CN104003918A - Diaryl thioether compound, preparation method and antitumor application thereof - Google Patents

Abstract

The present invention relates to the field of new drug research and development, in particular to a diaryl sulfide compound, a preparation method and its anti-tumor application. The structure of the diaryl sulfide compound is shown in general formula I: A is selected from structural formula II substituted phenyl substituted by 4-nitro, substituted phenyl substituted by 4-alkoxy shown in structural formula III, substituted heterocyclic ring shown in structural formula IV or substituted heterocyclic ring shown in structural formula V, wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R 15 , R ₁₆ , _{R 17 ,} R ₁₈ and X, Y are all as described in the specification. The compound has an action mechanism of resisting abnormal new blood vessels and inhibiting tubulin polymerization, and has the advantages of high activity and low toxicity.

Description

A kind of diaryl sulfide compound, preparation method and antitumor application thereof

technical field

The invention relates to the field of research and development of new drugs, in particular to the field of research and development of new anti-tumor drugs.

Background technique

Combretum caffrum (Combretaceae) is a plant of the genus Combretum in South Africa, which has very important medical application value. In 1988, Pettit et al. isolated a series of active group Combretastatins from it. This series of compounds can bind to the colchicine site to inhibit tubulin polymerization, among which Combretastatin A4 (Combretastatin A4, CA4) has the best activity. CA4 has a simple structure, which is somewhat similar to the structure of colchicine. It has a high inhibitory activity on cell mitosis. At the same time, it has a competitive inhibitory effect on the microtubule polymerization promoted by colchicine, and it can inhibit the mitosis of tumor cells. , disrupting the formation of the mitotic spindle, thereby causing G ₂ /M arrest. At the same time, CA4 can selectively target the neovascularization of growing tumor tissue. Studies have shown that it can close tumor-related blood vessels within a few minutes of administration, thereby effectively inhibiting blood from entering and flowing out of tumor cells. Within a few hours, new blood vessels close and die. Its mechanism of action is preliminarily considered: endothelial cell shrinkage, rounding, foaming, and endothelial permeability increase, resulting in increased blood flow resistance, and the increased permeability of tumor blood vessels may lead to impaired vascular function; plasma protein infiltrates from blood vessels It interferes with the internal and external osmotic pressure of blood vessels, causing a short-term increase in interstitial fluid pressure, resulting in vascular necrosis. The loss of fluid in blood vessels then causes blood viscosity to increase, blood flow velocity to slow down, and red blood cells to accumulate, further increasing vascular resistance, and blood flow. Slow down. In addition, the damage of endothelial cells can make the basement membrane contact with the blood flow, causing coagulation. In addition to these effects, tumor blood vessels themselves have many intersecting microvessels, which slow down blood circulation in tumor blood vessels, reduce or even stagnate blood flow. But CA4 has no toxic side effects on normal blood vessels. The reason for this selectivity may be the increased permeability caused by the morphological and functional changes of the vascular endothelial cells of the tumor tissue. The increased cell permeability will destroy the tubulin, that is, the cytoskeleton, while the normal endothelial cells will be affected. much smaller.

However, CA4 also has its weakness—poor water solubility, so Pettit et al. used it as a lead compound to synthesize its phosphate prodrug CA4P (US5561122), which greatly improved its water solubility, and is currently conducting phase III clinical trials. Ohsumi et al. synthesized AVE-8063 (EP0641767) by substituting the 3-position hydroxyl group with an isostere amino group. The activity was enhanced to a certain extent, and then condensed with amino acids to obtain AC-7700 with better water solubility (Ohsumi K.J Med Chem, 1998, 41(16), 3022-3032).

Although CA4 has not been discovered for a long time, it has aroused widespread interest due to its good anti-tumor activity, simple structure and relatively low side effects, and many new effective anti-tumor compounds have been synthesized. There has also been some research, and it is found that the A ring is an important group to maintain activity, the 4-methoxy group of the B ring is also very important but not irreplaceable, the 3-hydroxyl is an important modification site, and the double bond bridge part The cis-form is the active structure, the trans-form is inactive, and the double bond can be replaced by other structures such as heterocycles or other chain structures with a length of 1 to 3 atoms. Such as Welsh W.J. synthesized a series of CA4 derivatives (WO2006047631) with 1,2,4-triazole rings replacing double bonds, and Tracey et al. synthesized a series of compounds with furan rings replacing double bonds (Pirali Tracey etc.J.Med.Chem .2006,49(17),5372-5376.), Wang Le et al. synthesized a series of CA4 derivatives containing imidazole ring bridge chain (Wang Le etc.J.Med.Chem.2002,45(8),1697 -1711.), all have good anti-microtubule activity and cytotoxicity. Pettit G.R. obtained a series of compounds (WO9934788) in 1999 by replacing double bonds with carbonyl groups according to the bioelectronic isosteric principle, and Lawrence et al. obtained a series of compounds with oxygen atoms replacing double bonds (Lawrence N.J.Bioorg.Med.Chem. Lett.2001, 11(1), 51-54.), all have good antitumor activity.

