good inhibitory effect on liver cancer cells; moreover, the preparation method of the compound of the present invention is simple, the reaction conditions are mild, it is easy to operate and control, the energy consumption is small, the yield is high, the cost is low, and it is suitable for industrial production.

Description

3-(ureido-methyl)-4-aryl-pyridine derivative and preparation method thereof and application as anti-cancer drug

technical field

The present invention relates to a 3-(ureido-methyl)-4-aryl-pyridine derivative, its preparation method and its application as an anti-cancer drug.

Background technique

Tumor is one of the main factors threatening human life, and liver cancer is a malignant tumor with high morbidity and high lethality worldwide. Due to the influence of hepatitis B virus (HBV), hepatitis C virus (HCV) and alcoholic fatty liver disease, the incidence and mortality of liver cancer in my country ranks first in the world, which seriously threatens the life and health of our people. However, due to the lack of effective methods for early diagnosis of liver cancer, patients are usually in the middle or late stage when they are clinically diagnosed, and they lose the best chance of surgical cure, which makes drug therapy play an important role in the treatment of middle and advanced liver cancer.

At present, the main clinical drugs include traditional cytotoxic chemotherapy drugs and small molecule targeted drugs that have shown positive clinical efficacy in recent years. Traditional cytotoxic drugs play a crucial role in the systemic treatment of advanced liver cancer, but most patients with advanced liver cancer are accompanied by liver cirrhosis, impaired liver function and thrombocytopenia. Tolerability is often poor, so the use of traditional cytotoxic chemotherapeutics is greatly limited. In recent years, with the in-depth research on the pathogenesis and molecular biological mechanism of liver cancer, the research on small molecule targeted drugs for liver cancer has made substantial progress and has become an effective choice for liver cancer treatment. However, the only small-molecule targeted drug in clinical use so far is the FDA-approved multi-targeted small-molecule drug Sorafenib, which is expensive to use and has disadvantages such as limited efficacy and drug resistance. Therefore, the development of small molecule targeted drugs for liver cancer is still urgently needed.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel 3-(ureido-methyl)-4-aryl-pyridine derivative with medicinal value: the compound represented by formula I.

The compound represented by formula I provided by the present invention or its pharmaceutically acceptable salt, crystal form and solvate:

in,

X is O or S;

n is an integer from 0 to 3;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are independently selected from H, cyano, COOR ₁₁ , C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, trifluoromethyl, amino , nitro, hydroxyl, mercapto or halogen, R ₁₁ is selected from H or C ₁ -C ₄ alkyl.

Further, X is S; n is 0, 1 or 2.

Further, the R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are all H.

Further, the compound is:

Further, among the R ₁ , R ₂ , R ₃ , R ₄ and R ₅ , at least one is cyano, COOCH ₃ or COOCH ₂ CH ₃ , and the rest are selected from H, methyl, ethyl, methyl oxy, ethoxy, trifluoromethyl or halogen;

Preferably, one or two of the R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are cyano, COOCH ₃ or COOCH ₂ CH ₃ , and the rest are selected from H, methyl or ethyl ;

More preferably, only one of said R ₁ , R ₂ , R ₃ , R ₄ and R ₅ is cyano, COOCH ₃ or COOCH ₂ CH ₃ , and the rest are H.

Further, the compound is:

The present invention also provides a method for preparing the above compound, which comprises the following steps: compound 5 and compound 7

React in an organic solvent, separate and purify to obtain the compound shown in formula I:

in,

X is O or S;

n is an integer from 0 to 3;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are independently selected from H, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, trifluoromethyl, COOR ₁₁ , cyano, amino , nitro, hydroxyl, mercapto or halogen, R ₁₁ is selected from H or C ₁ -C ₄ alkyl;

The molar ratio of the compound 5 to the compound 7 is 1:1 to 1.5; the reaction temperature is 0°C to 60°C;

The organic solvent is selected from any one or two or more of dichloromethane, tetrahydrofuran, toluene, ethyl acetate and acetonitrile.

