CN106083836B - H based on quinazoline structure2S donor compound and application thereof - Google Patents

Abstract

The invention discloses a quinazoline structure-based H₂An S donor compound and application thereof, which is a compound shown in a formula (I) or pharmaceutically acceptable salt thereof, wherein Ar is substituted or unsubstituted phenyl, and substituent groups of Ar are selected from: halogen, nitro, C_1‑4Alkyl radical, C_1‑4Haloalkyl, C_1‑4Alkoxy radical, C_1‑4One or more of halogenated alkoxy; x is C_1‑4Alkoxy, B-NH-or A-CH₂A CO-NH-group, Y being H, B-C_nH_2nO-or A-C_nH_2nO-group, A or B independently of one another being H₂An S donor group, n is an integer of 1 to 5, and X is C_1‑3Y is not H in the case of alkoxy. The invention relates to a series of H based on 4-anilino quinazoline structure₂S donor compound by H₂The S and the 4-anilino quinazoline derivative have synergistic effect, so that the antitumor activity of the medicament is improved.

Description

H based on quinazoline structure2S donor compound and application thereof

Technical Field

The invention belongs to the field of pharmaceutical compounds, and particularly relates to a quinazoline structure-based H₂S donor compound and its preparation method and application.

Background

As is well known, hydrogen sulfide (H)₂S) is a gas with the smell of rotten eggs, H, for centuries₂S is considered to be a highly toxic air pollutant and inhalation of large quantities can lead to poisoning. However, recently such gas molecules have been considered as a further important gas transmitter following nitric oxide and carbon monoxide. Endogenous H₂S production, associated with at least three enzymes: cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and 3-mercaptopyruvate thiotransferase (MPST). In different tissues or organs, these enzymes convert cysteine or cysteine derivatives into H₂S, and H₂S has a variety of biological effects including vasodilation, anti-inflammatory, anti-cancer and cardioprotection, among others.

It is reported that H₂S results in the hydrolysis of the disulfide bonds of the protein molecules (i.e., -S-SH formation), and thus, H₂S may alter the biological functions of various proteins and enzymes. At the same time, H₂S also reacts with S-nitrosothiols to produce mercaptonitrous acid (HSNO), and small amounts of S-nitrosothiols are free to pass through cell membranes, promoting nitrosation of proteins. The results of the above studies indicate that H can be regulated₂Levels of S may have greater utility in the treatment of disease.

It has now been found that exogenous H₂S induces tumor cell apoptosis by activating MAP kinase and caspase-3, thereby inhibiting tumor growth. In liver cancer, exogenous H₂S blocks STAT3 signal channel, blocks cell cycle, induces apoptosis, and inhibits tumor growth. In human colon cancer, exogenous H₂S inhibits the proliferation of tumors by inhibiting the NF-kB signaling pathway and the activity of Trx/TrxR. In breast cancer, exogenous H₂S induces apoptosis in tumor cells by cleaving PARP and activating caspase-7. In gastric cancer, exogenous H₂S inhibits tumor proliferation by regulating and controlling related apoptosis proteins Bax, CytC and caspase-3. It can be seen that exogenous H₂S has an inhibiting effect on tumors, which provides a possible new way for treating tumors.

In the field of hydrogen sulfide, H₂The S donor is an important research tool. Meanwhile, the results of the study confirmed that H₂S has an inhibitory effect in many tumors. In these H₂Among the S donors, the most commonly used are the sulfide salts, including sodium sulfide (Na)₂S) and sodium hydrosulfide (NaHS). These inorganic salts have excellent water solubility, are nontoxic to cells at specific concentrations, and can rapidly increase H₂The concentration of S. However, they spontaneously release H in aqueous solution₂S, it is therefore difficult to precisely control H₂The concentration of S. In addition, due to H₂The concentration of S gas in the aqueous solution is rapidly reduced by volatilization of S gas, so that the use of sodium sulfide and sodium hydrosulfide is greatly limited.