Some early studies have found that some simple diaryl sulfide compounds have biological activities such as antimalarial and antibacterial (Chan, J.H.etc J.Med.Chem.1995,38,3608-3616; Singh, T.etcJ.Med . Chem. 1970, 326-327). During the structural modification of CA4, Chen et al. found that the diarynylaryl sulfide compound 9 obtained by introducing a thioether bridge into the molecule retained certain antitumor activity (Chen, Z.etc J.Org.Chem 2000 , 65, 8811-8815.) Recently, Barbosa, E.G, etc. also found that thioether compound 10 has strong activity of inhibiting tubulin and strong activity of inhibiting breast cancer cell line MCF-7 (Barbosa, E.G.etc Eur .J.Med.Chem.2009,44,2685-2688) In addition, a patent published by Serenex INC in 2008 also involves several such compounds with certain anti-proliferation activity (WO2008024963).

In view of the better antitumor activity of diaryl sulfide CA4 analogs, the present invention has carried out more in-depth research on this type of compound, and synthesized a series of new diaryl sulfide CA4 analogs, and the test results show that among them Several compounds have stronger antitumor activity.

Contents of the invention

The present invention aims to provide a diaryl sulfide CA4 derivative, the structure of the compound is as general formula I.

Another object of the present invention is to provide the preparation method of the compound of general formula I.

Another object of the present invention is to provide the medical use of the compound of general formula I.

One of technical solutions of the present invention is:

A compound with structural formula I and pharmaceutically acceptable salts thereof,

A is selected from 4-nitro substituted substituted phenyl shown in structural formula II, 4-alkoxy substituted substituted phenyl shown in structural formula III, substituted heterocycle shown in structural formula IV or substituted heterocyclic ring shown in structural formula V ring;

Where R ₁ , R ₂ , R ₃ , R 4 , R ₅ , R ₆ , _{R 7 ,} R ₈ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R _{14, , R 15} , _{R 16 ,} R ₁₇ , _R18 are independently selected from hydrogen, halogen, hydroxyl, amino, substituted amino, cyano, alkyl, alkoxy, nitro, amido, haloalkyl, acyl, aldehyde, carboxyl, alkoxycarbonyl or acyl Oxygen, and R ₅ , R ₆ , R ₇ , R ₈ are not hydrogen at the same time;

_R9 is selected from an alkyl group of 1-3 carbon atoms or a substituted alkyl group of 1-3 carbon atoms;

X, Y are independently selected from nitrogen, oxygen or sulfur;

Wherein the alkyl group is an alkyl group with 1-6 carbon atoms; the alkoxy group is an alkyloxy or oxyalkyl group with 1-6 carbon atoms; the haloalkyl group is substituted by F, Cl or Br The alkyl group of 1-6 carbon atoms; the alkoxycarbonyl group is the alkoxycarbonyl group of 1-6 carbon atoms.

When A is substituted phenyl II, wherein R ₁ , R ₂ , R ₃ , and R ₄ independently refer to hydrogen, halogen, hydroxyl, amino, methyl, and ethyl. Preferably at least one of the R ₁ , R ₂ , R ₃ and R ₄ is a hydroxyl or an amino group, and the hydroxyl or amino group is further substituted with its corresponding phosphate, carboxylate, sulfonate, amide, peptide Classes and corresponding organic or inorganic salt derivatives.

When A is substituted phenyl III, wherein R ₅ , R ₆ , R ₇ , and R ₈ independently refer to hydrogen, halogen, hydroxyl, amino, methyl, ethyl, benzyl, alkoxy, nitro , acyloxy group or amido group, and R ₅ , R ₆ , R ₇ , R ₈ are not hydrogen at the same time. The R ₅ , R ₆ , R ₇ and R ₈ are preferably at least one of a hydroxyl or an amino group, and the hydroxyl or amino group is further substituted with its corresponding phosphate, carboxylate, sulfonate, amide, peptide Classes and corresponding organic or inorganic salt derivatives.

When A is a substituted heterocycle IV or a substituted heterocycle V, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , and R ₁₈ are independently selected from hydrogen, halogen, and hydroxyl , amine, substituted amine, cyano, alkyl, alkoxy.

The compound of general formula I is selected from the following compounds:

3,4,5-Trimethoxy-1-[(3-amino-4-nitrophenyl)thio]benzene,

3,4,5-Trimethoxy-1-[(3-acetamido-4-nitrophenyl)thio]benzene,

3,4,5-Trimethoxy-1-[(3-nitro-4-methoxyphenyl)thio]benzene,

3,4,5-Trimethoxy-1-[(3-amino-4-methoxyphenyl)thio]benzene,

3,4,5-trimethoxy-1-[(3-fluoro-4-methoxyphenyl)thio]benzene,

3,4,5-Trimethoxy-1-[(4-ethoxyphenyl)thio]benzene,

3,4,5-Trimethoxy-1-[(3-benzyloxy-4-methoxyphenyl)thio]benzene,

5-trifluoromethyl-2-[(3,4,5-trimethoxyphenyl)thio]pyridine,

3-[(3,4,5-Trimethoxyphenyl)thio]benzothiophene-2-carboxylic acid methyl ester,

2-Fluorenylmethoxyamido-3-acetoxy-N-[2-methoxy-5-(3,4,5-trimethoxyphenyl)thio]propionamide,

2-Amino-3-hydroxy-N-[2-methoxy-5-(3,4,5-trimethoxyphenyl)thio]propionamide,

3,4,5-Trimethoxy-1-[(3-hydroxy-4-methoxyphenyl)thio]benzene.