Further, the compound 5 is prepared by the following steps:

①, protect the amino group of 3-pyridine methylamine to obtain compound 2;

②, compound 2 reacts with halogen element to obtain compound 3;

反应，得到化合物4；③, compound 3 and

reaction to obtain compound 4;

④ Compound 4 removes the amino protecting group to obtain compound 5;

in,

R _a is tert-butoxycarbonyl, benzyloxycarbonyl, fluorenemethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, phthaloyl, p-toluenesulfonyl, trimethylbenzene Fluoroacetyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, pivaloyl, benzoyl, trityl, benzyl, 2,4-dimethoxybenzyl or p-methoxybenzyl;

The halogen element is fluorine element, chlorine element, bromine element or iodine element;

R _b is fluorine, chlorine, bromine or iodine;

X is O or S.

The present invention also provides the use of the above compounds or their pharmaceutically acceptable salts, crystal forms and solvates in the preparation of medicines for treating and/or preventing liver cancer.

The present invention also provides a pharmaceutical composition for treating and/or preventing liver cancer, which is prepared by using the above-mentioned compound or a pharmaceutically acceptable salt, crystal form or solvate thereof as an active ingredient, and adding commonly used pharmaceutically acceptable excipients. obtained preparation.

The half inhibitory concentration (IC ₅₀ ) of the compound of the present invention is small, and it has a good inhibitory effect on liver cancer cells; moreover, the preparation method of the compound of the present invention is simple, the reaction conditions are mild, the operation and control are convenient, the energy consumption is low, and the yield is high , low cost, suitable for industrial production.

The compounds and derivatives provided in the present invention may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions of groups or terms provided herein apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be based on the disclosure and context. , give their meanings that those skilled in the art can give them.

"Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

Minimum and maximum carbon content in a hydrocarbon group are indicated by prefixes, eg, the prefix C _a to C _b alkyl denotes any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C ₁ -C ₄ alkyl refers to an alkyl group containing 1 to 4 carbon atoms, in other words, C ₁ -C ₄ alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The terms "salts" and "pharmaceutically acceptable salts" refer to the above-mentioned compounds or their stereoisomers, acid and/or base salts with inorganic and/or organic acids and bases, and also zwitterionic salts (internal). salts), also including quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (for example, an equivalent amount). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, parenteral (intravenous, intramuscular or subcutaneous), and topical.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.

Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The pharmaceutically acceptable adjuvants in the present invention refer to the substances contained in the dosage form other than the active ingredients.

The pharmaceutically acceptable auxiliary component of the present invention has certain physiological activity, but the addition of the component will not change the dominance of the above-mentioned pharmaceutical composition in the process of disease treatment, but only exert auxiliary effects, and these auxiliary effects are only It is the utilization of the known activity of the ingredient and is a commonly used adjuvant therapy in the field of medicine. If the above-mentioned auxiliary components are used in combination with the pharmaceutical composition of the present invention, it still falls within the protection scope of the present invention.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 is the ¹ H NMR chart of the compound 8b of the present invention.

Figure 2 is a ¹ H NMR chart of the compound 8d of the present invention.

Figure 3 is a ¹ H NMR chart of the compound 8i of the present invention.

Detailed ways

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.

in,

Compound 5 can be obtained by purchasing a commercially available product, or it can be prepared by the following method:

(1) Synthesis of compound 2 (N-tert-butoxycarbonyl-3-pyridinemethylamine)

Compound 1: Amine, there are commercially available products from Tixiai (Shanghai) Chemical Industry Development Co., Ltd.;

DMAP: 4-dimethylaminopyridine; PE: petroleum ether; EA: ethyl acetate;

At room temperature, compound 1 (1.08 g, 10 mmol) was dissolved in acetonitrile (40 ml), di-tert-butyl dicarbonate (2.40 g, 11 mmol) and DMAP (0.12 g, 1 mmol) were added and stirred at room temperature for 2 h, and detected by TCL The reaction of the raw materials was completed, and the crude product was purified by column chromatography (PE:EA=5:1) after concentration under reduced pressure to obtain compound 2 (1.25 g, yield 60%) as a colorless oil.