In view of these disadvantages, synthesis H₂The S donor has received considerable attention, and has been reported in the literature as H₂An S donor compound. E.g. H₂The S donor, GYY4137, is a derivative of Lawesson' S reagent. GYY4137 has been shown to release H both in vitro and in vivo₂S, and can pass through slowly released H₂S, the STAT3 signal channel is blocked, so that the inhibition effect on the liver cancer cells is generated. Other H reported so far₂The S donor mainly comprises DATS, Z-ajoene, S-propylcysteine sulfoxide, ADT-OH, ACS-81, ACS-60 and the like. Above H₂The S donor can be combined with a parent compound with biological activity to obtain H with better activity₂S donor compounds such as HS-sublindac, HS-ibuprofen, HS-naproxen, HS-aspirin and HS-ASA, etc. After the compound enters the body, H is slowly released under the action of in-vivo enzyme₂S and the parent compound or the structural analogue thereof exert the synergistic effect of the S and the parent compound, thereby showing better treatment effect or lower toxic and side effect than the parent compound. Researches find that the compounds have inhibiting effect on lung cancer, colon cancer, breast cancer and pancreatic cancer.

Quinazolines are the most studied structures in medicinal chemistry. The quinazoline alkaloid has potential antimalarial, antitumor, antibacterial, anti-inflammatory, antihypertensive and other activities. Over the last 15 years, the FDA has approved several anti-cancer drugs of 4-anilinoquinazoline derivatives, such as gefitinib, erlotinib, and lapatinib, among others. These 4-anilinoquinazoline compounds are all capable of inhibiting the activity of EGFR kinase. In addition, some kinase inhibitors using 4-anilinoquinazoline as a parent nucleus have been increasingly studied, and some of them have entered clinical research.

The Epidermal Growth Factor Receptor (EGFR) is a member of receptor tyrosine kinase families, mainly comprises subtypes of HER1 (namely EGFR), HER2, HER3, HER4 and the like, the high expression of the EGFR is closely related to epithelial cell tumors, the high expression tumor cells have strong invasiveness, are easy to transfer and have poor curative effect, and patients are poor after being cured.

Gefitinib (Gefitinib) has a 4-anilinoquinazoline structure, is a small molecule inhibitor targeting EGFR developed by Astra ZenecaAnd (3) preparing. Non-small cell lung cancer, which is not operable or relapsed, was first marketed in japan in 2002, and was approved as a three-line monotherapy for advanced non-small cell lung cancer (NSCLC) in the united states and australia in 2003. Experimental study shows that the compound can inhibit IC of EGFR in vitro₅₀0.037. mu.M; erlotinib (Erlotinib) a 4-anilinoquinazoline class of EGFR small molecule inhibitors developed by OSI. US FDA approval to market for locally advanced or metastatic non-small cell lung cancer (NSCLC) at 11 months 2004, which inhibits the IC of EGFR₅₀0.002. mu.M; lapatinib (Lapatinib), developed by the company glatiramer as an EGFR/HER2 dual inhibitor, is a 4-anilinoquinazoline compound that was approved by the FDA in the united states for sale in 2007 for the treatment of advanced or metastatic breast cancer. Research shows that in some cancer cell lines (A549, PC3, MCF7 and the like), lapatinib shows high antiproliferative activity, and IC of the lapatinib inhibits EGFR₅₀It was 0.010. mu.M. In addition, some Lapatinib analogues have inhibition effect on EGFR and HER-2 in preclinical research stage, such as compound 1, and IC thereof₅₀Values were less than 0.05 and 0.03nM, respectively.

Aurora kinase is an important serine/threonine kinase that plays an important regulatory role in cell mitosis. Aurora kinase expression is abnormal, interferes with mitosis, and may lead to gene instability and tumor growth.