The second technical scheme of the present invention:

The preparation method of compound described in general formula I and pharmaceutically available salt thereof, concrete steps are:

Using thiophenol compound 7 as a raw material, dissolving it with aromatic chlorinated compound 8 or aromatic iodo compound 6 under the action of a basic catalyst to obtain diaryl sulfide derivatives in the compound of general formula I; wherein aromatic iodo compound 6 for Thiophenol compound 7 is Aromatic chlorinated compound 8 is Wherein, the structure of A is as above.

The synthesis method is further explained and illustrated below:

The thiophenol compound 7 in the above synthesis method is a known compound, which can be synthesized by existing methods or purchased.

Concrete synthetic route of the present invention is as follows:

Where: THF stands for tetrahydrofuran, DMF stands for N,N-dimethylformamide, i-PrOH stands for isopropanol, EG stands for ethylene glycol, t-BuOK stands for potassium tert-butoxide, and HAc stands for acetic acid. con.HNO ₃ means concentrated nitric acid, con.HCl means concentrated hydrochloric acid.

Specific steps include:

a. Compound 1 is decarboxylated and nitrated to obtain compound 2, then reduced to compound 3, then diazotized, and reacted with ethyl xanthate potassium to generate compound 4;

b. Diazotizing the aromatic amine 5, and then reacting with sodium iodide to synthesize the aromatic iodo product 6;

c. first reducing compound 4 to thiophenol 7, and then dissolving it with aromatic chlorinated compound 8 or aromatic iodo compound 6 under the action of a basic catalyst to obtain diaryl sulfide derivatives in the compound of general formula I;

Wherein said compound 1 is: Compound 2 is Compound 3 is Compound 4 is Aromatic amine 5 is Aromatic iodo compound 6 is Thiophenol 7 is Aromatic chlorinated compound 8 is

Wherein, the structure of A is as above.

In order to prepare the CA4 derivative of the thioether bridge chain in the compound of general formula I, specific conditions are as follows:

The reaction temperature of decarboxylation and nitration in step a is 0-100°C; the reducing agent used in the reduction reaction can be zinc powder, iron powder, magnesium powder, the acid can be acetic acid, hydrochloric acid, and the reducing agent can also be palladium carbon/hydrazine hydrate, magnesium Powder/hydrazine hydrate, iron powder/hydrazine hydrate, palladium carbon/hydrogen, etc., the reaction temperature is 0-100°C; the reagent in the diazotization step can be sodium nitrite, potassium nitrite, nitrobutane, and the acid can be hydrochloric acid , sulfuric acid, phosphoric acid and acetic acid, the reaction temperature is -20-10°C; the dithiocarbonation reagent can be various alkyl xanthates, and the reaction temperature is 0-120°C;

The diazotization step in step b can be reduced with lithium tetrahydrogen aluminum/tetrahydrofuran, or can be hydrolyzed with aqueous sodium hydroxide solution, and the reaction temperature is 0-100 ° C; and the reaction solvent of the iodo reaction can be ethanol, methanol, isopropyl Alcohol, ethylene glycol and other alcohols, benzene, toluene, acetone, tetrahydrofuran, dioxane, N,N-dimethylformamide, pyridine, dimethyl sulfoxide, dichloromethane, chloroform, dichloroethane wait.

The basic catalyst used in step c is selected from inorganic basic compounds, such as potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, etc., and the reaction temperature is 0-200°C.

In another preferred embodiment, it comprises the steps of:

(1) Compound 1 is decarboxylated and nitrated under the action of concentrated nitric acid and acetic acid to obtain compound 2, and then reduced to compound 3 with zinc powder/acetic acid or palladium carbon/hydrazine hydrate, then made diazonium salt solution with sodium nitrite and hydrochloric acid, and then React with potassium ethyl xanthate to generate compound 4;

(2) making aromatic amine 5 into diazonium salt solution with sodium nitrite and hydrochloric acid, and then reacting with sodium iodide to synthesize aromatic iodo product 6;

(3) Compound 4 is first dissolved in tetrahydrofuran, reduced to thiophenol 7 with tetrahydrolithium aluminum, then dissolved in solvents such as ethylene glycol and isopropanol or toluene with aromatic iodide compound 6, and dissolved in potassium carbonate and iodide Synthesis of diaryl sulfide derivatives under the action of cuprous.