(2) Synthesis of compound 3 (N-tert-butoxycarbonyl-4-iodo-3-pyridinemethanamine)

THF: tetrahydrofuran; tert-BuLi: tert-butyl lithium;

A solution of compound 2 (0.954 g, 4.59 mmol) in THF (8 ml) was cooled to -78 °C, and tert-BuLi (10.6 ml, 13.77 mmol) was added at -78 °C, the reaction was stirred for 30 min and then I ₂ (1.746 mmol) was added. g, 6.88 mmol) in THF (7 ml) solution, stirred for 1 h at -78 °C, and then slowly raised to -20 °C for 0.5 to 2 h, the reaction was complete as detected by TLC, washed with saturated Na ₂ S ₂ O ₃ solution The excess I ₂ was extracted three times with ethyl acetate, the organic layer was dried with anhydrous MgSO ₄ , concentrated under reduced pressure, and subjected to column chromatography to obtain compound 3 (0.31 g, yield 20%) as a yellow oil.

(3) Synthesis of compound 4

a. Synthesis of compound 4a (N-tert-butoxycarbonyl-4-(3-thiophene)-3-pyridinemethanamine)

3-thiophene boric acid: Bailingwei Technology Co., Ltd. has a commercially available product;

Pd(PPh ₃ ) ₄ : tetrakis(triphenylphosphine) palladium, available from Shanghai Yinggong Biotechnology Co., Ltd.;

Compound 3 (0.10 g, 0.31 mmol) and 3-thiopheneboronic acid (0.048 g, 0.37 mmol), potassium carbonate (57.05 mg, 0.413 mmol) were dissolved in toluene (3 ml) and ethanol (1 ml) and degassed for 10 min, Then, Pd(PPh ₃ ) ₄ (35.8 mg, 0.031 mmol) was added to it, and the reaction was refluxed overnight. The reaction was completed by TCL, and the inorganic salt was removed by filtration. After concentration under reduced pressure, the crude product was subjected to column chromatography (PE:EA=8: 1) Purified to obtain yellow solid compound 4a (0.08 g, yield 93%).

b. Synthesis of compound 4b (N-tert-butoxycarbonyl-4-(2-thiophene)-3-pyridinemethanamine)

According to the above synthesis method of compound 4a, 2-thiophene boronic acid was used to replace 3-thiophene boronic acid in the raw material to prepare compound 4b (yield 89%);

(4) Synthesis of compound 5

a. Synthesis of compound 5a ((4-(thiophen-3-yl)pyridin-3-yl)methanamine)

Compound 4a (0.21 g, 0.74 mmol) was dissolved in 5M HCl in ethyl acetate solution (EA.HCl) (20 ml), and the reaction was stirred at room temperature overnight. After the complete reaction of the raw materials was detected by TCL, it was adjusted to basicity with saturated NaHCO ₃ , extracted three times with ethyl acetate, the organic layer was dried and concentrated to obtain compound 5a (0.12 g, yield 86%) as a yellow oil.

b. Synthesis of compound 5b

According to the synthesis method of compound 5a above, compound 5b was prepared by using compound 4b as raw material.

c. Synthesis of compound 5c

According to the synthesis method of compound 5a above, compound 5c was prepared by using compound 4c as raw material.