AZD1152 is a 4-anilinoquinazoline Aurora B selective inhibitor developed by Astra Zeneca, Inc. that inhibits the IC of Aurora B₅₀0.37nM, and inhibition of Aurora A IC₅₀1368 nM. The study shows that AZD1152 can induce cycle arrest and apoptosis of human acute lymphocyte and leukemia cell, and the clinical phase II evaluation of the AZD for treating blood tumor, malignant solid tumor and advanced solid tumor. ZM447439 is the first reported Aurora A and Aurora B inhibitor, and is the first 4-anilinoquinazoline with Aurora A/B kinase inhibitory activity screened from 250000 compounds by Astra ZenecaLeads, IC's of Aurora A and B inhibition₅₀110 and 130nM, respectively. Since 2011, quinazoline Aurora kinase inhibitors have been reported in a number of patents. Among them, a series of quinazoline derivatives are designed and synthesized in a patent (WO2011144059), and show a dual-inhibition effect on Aurora B and EGFR, for example, when the compound 3 is at 200 μ M, the inhibition rates on Aurora B and EGFR reach 96% and 98%. The compound 4 designed and synthesized in the Chinese patent CN10409855 has high homology with AZD1152 structure, has better Aurora kinase inhibitory activity and inhibits the IC of Aurora A₅₀IC for inhibition of Aurora B at 82nM₅₀It was 0.15 nM.

European patent EP2708532 describes a series of derivatives related to 4-anilinoquinazoline, which primarily inhibit HDAC-1 enzymes. Wherein IC of Compound 5₅₀The value was 178 nM. The US20120094997 patent found that when a hydroxamic acid substituent is present at the C-6 position of the quinazoline, the selectivity for HDAC-6 enzyme is higher than that for HDAC-1 enzyme.

In conclusion, the 4-anilinoquinazoline derivatives as EGFR inhibitors, Aurora kinase inhibitors, histone deacetylase inhibitors and the like show good antitumor activity.

In recent years, the incidence of tumors has increased year by year, and the research and development of novel safe, efficient and low-toxicity antitumor agents is imminent.

Disclosure of Invention

The invention aims to design and synthesize a series of H based on 4-anilino quinazoline structure based on the prior art₂S donor compound by H₂The S and the 4-anilino quinazoline derivative have synergistic effect, so that the antitumor activity of the medicament is improved.

Another object of the present invention is to provideFor one kind of the above-mentioned H based on quinazoline structure₂A method for preparing an S donor compound.

The third purpose of the invention is to provide the H based on the quinazoline structure₂The S donor compound is applied to the anti-tumor aspect.

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

quinazoline structure-based H₂An S donor compound which is a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,

wherein the content of the first and second substances,

ar is substituted or unsubstituted phenyl, and the substituents are selected from: halogen, nitro, C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Alkoxy radical, C_1-4One or more of halogenated alkoxy;

x is C_1-4Alkoxy, B-NH-or A-CH₂A group of CO-NH-or a group of NH-,

y is H, B-C_nH_2nO-or A-C_nH_2nA group of O-is selected from the group consisting of,

a or B is independently H₂An S donor group, a N-substituted phenyl group,

n is an integer of 1 to 5,

and X is C_1-3Y is not H in the case of alkoxy.

In a preferred embodiment, Ar is a substituted or unsubstituted phenyl group, wherein the substituents are selected from the group consisting of: fluorine, chlorine, bromine, nitro, C_1-3Alkyl radical, C_1-3Haloalkyl, C_1-3One or more of alkoxy.

In a more preferred embodiment, Ar is a substituted or unsubstituted phenyl group, the substituents of which are selected from the group consisting of: one or more of fluorine, chlorine, bromine, nitro, methyl, ethyl, methoxy, ethoxy and trifluoromethyl.

In the invention, A or B is respectively H₂S donor group, in a preferred embodiment, A is

B is

N in the present invention may be 1,2, 3,4 or 5, preferably 2, 3 or 4, most preferably 3.

In a preferred embodiment, X is C_1-3Alkoxy, Y is B-C_nH_2nO-or A-C_nH_2nAn O-group.

In another preferred embodiment, X is B-NH-or A-CH₂A CO-NH-group and Y is H.