In order to prepare the diaryl sulfide compounds in the compound of general formula I, the reaction temperature of the decarboxylation and nitration step in step a is 0-100° C., the reducing agent used in the reduction reaction can be zinc powder, iron powder, magnesium powder, and the acid can be Acetic acid, hydrochloric acid, the reducing agent can also be palladium carbon/hydrazine hydrate, magnesium powder/hydrazine hydrate, iron powder/hydrazine hydrate, palladium carbon/hydrogen, etc., the reaction temperature is 0-100 ° C, the diazotization reagent can be sodium nitrite , Potassium nitrite, nitrobutane, the acid used can be hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid, the reaction temperature is -20-10 ° C, the dithiocarbonation reagent can be various alkyl xanthates, The reaction temperature is 0-120°C;

The diazotization reagent used in the diazotization in the b step can be sodium nitrite, potassium nitrite, nitrobutane, and the acid used can be hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid, and the reaction temperature is-20-10°C; For the reaction, the iodine reagent can be sodium iodide, potassium iodide, elemental iodine, or iodine chloride, and the reaction temperature is 0-100°C;

The reduction step in the c step can be reduced with lithium tetrahydrogen aluminum/tetrahydrofuran, and can also be hydrolyzed with aqueous sodium hydroxide solution, the reaction temperature is 0-100 ° C, and the reaction solvent of the iodo reaction can be ethanol, methanol, isopropanol, Alcohols such as ethylene glycol, benzene, toluene, acetone, tetrahydrofuran, dioxane, N,N-dimethylformamide, pyridine, dimethyl sulfoxide, dichloromethane, chloroform, dichloroethane or mixed Solvent, etc., the basic catalyst used is potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, various potassium alcohols or sodium alcoholate, etc., the catalyst is cuprous iodide, copper iodide, and the reaction temperature is 0- 200°C.

The third technical solution of the present invention is:

The application of the compound described in the general formula I and the pharmaceutically acceptable salt thereof is specifically the application in the preparation of tubulin polymerization inhibitors and the application in the preparation of drugs for treating tumors related to abnormal blood vessel growth.

The tumor is selected from lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, breast cancer, liver cancer, gastric cancer, colon cancer, lymphoma, leukemia, pancreatic cancer, bone cell cancer, prostate cancer, testicular cancer, bladder cancer, Cervical cancer, basal cell carcinoma, multiple myeloma, neuroblastoma, kidney cancer, pineal tumor, hematoblastic carcinoma, rectal cancer, fibrosarcoma, liposarcoma, leiomyoma, chondrosarcoma, seminoma , malignant hemangioendothelioma or osteosarcoma.

Accordingly, the present invention provides a new class of diaryl sulfide CA4 derivatives, which have the advantages of high activity and low toxicity because of their mechanism of anti-abnormal neovascularization and inhibition of tubulin polymerization.

Detailed ways

Below in conjunction with specific embodiment, further set forth the invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The percentages in this application all refer to mass percentages.

Example 1

Preparation of O-ethyl-S-(3,4,5-trimethoxyphenyl)dithiocarbonate (ZLM-1)

Dissolve 3,4,5-trimethoxyaniline (22.3g) in 30.5mL of concentrated hydrochloric acid and 250mL of water, cool in an ice bath to about 0°C, slowly add NaNO ₂ aqueous solution (9.2g) dropwise under stirring, and control the temperature not to exceed 5°C, the reaction was continued for 15 min after the dropwise addition, and then AcONa (20.0 g) was added. The diazonium salt solution was slowly added dropwise to KS ₂ COEt aqueous solution (48.7 g dissolved in 120 mL water) at about 80°C, and reacted at 70-80°C for 1 h. After the reaction was completed, cool to room temperature, extract 100 mL×3 with EA, and combine the organic phases. Then dry with anhydrous sodium sulfate. Suction filtration to obtain the filtrate, spin off the solvent to obtain 30.4g reddish-brown oily substance, and silica gel column separation to obtain 11.8g light yellow-white viscous solid with a yield of 34%. ¹ H NMR (400MHz, CDCl ₃ ): δ1.34 (t,J=7.2Hz,3H),3.84(s,3H),3.85(s,6H),3.88(s,3H),4.61(dd,J=7.2,7.2Hz,1H),6.73(s, 1H).

Example 2

Preparation of 3,4,5-trimethoxy-1-[(3-amino-4-nitrophenyl)thio]benzene (ZLM-2)

Dissolve O-ethyl-S-(3,4,5-trimethoxyphenyl)dithiocarbonate ZLM-1 (4.0g) in THF (66mL), slowly add LiAlH4 (2.11g ), refluxed for 1 h, cooled to room temperature, adjusted to pH=5 with 10% HCl, then extracted 100 mL×3 with EA, combined the organic phases, added anhydrous sodium sulfate to dry for 1 h, and then suction filtered to obtain the filtrate, which was spun off from the solvent. In 3,4,5-trimethoxythiophenol. Under the protection of nitrogen, first add K ₂ CO ₃ (5.8g) and 5-chloro-2-nitroaniline (1.2g) into a 50mL two-necked bottle, and then add the 3,4,5-trimethoxy Thiophenol was dissolved in DMF (23.7 mL) and added to the reaction flask, refluxed at 120°C, and monitored by TLC (developing solvent: PE:EA=4:1). The raw materials disappeared, the reaction was stopped, cooled to room temperature, diluted with 200 mL of water, extracted with EA (200 mL×4), combined organic phases, and dried with anhydrous sodium sulfate for 1 h. Suction filtration to obtain the filtrate, spin off the solvent, and separate on a silica gel column to obtain 1.2 g of a yellow solid with a yield of 51%, mp168.1-170.2°C. ¹ H NMR (400 MHz, CDCl ₃ ): δ3.86 (s, 6H) ,3.91(s,3H),6.09(br,2H),6.33(d,J=1.6Hz,1H),6.43(dd,J=7.2,2.0Hz,1H),6.79(s,2H),7.99( d,J＝9.6Hz,1H). ¹³ C NMR(400MHz,CDCl3):δ56.3,61.0,112.2,113.5,114.9,124.1,126.6,129.7,139.3,144.9,149.8,153.9.MS(EI)m /z:336(M ⁺ ).