Compound 7 can be obtained by purchasing commercially available products (for example: phenyl isocyanate can be obtained by purchasing the commercially available products of Hunan Dejia Biochemical Technology Co., Ltd.), also can be prepared by the following methods:

a. Synthesis of compound 7a (phenyl isocyanate)

DCM: dichloromethane;

Aniline (0.12 g, 1.25 mmol) was added to the reaction flask and dissolved in DCM (5 ml), saturated NaHCO ₃ (8.75 ml) solution was added, cooled to 0°C, followed by trichloromethyl carbonate (0.12 g, 0.41 ml) mmol) and stirred the reaction for 15 min, after TCL detection showed that the reaction was complete, it was extracted three times with dichloromethane, and the organic layer was dried and concentrated to obtain a colorless liquid compound 7a.

b. Synthesis of compound 7b

Compound 7b was prepared according to a similar method as above, replacing aniline in the starting material with ethyl m-aminobenzoate.

c. Synthesis of compound 7c

Compound 7c was prepared according to a similar method as above, substituting ethyl p-aminobenzoate for aniline in the starting material.

d. Synthesis of compound 7d

According to a method similar to the above, the aniline in the starting material is replaced with ethyl m-aminomethyl benzoate (which can be obtained by reacting m-aminomethyl benzoic acid with ethanol) to prepare compound 7d.

e. Synthesis of compound 7e

According to the method similar to the above, the aniline in the starting material was replaced with ethyl p-aminomethyl benzoate to prepare compound 7e.

Example 1. Synthesis of compound 8a (1-phenyl-3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)urea)

Compound 5a (0.14 g, 0.74 mmol) and compound 7a (0.13 g, 1.11 mmol) were dissolved in dichloromethane (4 ml), and the reaction was stirred at 0 °C overnight. After the reaction was detected by TLC, the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH=20:1) to give compound 8a (0.12 g, yield 53%) as a white solid, LC-MS m/z: 310.09 [M+H]. Example 2. Synthesis of compound 8b (3-(3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)ureido)benzoic acid

According to the method similar to Example 1, compound 7a was replaced with compound 7b to obtain compound 8ba (yield 51%).

Compound 8ba (0.5 g, 1.30 mmol) was dissolved in a mixture of ethanol (15 ml) and water (15 ml), NaOH was added, refluxed at 100°C for 1 to 2 hours, and the reaction was completed by TLC, and 1N HCl solution was added to adjust the pH When it became acidic, the precipitated solid was filtered and dried in vacuo to obtain compound 8b (0.42 g, yield 92%) as a white solid.

¹ H NMR (400MHz, DMSO-d6): δ12.81(s, br 1H), 8.86(s, 1H), 8.63(s, 1H), 8.50(d, J=4.8Hz, 1H), 8.05(t ,J=1.6Hz,1H),7.83(dd,J=1.2Hz,1.6Hz,1H),7.76-7.74(m,1H),7.60-7.58(m,1H),7.49(dt,J=1.2Hz ,1.2Hz,1H),7.40(dd,J=1.2Hz,1.2Hz,1H),7.38-7.32(m,2H),6.76-6.75(m,1H),4.42(d,J=5.6Hz,2H) ) ppm, see Figure 1; LC-MS m/z: 352.09 [MH].

Example 3. Synthesis of compound 8c (4-(3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)ureido)benzoic acid

According to the method similar to Example 2, the compound 7b in the starting material was replaced by the compound 7c, and the compound 8c was prepared (the yield was 55%).

¹ H NMR (400MHz, DMSO-d6): δ12.50(s, 1H), 9.01(s, 1H), 8.63(s, 1H), 8.50(d, J=4.0Hz, 1H), 7.83-7.75( m, 3H), 7.50-7.40 (m, 3H), 6.83 (s, 1H), 4.43 (d, J=4.8 Hz, 2H) ppm, LC-MS m/z: 352.09 [MH].

Example 4. Synthesis of compound 8d (3-((3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)ureido)methyl)benzoic acid)

According to the method similar to Example 2, the compound 7b in the starting material was replaced by the compound 7d, and the compound 8d was prepared (the yield was 53%).

¹ H NMR (400MHz, DMSO-d6): δ 12.92(s,br 1H), 8.58(s, 1H), 8.48(d, J=5.2Hz, 1H), 7.87(s, 1H), 7.82-7.78 (m,2H),7.73-7.71(m,1H),7.51-7.42(m,2H),7.38-7.35(m,2H),6.68-6.64(m,2H),4.34(d,J=8.0Hz , 2H), 4.29 (d, J=8.0 Hz, 2H) ppm, see Figure 2; LC-MS m/z: 366.09.08 [MH].