In one embodiment, the compound of the present invention may be selected from compounds represented by formula (II) or formula (III),

wherein the content of the first and second substances,

R₃is a group A or a group B,

R₄is A-CH₂CO-or B groups.

The invention relates to a compound or a pharmaceutically acceptable salt thereof, wherein the compound can be selected from the following compounds:

the invention provides a synthetic route of a compound shown in a formula (II), which comprises the following steps:

the invention provides a synthetic route of a compound shown in a formula (III), which comprises the following steps:

the invention provides a pharmaceutical composition, which takes the compound or the pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and is assisted by pharmaceutically acceptable auxiliary materials.

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

"alkyl" in the present invention means a saturated aliphatic group of 1 to 20 carbon atoms, including straight and branched chain groups (the numerical range mentioned in this application, e.g. "1 to 20" means that the group, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). Alkyl group having 1 to 4 carbon atoms (C)_1-4Alkyl) is referred to as lower alkyl. When a lower alkyl group has no substituent, it is referred to as unsubstituted lower alkyl. More preferably, the alkyl group is a medium size alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, and the like. Preferably, the alkyl group is a lower alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, or the like. Alkyl groups may be substituted or unsubstituted.

"halogen" in the present invention means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

The "nitro group" in the present invention means "-NO₂"group

"haloalkyl" in the context of the present invention denotes halogen-substituted alkyl, preferably halogen-substituted lower alkyl as defined above, which is substituted by one or more halogen atoms which may be the same or different (e.g. C)_1-4Haloalkyl), e.g. -CH₂Cl、-CF₃、-CH₂CF₃、-CH₂CCl₃And the like.

"alkoxy" in the present invention means-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

"haloalkoxy" in the present invention means an alkoxy group substituted with one or more of the same or different halogen atoms. Representative examples include, but are not limited to, trifluoromethoxy, chloromethoxy, and the like.

By "pharmaceutically acceptable salts" in the context of this invention is meant those salts which retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) salts with acids are obtained by reaction of the free base of the parent compound with inorganic acids including hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid, perchloric acid and the like, or with organic acids including acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, benzoic acid, hydroxybenzoic acid, γ -hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid or malonic acid and the like.

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

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

The compound or the pharmaceutically acceptable salt thereof can be applied to the preparation of anti-cancer drugs, in particular to the anti-liver cancer or liver cancer drugs.

The invention relates to a series of H based on 4-anilino quinazoline structure₂S donor compound by H₂The S and the 4-anilino quinazoline derivative have synergistic effect, so that the antitumor activity of the medicament is improved. Researching the in vitro anti-HepG 2 and A-549 cell proliferation activity of the compound; on the basis of the detection, the in-vitro release H of the compound is detected₂S level; the molecular level EGFR inhibitory activity of the target compound is detected so as to discover a lead compound with higher comprehensive evaluation and provide a new way for the research and development of novel antitumor drugs.

Drawings

FIG. 1 shows the QHS-6 qualitative release H of the compound of the present invention₂S level.

In the figure, the left panel A is: the fluorescent probe NIR-HS (10 μ M) was incubated with compound QHS-6(0, 5, 10,50,100,200 μ M) in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃) and the fluorescence intensity was measured. The right panel B is: the fluorescence intensity of compound QHS-6(0, 5, 10,50,100, 200. mu.M) was measured at 723 nm.

FIG. 2 shows the QHS-13 qualitative release H of the compound of the present invention₂S level.

In the figure, the left panel A is: the fluorescent probe NIR-HS (10 μ M) was incubated with compound QHS-13(0, 5, 10,50,100,200 μ M) in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃) and the fluorescence intensity was measured. The right panel B is: the fluorescence intensity of compound QHS-13(0, 5, 10,50,100, 200. mu.M) was measured at 723 nm.

FIG. 3 shows qualitative release of H from LHS-1 of the compound of the invention₂S level.