Example 3

Preparation of 3,4,5-trimethoxy-1-[(3-acetamido-4-nitrophenyl)thio]benzene (ZLM-3)

Add 3,4,5-trimethoxy-1-[(3-amino-4-nitrophenyl)thio]benzene ZLM-2 (50 mg) to a mixture of acetic anhydride (17 μL) and acetic acid (9 μL) In the solvent, reflux at 85-90°C for 2h. Low boiling point substances were distilled off under reduced pressure to obtain 61 mg of yellow solid with a yield of 93%, mp168.1-170.2°C. ¹ H NMR (400 MHz, CDCl ₃ ): δ2.26 (s, 3H), 3.87 (s, 6H) ,3.92(s,3H),6.73(dd,J=7.2,1.8Hz,1H),6.81(s,2H),8.07(d,J=8.8Hz,1H),8.61(d,J=6.0Hz, ⁽ EI) m/z: 378 (M ⁺ ).

Example 4

Preparation of 3-nitro-4-methoxyiodobenzene (ZLM-4)

Dissolve 3-nitro-4-methoxyaniline (860 mg) in dilute hydrochloric acid (diluted with 0.5 mL concentrated hydrochloric acid and 9 mL water), cool to 0 ° C, start to drop NaNO ₂ aqueous solution (424 mg,), and then React at high temperature for 20 minutes to obtain the diazonium salt solution. Then the diazonium salt solution was slowly added dropwise to NaI aqueous solution (921mg) at 0°C, after the drop was completed, the mixture was raised to room temperature and stirred for 12h. EA extracted 50mL×3, combined the organic phases, added anhydrous sodium sulfate to dry for 1 h, and then suction filtered to obtain the filtrate, which was spun to remove the solvent and separated on a silica gel column to obtain 573 mg of a yellow solid with a yield of 40%, mp95.8-97.3°C ( Literature value: 97-99°C).

Example 5

Preparation of 3-fluoro-4-methoxyiodobenzene (ZLM-5)

According to Example 4, 3-nitro-4-methoxyaniline was replaced with 3-fluoro-4-methoxyaniline (600 mg) to obtain 549 mg of light yellow solid, yield 56%, mp32.8-34.1 ° C ( Literature value: 34°C).

Example 6

Preparation of 3,4,5-trimethoxy-1-[(3-nitro-4-methoxyphenyl)thio]benzene (ZLM-6)

Dissolve O-ethyl-S-(3,4,5-trimethoxyphenyl) dithiocarbonate ZLM-1 (2.42g) in THF (32mL), slowly add LiAlH4 (1.3g ), refluxed for 1 h, cooled to room temperature, adjusted to pH = 5 with 10% HCl, then extracted 20 mL×4 with EA, combined the organic phases, dried with anhydrous sodium sulfate for 1 h, and then suction filtered to obtain the filtrate, which was spun off to remove the solvent. A yellow oil was obtained. Under nitrogen protection, first add potassium carbonate (1.16g, 8.40mmol, 2.0eq), CuI (400mg, 2.10mmol, 0.5eq) and ethylene glycol (0.49mL, 8.40mmol, 2.0eq) into a 25mL two-necked flask , and then dissolve the yellow oil obtained in the previous step in isopropanol (4.0 mL), then add it to the reaction flask, reflux at 80 ° C for 20 h, cool to room temperature, filter with suction, and wash the filter residue with EA several times to obtain the filtrate. The solvent was spun off and separated on a silica gel column to obtain 597mg of a yellow solid with a yield of 40%, mp168.1-170.2°C. ¹ H NMR (400MHz, CDCl ₃ ): δ3.81(s, 6H), 3.85(s, 3H) ,3.95(s,3H),6.61(s,2H),7.03(d,J=8.8Hz,1H),7.48(dd,J=6.4,2.2Hz,1H),7.81(d,J=6.4Hz, 1H). ¹³ C NMR (400MHz, CDCl3): δ56.3, 61.0, 112.2, 113.5, 114.9, 124.1, 126.6, 129.7, 139.3, 144.9, 149.8, 153.9. MS (EI) m/z: 351 (M ⁺ ).