Example 5. Synthesis of compound 8e (4-((3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)ureido)methyl)benzoic acid)

According to the method similar to Example 2, the compound 7b in the starting material was replaced by the compound 7e, and the compound 8e was prepared (the yield was 52%).

¹ H NMR (400MHz, DMSO-d6): δ 12.85 (S, 1H), 8.59 (s, 1H), 8.48 (d, J=5.2Hz, 1H), 7.89 (d, J=8.0Hz, 2H) ,7.80-7.79(m,1H),7.73-7.71(m,1H),7.39-7.35(m,4H),6.70(s,2H),4.34(d,J＝6.0Hz,2H),4.30(d , J=6.4 Hz, 2H) ppm, LC-MS m/z: 366.09.08 [MH].

Example 6. Synthesis of compound 8f (1-(3-cyanophenyl)-3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)urea)

m-aminobenzonitrile (0.08 g, 0.69 mmol) was dissolved in THF (7 ml), triethylamine (0.14 g, 1.37 mmol) was added, cooled to 0 °C, and trichloromethyl carbonate ( 0.07 g, 0.23 mmol), react for 3 h, add compound 5a (0.09 g, 0.46 mmol) at 0° C. and move to room temperature to react overnight. The reaction is complete as detected by TLC, concentrated under reduced pressure, and subjected to column chromatography (CH ₂ Cl ₂ : CH ₃ OH=10:1) was purified to give compound 8f as a white solid (0.07 g, 44% yield).

¹ H NMR (400MHz, DMSO-d6): δ8.98 (s, 1H), 8.62 (s, 1H), 8.50 (d, J=4.8Hz, 1H), 7.93 (t, J=1.6Hz, 1H) ,7.83-7.82(m,1H),7.76-7.74(m,1H),7.61-7.58(m,1H),7.46-7.34(m,4H),6.87(t,J=5.6Hz,1H),4.43 (d, J=5.6 Hz, 2H) ppm, LC-MS m/z: 335.08 [M+H].

Example 7. Synthesis of compound 8g (1-(4-cyanophenyl)-3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)urea)

According to the method similar to Example 6, compound 6f was replaced with compound 6g to obtain compound 8g (yield 38%), LC-MS m/z: 335.08 [M+H].

Example 8. Synthesis of compound 8h (1-phenyl-3-((4-(thiophen-2-yl)pyridin-3-yl)methyl)urea)

According to the method similar to Example 1, the compound 5a in the starting material was replaced by the compound 5b, and the compound 8h was prepared (the yield was 56%).

¹ H NMR (400MHz, DMSO-d6): δ 8.63 (d, J=12.4Hz, 2H), 8.51 (d, J=5.2Hz, 1H), 7.81 (dd, J=1.2, 0.8Hz, 1H) ,7.48(dd,J＝1.2,1.2Hz,1H),7.46(d,J＝5.2Hz,1H),7.40-7.38(m,2H),7.27-7.20(m,3H),6.92-6.88(m , 1H), 6.66 (t, J=5.6 Hz, 1H), 4.50 (d, J=5.6 Hz, 2H) ppm, LC-MS m/z: 310.09 [M+H].

Example 9. Synthesis of compound 8i (4-(3-((4-(thiophen-2-yl)pyridin-3-yl)methyl)ureido)benzoic acid

According to the method similar to Example 2, the compound 5a in the starting material was replaced with compound 5b, and the compound 7b in the starting material was replaced with compound 7c to prepare compound 8i (yield 48%).