In the figure, the left panel A is: the fluorescent probe NIR-HS (10 μ M) was incubated with compound LHS-1(0, 5, 10,50,100,200 μ M) in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃) and the fluorescence intensity was measured. The right panel B is: the fluorescence intensity of compound LHS-1(0, 5, 10,50,100, 200. mu.M) was measured at 723 nm.

FIG. 4 is Na₂S standard curve.

In the figure, the left panel A is: fluorescent probes NIR-HS (10. mu.M) and Na₂S (0,2,4,6, 8. mu.M) was incubated in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃ C.), and the fluorescence intensity was measured. The right panel B is: na (Na)₂S standard curve.

FIG. 5 is Compound QHS-6, QHS-13 and LHS-1 release H quantitatively₂S level.

Detailed Description

Some aspects and embodiments of the invention are further illustrated by the following specific examples. The compounds encompassed by the present invention can be synthesized by known conventional techniques. These compounds can be conveniently synthesized from readily available starting materials. The following is a general synthetic scheme for the compounds synthesized in the present invention. The schemes disclosed herein are illustrative and are not meant to limit the ability of those skilled in the art to synthesize compounds using other possible methods. Various methods are conventional in the art. In addition, different synthetic steps may be applied to synthesize the target compound in different schemes. All documents cited herein are incorporated by reference herein.

The following representative examples are intended to aid in the illustration of the present invention and are not intended to, and should not be construed to, limit the scope of the present invention. Indeed, the entire contents of the documents in this invention, including examples in accordance with the scientific literature and patents cited herein, as well as various modifications and numerous further variations thereof, will be readily apparent to those skilled in the art, except to those shown and described herein. It should also be understood that the citation of these references is helpful in setting forth the disclosure herein. The following examples contain important supplementary information, examples and guidance, and are adaptable to various variations and the like in the present invention.

Synthetic examples

The compounds of the present invention are primarily directed to two classes: QHS series and LHS series. Wherein, the QHS series comprise ether bond QHS-a (QHS-1, QHS-2, QHS-3, QHS-4, QHS-5 and QHS-6) and ester bond QHS-b (QHS-13 and QHS-15), and the LHS series comprise LHS-a (FHS-1, LHS-1 and XHS-1) and LHS-b (FHS-2, LHS-2 and XHS-2). Specific compounds are shown in tables 1 and 2.

TABLE 1 QHS series of compounds

TABLE 2 LHS series of compounds

Overall synthetic route and steps of QHS series compounds

4-hydroxy-3-methoxybenzoic acid is used as a raw material, and a series of reactions such as esterification, nitration, reduction, ring closure, chlorination and the like are carried out to synthesize an intermediate (7a,7b,7c,7d,7e,7f), and then H is connected₂And 8 QHS series compounds are finally obtained by an S donor, and the specific synthetic route is as follows.

Synthesis of methyl 4-hydroxy-3-methoxybenzoate (1)

To a 500mL three-necked flask were added 4-hydroxy-3-methoxybenzoic acid (20.0g, 0.19mol), 250mL of methanol, and 0.8mL of concentrated sulfuric acid, and the mixture was heated to reflux for about 48 hours, after which the reaction was stopped. The methanol is distilled off, water is added to the residue, saturated K₂CO₃Neutralizing the solution, extracting with ethyl acetate, drying the organic layer with anhydrous sodium sulfate, filtering, concentrating the mother liquor under reduced pressure to obtain brown oily substance, adding petroleum ether: ethyl acetate (8:1) was recrystallized to give (1) as a white solid (19.6g, 90.5%) Mp:62-63 ℃.

Synthesis of methyl 3-methoxy-4- (3-chloropropyloxy) benzoate (2)

4.22g (23mmol) of methyl 4-hydroxy-3-methoxybenzoate (1), 5.06g (32.4mmol) of 1-bromo-3-chloropropane are dissolved in 20ml of DMF, 10.24g (74.2mmol) of potassium carbonate are added, and the reaction is carried out at 100 ℃ for 12 hours. After the reaction solution was cooled to room temperature, the reaction solution was poured into 500mL of ice water and vigorously stirred for 30 min. Suction filtration gave 5.85g of off-white solid. Recrystallization from ethyl acetate gave 4.85g of the white target product in 80.7% yield, Mp: 103-.