Example 7

Preparation of 3,4,5-trimethoxy-1-[(3-amino-4-methoxyphenyl)thio]benzene (ZLM-7)

According to Example X, replace 1-(3'- Nitro-4'-methoxyphenyl)-2-methyl-5-(3',4',5'-trimethoxyphenyl)pyrrole ZLM-12, 181 mg white solid was obtained, yield 66% , mp168.1-170.2℃. ¹ H NMR (400MHz, CDCl ₃ ): δ3.77(s, 6H), 3.81(s, 3H), 3.86(s, 3H), 6.50(s, 2H), 6.75( d, J = 8.4Hz, 1H), 6.78 (d, J = 2.4Hz, 1H), 6.83 (dd, J = 6.4, 2.0Hz, 1H). ¹³ C NMR (400MHz, CDCl3): δ55.5, 56.0 , 60.9, 106.4, 110.7, 118.6, 122.9, 125.3, 132.6, 136.4, 136.8, 147.2, 153.3. MS (EI) m/z: 321 (M ⁺ ).

Example 8

Preparation of 3,4,5-trimethoxy-1-[(4-nitrophenyl)thio]benzene (ZLM-8)

According to Example 6, p-nitroiodobenzene (200 mg) was used instead of 3-nitro-4-methoxy iodobenzene to obtain 194 mg of a light yellow solid with a yield of 76%, mp168.1-170.2 ° C. ¹ H NMR ( 400MHz, CDCl ₃ ):δ3.85(s,6H),3.91(s,3H),6.79(s,2H),7.17(d,J＝8.8Hz,2H),8.09(d,J＝8.8Hz, 2H). ¹³ C NMR (400MHz, CDCl3): δ56.3, 61.0, 112.0, 124.1, 124.2, 125.9, 138.4, 145.1, 149.1, 154.1. MS (EI) m/z: 321 (M ⁺ ).

Example 9

Preparation of 3,4,5-trimethoxy-1-[(3-fluoro-4-methoxyphenyl)thio]benzene (ZLM-9)

According to Example 6, 3-nitro-4-methoxyiodobenzene was replaced with 3-fluoro-4-methoxyiodobenzene (282 mg) to obtain 222 mg of white solid, yield 69%, mp168.1-170.2 ° C ^.1 H NMR(400MHz,CDCl ₃ ):δ3.80(s,6H),3.84(s,3H),3.90(s,3H),6.55(s,2H),6.91-6.95(m,1H), 7.10-7.14(m,2H) ^.13C NMR(400MHz,CDCl3):δ56.1,56.2,60.9,107.8,113.6,119.1,119.2,126.9,127.0,127.6,130.6,137.2,147.2,147.3,151.0, 153.5. MS (EI) m/z: 324 (M ⁺ ).

Example 10

Preparation of 3,4,5-trimethoxy-1-[(4-ethoxyphenyl)thio]benzene (ZLM-11)

According to Example 6, 4-ethoxy iodobenzene (248 mg) was used instead of p-methoxy iodobenzene to obtain 89 mg of a pale yellowish white solid with a yield of 27.8%, mp91.4-93.3°C. ¹ H NMR (400 MHz, CDCl ₃ ): δ1.43(t,J=7.0Hz,3H),3.76(s,6H),3.81(s,3H),4.04(q,J=7.2Hz,3H),6.45(s,2H), 6.88 (d, J=8.8Hz, 2H), 7.38 (d, J=8.8Hz, 2H). ¹³ C NMR (400MHz, CDCl ₃ ): δ14.7, 56.0, 60.9, 63.5, 106.1, 115.3, 124.7, 132.8, 134.5, 136.5, 153.4, 159.0. MS (EI) m/z: 320 (M ⁺ ).

Example 11

Preparation of 3,4,5-trimethoxy-1-[(3-benzyloxy-4-methoxyphenyl)thio]benzene (ZLM-12)

According to Example 6, 3-benzyloxy-4-methoxyiodobenzene (1.02g) was used instead of p-methoxyiodobenzene to obtain 728mg of a white solid with a yield of 58.9%, mp100.6-101.1°C. ¹ H NMR(400MHz, CDCl ₃ ): δ3.73(s,6H),3.83(s,3H),3.90(s,3H),5.10(s,2H),6.43(s,2H),6.86(d,J ＝8.8Hz, 1H), 6.95(d, J＝2.0Hz, 1H), 7.02(dd, J＝6.8, 2.0Hz, 1H), 7.28-7.38(m, 5H). ¹³ CNMR(400MHz, CDCl ₃ ) :δ56.0,56.1,60.9,70.9,106.7,112.1,117.8,125.5,125.6,127.2,127.9,128.5,132.0,136.5,136.7,148.4,149.6,153.4.MS(EI)m/z:412(M ⁺ ).

Example 12

Preparation of 5-trifluoromethyl-2-[(3,4,5-trimethoxyphenyl)thio]pyridine ((ZLM-13)

Referring to the synthesis of ZLM-2, 2-chloro-5-trifluoromethylpyridine (127mg) was used instead of 5-chloro-2-nitroaniline, and the reaction temperature was lowered to 80°C to obtain 206mg of white solid with a yield of 59.7% , mp83.8-84.5°C. ¹ H NMR (400MHz, CDCl ₃ ): δ3.86(s,6H),3.91(s,3H),6.85(s,2H),6.94(d,J＝8.8Hz,1H),7.66(dd,J ＝6.4, 2.0Hz, 1H), 8.67(s, 1H). ¹³ CNMR (400MHz, CDCl ₃ ): δ56.6, 61.3, 112.8, 120.1, 122.7, 123.0, 123.7, 125.3, 133.8, 133.9, 139.9, 146.6 , 146.7, 154.3, 167.6. MS (EI) m/z: 345 (M ⁺ ).