¹ H NMR (400MHz, DMSO-d6): δ 12.46 (s, 1H), 9.08 (d, J=19.2 Hz, 1H), 8.65 (s, 1H), 8.51 (d, J=4.8 Hz, 1H) ,7.83-7.80(m,3H),7.51-7.48(m,3H),7.46(d,J=5.2Hz,1H)7.28-7.26(m,1H),6.87-6.85(m,1H)4.51(d , J=8.0 Hz, 2H) ppm, see Figure 3; LC-MS m/z: 352.09 [MH].

Example 10. Synthesis of compound 8j (4-((3-((4-(thiophen-3-yl)pyridin-3-yl)methyl)ureido)methyl)ethyl benzoate)

According to the method similar to Example 1, the compound 7a in the starting material was replaced by the compound 7e to prepare the compound 8j (yield 63%), LC-MS m/z: 396.12 [M+H].

Example 11. Synthesis of compound 8k(1-(3-cyanophenyl)-3-((4-(thiophen-2-yl)pyridin-3-yl)methyl)urea)

According to the method similar to Example 6, the compound 5a in the starting material was replaced by the compound 5b, and the compound 8k was prepared (the yield was 33%).

¹ H NMR (400MHz, DMSO-d6): δ9.02 (s, 1H), 8.64 (s, 1H), 8.51 (d, J=4.8Hz, 1H), 7.94 (t, J=1.6Hz, 1H) ,7.82(dd,J＝1.2Hz,1.2Hz,1H),7.61-7.58(m,1H),7.49-7.42(m,3H),7.37-7.34(m,1H),7.28-7.26(m,1H) ), 6.88 (t, J=5.6 Hz, 1H), 4.51 (d, J=5.6 Hz, 2H) ppm, LC-MS m/z: 335.08 [M+H].

Example 12. Synthesis of compound 81 (1-(4-cyanophenyl)-3-((4-(thiophen-2-yl)pyridin-3-yl)methyl)urea)

According to the method similar to Example 6, the compound 6f in the starting material was replaced with compound 6g, and the compound 5a in the starting material was replaced with compound 5b to prepare compound 81 (the yield was 30%).

¹ H NMR (400MHz, DMSO-d6): δ9.19 (s, 1H), 8.64 (s, 1H), 8.52 (d, J=5.2Hz, 1H), 7.82 (dd, J=1.2Hz, 1.2Hz ,1H),7.69-7.66(m,2H),7.59-7.57(m,2H),7.49-7.46(m,2H),7.27-7.25(m,1H),6.91(t,J=5.6Hz,1H ), 4.51 (d, J=5.6 Hz, 2H) ppm, LC-MS m/z: 335.08 [M+H].

The beneficial effects of the compounds of the present invention are illustrated below through test examples.

Test Example 1. Inhibitory activity test of the compounds of the present invention on liver cancer cell lines (Huh7 and SMMC7721)

(1) Experimental materials

Main reagents:

RPMI-1640, fetal bovine serum, pancreatin, etc. were purchased from Gibco BRL (Invitrogen Corporation, USA);

IMDM medium was purchased from ATCC (American Type Culture Collection);

Tetramethylazolium salt (MTT) and dimethyl sulfoxide (DMSO) are products of Sigma Company (USA);

Human hepatoma cell lines Huh7 and SMMC7721.

When the compounds of the present invention are subjected to in vitro experiments, 100% DMSO is used to prepare a 10 mM stock solution, which is stored in a -20°C refrigerator in the dark for future use, and is diluted to a desired concentration with a complete culture solution for temporary use.

(2) Experimental method

A cell suspension with a cell concentration of 1-2×10 ⁴ cells/mL was adjusted with the complete cell culture medium, and inoculated into a 96-well plate with 200 μl of the cell suspension per well, and cultured overnight. The next day, the supernatant was aspirated (the supernatant was aspirated after the suspension cells were centrifuged), and then the cells were treated with a gradient concentration of the compound of the present invention (Sorafenib as a positive control), respectively. At the same time, a drug-free negative control group and an equal volume of solvent control group were set up, the concentration of DMSO was 0.1%, and each dose group was set up with 3 duplicate wells, which were cultured at 37°C and 5% _CO2 . After 72 hours, add 20 μl of MTT reagent with a concentration of 5 mg/mL to each well, and after culturing for 2-4 hours, discard the supernatant, add 150 μL of DMSO to each well, shake and mix for 15 min, and measure with a microplate reader (λ=570 nm). Absorbance (A) value (A value is proportional to the number of viable cells), take the average value. Relative cell proliferation inhibition rate=(negative control group A _{570 -experimental} group A ₅₇₀ )/negative control group A ₅₇₀ ×100%. The experiment was repeated at least 3 times.