Synthesis of methyl 2-nitro-5-methoxy-4- (3-chloropropyloxy) benzoate (3)

9.3g (12.8mmol) of methyl 3-methoxy-4- (3-chloropropyloxy) benzoate (2) are dissolved in 30mL of acetic acid and 3mL of acetic anhydride at 0 to 5 ℃, concentrated nitric acid (8.45mL, 66%) is added dropwise, and the mixture is stirred at room temperature for 6 hours. The reaction solution was poured into 500mL of ice water, and extracted with ethyl acetate. The organic layers were combined, washed with a saturated aqueous sodium bicarbonate solution and brine in this order, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness to give a yellow oil (13.0 g). Recrystallization from ethyl acetate/petroleum ether gave 8.13g of a bright yellow solid in 75% yield, Mp 57-59 ℃. Synthesis of methyl 2-amino-5-methoxy-4- (3-chloropropyloxy) benzoate (4)

After 5.0g (89.3mmol) of reduced iron powder was added to 40mL of acetic acid and stirred at 50 ℃ for 15min under nitrogen protection, 9.0g (25mmol) of methyl 2-nitro-5-methoxy-4- (3-chloropropyloxy) benzoate (3) in methanol (30mL) was slowly added, and the reaction mixture was reacted at 50-60 ℃ for 30 min. The iron mud in the reaction solution is filtered while the solution is hot, the filtrate is decompressed and distilled to remove the solvent until no distillate exists, the residue is poured into ice water, and ethyl acetate is used for extraction. The organic layer was washed with saturated potassium carbonate and brine in this order, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give a brown solid, which was recrystallized from ethyl acetate/petroleum ether to give 6.34g of a bright brown solid in 77% yield, Mp:67-69 ℃.

Synthesis of 6-methoxy-7- (3-chloropropoxy) quinazolin-4 (3H) -one (5)

1.7g (6mmol) of methyl 2-amino-5-methoxy-4- (3-chloropropyloxy) benzoate (4) are dissolved in 20mL of absolute ethanol, 0.97g (9mmol) of formamidine acetate are added and the mixture is refluxed for 24 h. Cooling, standing in a refrigerator, filtering, washing with cold ethanol to obtain 1.52g of white powdery solid, with the yield of 94.4 percent and the Mp of 113 and 115 ℃.

Synthesis of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6)

1.0g (3.15mmol) of 6-methoxy-7- (3-chloropropoxy) quinazolin-4 (3H) -one (5) are added in portions to 8mL of thionyl chloride, and then 0.62mL of DMMF is slowly added dropwise. The reaction was refluxed for 4 h. The excess thionyl chloride was evaporated under reduced pressure to give a yellow solid which was dissolved in chloroform, washed with a saturated sodium carbonate solution and water in this order, and dried over anhydrous sodium sulfate. The organic solvent was evaporated under reduced pressure to obtain a white solid, which was recrystallized from ethyl acetate to obtain 0.91g of a white solid with a yield of 86%, Mp: 146-.

Synthesis of 4- (3-chloro-4-fluoroanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7a)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 2.1g (13.7mmol) of 3-chloro-4-fluoroaniline were added to 10mL of isopropanol and reacted for 3 hours under reflux. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.C to obtain yellow solid 2.73g, yield 93%, Mp: 170-.

Synthesis of 4- (4-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7b)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 2.36g (13.7mmol) of 4-bromoaniline are added to 10mL of isopropanol and reacted under reflux for 3 h. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.c to obtain yellow solid in 1.42g yield of 48% Mp 165-167 deg.c.

Synthesis of 4- (4-nitroanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7c)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 1.91g (13.7mmol) of 4-nitroaniline were added to 10mL of isopropanol and reacted under reflux for 3 h. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.C to obtain yellow solid 1.41g, yield 53%, Mp: 168-.