Example 13

Preparation of methyl 3-[(3,4,5-trimethoxyphenyl)thio]benzothiophene-2-carboxylate (ZLM-14)

Referring to the synthesis of ZLM-2, 5-chloro-2-nitroaniline was replaced with 3-chlorobenzothiophene-2-carboxylic acid (100 mg), and the reaction temperature was lowered to 80 ° C to obtain 45 mg of a yellow solid with a yield of 25.7% . ¹ H NMR (400MHz, CDCl ₃ ): δ3.69(s,6H),3.79(s,3H),3.95(s,3H),6.47(s,2H),7.35(t,J＝7.8Hz,1H ), 7.47(t, J=7.6Hz, 1H), 7.81(d, J=8.4Hz, 1H), 7.86(d, J=8.0Hz, 1H). ¹³ C NMR (400MHz, CDCl ₃ ): δ52. 6, 56.1, 60.9, 106.1, 122.7, 125.1, 125.4, 127.5, 130.6, 131.6, 139.7, 139.8, 153.4, 162.2. MS (EI) m/z: 390 (M ⁺ ).

Example 14

Preparation of 2-fluorenylmethoxyamido-3-acetoxy-N-[2-methoxy-5-(3,4,5-trimethoxyphenyl)thio]propionamide (ZLM-15)

Under nitrogen protection, 3,4,5-trimethoxy-1-[(3-amino-4-methoxyphenyl)thio]benzene ZLM-7 (140mg) and O-acetyl-N-Fmoc - Dissolve L-serine (148mg) in 5mL of anhydrous dichloromethane, add DCC (82mg) and HOBt (56mg), stir at room temperature for 24h, spin off the solvent, add 20mL of ethyl acetate to dissolve, filter with suction, and use the filter residue Wash with ethyl acetate several times, combine the organic phases, wash the reaction solution with water several times, dry the organic phase with anhydrous sodium sulfate, filter with suction to obtain the filtrate, and spin off the solvent to obtain 365 mg of a yellow-brown oil. Silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) gave 277 mg of a white solid with a yield of 80.6%. ¹ H NMR (400MHz, CDCl ₃ ): δ2.09(s, 3H), 3.78(s, 6H), 3.82(s, 3H), 3.83(s, 3H), 4.24(t, J＝6.8Hz, 1H ), 4.33(t, J=5.2Hz, 1H), 4.41-4.53(m, 3H), 4.64(d, J=4.0Hz, 1H), 5.73(d, J=6.8Hz, 1H), 6.57(s ,2H),6.83(d,J=8.4Hz,1H),7.13(dd,J=2.2,6.4Hz,1H),7.31-7.33(m,2H),7.39-7.42(m,2H),7.60( d, J = 6.8Hz, 2H), 7.71 (d, J = 7.6Hz, 2H), 8.44 (s, 1H), 8.49 (d, J = 2.4Hz, 1H).

Example 15

Preparation of 2-amino-3-hydroxy-N-[2-methoxy-5-(3,4,5-trimethoxyphenyl)thio]propionamide (ZLM-16)

2-Fluorenylmethoxyamido-3-acetoxy-N-[2-methoxy-5-(3,4,5-trimethoxyphenyl)thio]propionamide ZLM-15 (232mg) Dissolve in an appropriate amount of dichloromethane:methanol=1:1 mixed solvent, then add 0.36mL 2N aqueous sodium hydroxide solution, stir at room temperature for 2h, and spin off the solvent to obtain 256mg of yellow-brown oil. Separation on a silica gel column (dichloromethane:methanol=10:1) gave 56mg of a white solid with a yield of 41.6%. ¹ H NMR (400MHz, CDCl ₃ ): δ2.16(br,3H),3.62(t,J=2.6Hz,1H),3.78(s,6H),3.81(s,3H),3.90(s,3H ),3.99(dd,J=4.8,6.0Hz,1H),6.55(s,2H),6.84(d,J=8.4Hz,1H),7.11(dd,J=6.4,2.2Hz,1H),8.55 (d, J=2.0Hz, 1H), 9.91(s, 1H). ¹³ C NMR (400MHz, CDCl ₃ ): δ55.9, 56.2, 56.5, 60.9, 65.1, 107.4, 110.7, 123.6, 126.5, 127.6, 128.2, 132.0, 137.0, 148.3, 153.4, 171.5. MS (EI) m/z: 408 (M ⁺ ).