(3) Experimental results

Table 1 shows the median inhibitory concentration (IC ₅₀ ) of the compounds of the present invention on human hepatoma cell lines Huh7 and SMMC7721.

Table 1. The median inhibitory concentration (IC ₅₀ ) of the compounds of the present invention on Huh7 and SMMC7721

The above results show that the half inhibitory concentration (IC ₅₀ ) of the compound of the present invention is small, and it has a good inhibitory effect on liver cancer cells.

To sum up, the compound of the present invention has a low half inhibitory concentration (IC ₅₀ ) and has a good inhibitory effect on hepatoma cells; moreover, the compound of the present invention has a simple preparation method, mild reaction conditions, convenient operation and control, and energy consumption. Small, high yield, low cost, suitable for industrial production.

Claims (10)

1. A compound of formula I or a pharmaceutically acceptable salt thereof:

wherein,

x is S;

n is 0, 1 or 2;

R₁、R₂、R₃、R₄、R₅each independently selected from H, cyano, COOR₁₁，R₁₁Is selected from C₁～C₄An alkyl group; and said R is₁、R₂、R₃、R₄、R₅In which at least one is cyano, COOR₁₁And the balance of H.

2. The following compound, or a pharmaceutically acceptable salt thereof, characterized in that: the compound is as follows:

3. the compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: said R₁、R₂、R₃、R₄、R₅In which at least one is cyano, COOCH₃Or COOCH₂CH₃And the balance of H.

4. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, wherein: said R₁、R₂、R₃、R₄、R₅In which one or two are cyano, COOCH₃Or COOCH₂CH₃And the balance of H.

5. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein: said R₁、R₂、R₃、R₄、R₅In which only one is cyano, COOCH₃Or COOCH₂CH₃And the balance of H.

6. The compound according to claim 5, or a pharmaceutically acceptable salt thereof, wherein: the compound is as follows:

7. a process for preparing a compound according to any one of claims 1 to 6, characterized in that: it comprises the following steps: reacting the compound 5 with the compound 7 in an organic solvent, separating and purifying to obtain a compound shown in a formula I;

wherein,

the compound of formula I is a compound according to any one of claims 1 to 6;

the molar ratio of the compound 5 to the compound 7 is 1: 1 to 1.5; the reaction temperature is 0-60 ℃;

the organic solvent is selected from one or more of dichloromethane, tetrahydrofuran, toluene, ethyl acetate and acetonitrile.

8. The method of claim 7, wherein: the compound 5 is prepared by the following steps:

protecting amino of 3-pyridine methylamine to obtain a compound 2;

reacting the compound 2 with a halogen simple substance to obtain a compound 3;

③, Compound 3 and

reacting to obtain a compound 4;

fourthly, removing amino protecting groups from the compound 4 to obtain a compound 5;

wherein,

R_ais tert-butoxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, trimethylsilethoxycarbonyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, pivaloyl, benzoyl, trityl, benzyl, 2, 4-dimethoxybenzyl or p-methoxybenzyl;

the halogen simple substance is fluorine simple substance, chlorine simple substance, bromine simple substance or iodine simple substance;

R_bis fluorine, chlorine, bromine or iodine;

x is S.

9. Use of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of liver cancer.

10. A pharmaceutical composition for treating and/or preventing liver cancer, which is characterized in that: the compound or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 is used as an active ingredient, and is added with pharmaceutically common auxiliary materials to prepare the preparation.

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