Example 16

Preparation of 3,4,5-trimethoxy-1-[(3-hydroxy-4-methoxyphenyl)thio]benzene (ZLM-17)

Dissolve 3,4,5-trimethoxy-1-[(3-benzyloxy-4-methoxyphenyl)thio]benzene ZLM-12 (200mg) in dichloromethane: trifluoroacetic acid=1 :1 mixed solvent 4.8mL, stirred at room temperature for 15h, and removed the solvent to obtain a reddish-brown oil, which was separated on a silica gel column (petroleum ether:ethyl acetate=4:1) to obtain 32mg of a light yellow oil, with a yield of 20.7%. ¹ H NMR (400MHz, CDCl ₃ ): δ3.78(s,6H),3.82(s,3H),3.88(s,3H),5.70(br,1H),6.55(s,2H),6.82(d , J=8.4Hz, 2H), 6.93 (dd, J=1.8, 6.8Hz, 1H), 6.98 (d, J=2.0Hz, 1H). MS (EI) m/z: 322 (M ⁺ ).

Example 17

Compound Antitumor Activity Test

experimental method

1. Collect breast cancer MCF-7 cells and liver cancer HepG2 cells in the logarithmic phase, adjust the concentration of the cell suspension, add 100 μL to each well, and plate to adjust the density of the cells to be tested to 4000 per well, (the edge wells are filled with sterile PBS) .

2. Incubate at 5% carbon dioxide at 37°C until the cell monolayer covers the bottom of the well (96-well flat-bottomed plate), and add the drug with a concentration gradient. In principle, the drug can be added after the cells adhere to the wall, or two hours, or half a day Time, but we often lay the board the afternoon before and add medicine the next morning. Generally, there are 5-7 gradients, 100 μL per well, and 3-5 duplicate wells.

3. Incubate in 5% carbon dioxide at 37°C for 16-48 hours, and observe the cell growth under an inverted microscope.

4. Add 20 μL of MTT (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL, 0.5% MTT) to each well, continue Cultivate for 4h. If the drug can react with MTT, centrifuge first, then discard the culture medium, carefully rinse with PBS 2-3 times, and then add the culture medium containing MTT.

5. Terminate the culture, carefully aspirate the culture medium in the well.

6. Add 100 μL of dimethyl sulfoxide to each well, shake on a shaker at low speed for 10 minutes, and fully dissolve the crystals. The absorbance of each well was measured at 490 nm in an enzyme-linked immunosorbent assay.

7. At the same time set the zero adjustment wells (medium, MTT, dimethyl sulfoxide), control wells (cells, drug dissolution medium of the same concentration, culture solution, MTT, dimethyl sulfoxide).

8. Take the average value of the OD value of each duplicate hole to calculate the cell survival rate and inhibition rate

Survival rate = average value of OD value of the drug group / average value of OD value of the control group × 100%

Inhibition rate = 100% - survival rate

Draw a line graph with the inhibition rate as the ordinate and the drug concentration as the abscissa, and you can intuitively see the drug concentration roughly corresponding to the IC50 from the graph.

The test results are shown in Table 1. The results of the anti-human breast cancer MCF proliferative activity test show that ZLM-17, ZLM-9, ZLM-7, etc. have shown strong activity. The test results of anti-liver cancer HepG2 proliferative activity are shown in Table 2, ZLM-11, ZLM-16, ZLM-7, etc. have shown good activity.

Compound structure shown in Table 1 general formula I of the present invention and the IC50 value to human breast cancer MCF-7 cell line

Compound structure shown in the general formula I of table 2 of the present invention and the IC50 value to liver cancer HepG2 cell line

Claims (5)

1. a salt that there is the compound of structural formula I and pharmaceutically can use,

A is the substituted-phenyl that the 4-alkoxyl group shown in formula II I replaces;

Wherein R ₅, R ₆, R ₇, R ₈independently be selected from respectively hydrogen, hydroxyl, and R ₅, R ₆, R ₇, R ₈when different, be hydrogen; R ₉be selected from the alkyl of 1-3 carbon atom.

2. compound according to claim 1 and the salt that pharmaceutically can use thereof, is characterized in that, described compound of Formula I is 3,4,5-trimethoxy-1-[(3-hydroxyl-4-p-methoxy-phenyl) sulfo-] benzene.

3. compound according to claim 1 and 2 and the salt that pharmaceutically can use thereof, is characterized in that, described hydroxyl is further replaced to corresponding phosphoric acid ester, carboxylicesters and corresponding organic salt or inorganic salts derivative.

4. the described compound of one of claim 1-3 and the salt that pharmaceutically can use thereof, is preparing application aspect tubulin polymerization inhibitor and the application in the medicine of preparation treatment abnormal vascular growth correlation tumour.

5. application according to claim 4, it is characterized in that, described tumour is selected from lung cancer, nonsmall-cell lung cancer, melanoma, ovarian cancer, mammary cancer, liver cancer, cancer of the stomach, colorectal carcinoma, lymphoma, leukemia, carcinoma of the pancreas, bone cell cancer, prostate cancer, carcinoma of testis, bladder cancer, cervical cancer, rodent cancer, multiple myeloma, neuroblastoma, kidney, pinealoma, protometrocyte cancer, the rectum cancer, fibrosarcoma, liposarcoma, leiomyoma, chondrosarcoma, spermatogonium cancer, malignant hemangioendothelioma or osteosarcoma.