Synthesis of 4- (3-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7d)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 2.36g (13.7mmol) of 3-bromoaniline are added to 10mL of isopropanol and reacted under reflux for 3 h. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.c to obtain yellow solid in 0.92g yield of 31% and Mp: 157-.

Synthesis of 4- (3-trifluoromethylanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7e)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 2.21g (13.7mmol) of 3-trifluoromethylaniline were added to 10mL of isopropanol and reacted under reflux for 3 hours. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.C to obtain yellow solid 1.24g, yield 43%, Mp: 167-.

Synthesis of 4- (3, 4-dimethoxyanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7f)

2.0g (7mmol) of 4-chloro-6-methoxy-7- (3-chloropropoxy) quinazoline (6) and 2.0g (13.7mmol) of 3, 4-dimethoxyaniline are added to 10mL of isopropanol and reacted for 3h under reflux. The reaction was cooled to room temperature, cooled in a refrigerator overnight, filtered with suction, and the filter cake was washed with cold isopropanol. Vacuum drying at 60-70 deg.C to obtain yellow solid 1.41g, yield 50%, Mp 173-.

Synthesis of 5-p-hydroxyphenyl-3H-1, 2-dithiole-3-thione (ADT-OH)

To a single-neck flask was added 5-p-methoxyphenyl-3H-1, 2-dithiole-3-thione (0.24g, 1mmol) and pyridine hydrochloride

(1.20g, 10mmol) was mixed well. Carrying out melt reaction for 0.5h at 210 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, adding 1mol/L diluted hydrochloric acid, carrying out suction filtration to obtain a tan solid, and carrying out vacuum drying. The yield was 81%, Mp: 185-.

Synthesis of 3-allyldithiopropionic acid (ACS-81)

To a three-necked flask was added diallyl disulfide (5.85g, 40mmol), 3-mercaptopropionic acid (0.85g, 8mmol), methanol: diethyl ether (2: 1V/V) mixed solvent, then 10mol/L NaOH (0.24g, 6mmol) is added, nitrogen is introduced for 30min, and reaction is carried out for 24h at 25 ℃. The reaction was monitored by TLC, after completion of the reaction, the reaction solution was evaporated to dryness, 1mol/L HCl was added, extraction was performed with diethyl ether, and the solvent was evaporated under reduced pressure to give 1.2g of an off-white oil. The yield thereof was found to be 84%.

Synthesis example 1: synthesis of QHS-1

Adding 4- (3-chloro-4-fluoroanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7a) (0.395g, 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g, 2mmol), and catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g, 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction solution into ice water, stirring for 30min, performing suction filtration, performing vacuum drying, and separating dichloromethane through silica gel column chromatography: methanol (120:1) gave 0.12g of an orange solid in 20% yield, Mp: 104-.

¹H NMR(400MHz,CDCl₃)δ8.66(s,1H),7.91(s,1H),7.64-7.59(m,2H),7.31(d,J＝16.0Hz,5H), 7.22-7.18(m,1H),7.03(d,J＝8.0Hz,2H),4.36(d,J＝36.0Hz,4H),4.04(s,3H),2.46(s,2H).

¹³C NMR(101MHz,DMSO)δ215.24,174.23,172.49,162.39,156.54,154.05,152.95,152.48,149.58, 134.63,129.49,124.21,124.01,122.87,122.80,119.36,119.17,117.10,116.89,116.01,109.24,108.14, 102.40,65.59,65.27,56.78,28.70.

LC-MSm/z:585.8[M-H]^-.

Synthesis example 2: synthesis of QHS-2

Adding 4- (4-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7b) (0.423g and 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g and 2mmol), a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g and 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, performing suction filtration, performing vacuum drying, and separating dichloromethane by silica gel column chromatography: ethyl acetate (2:1) was added to 1mL of methanol to give 0.1g of an orange solid in 16% yield, Mp: 113-.

¹H NMR(400MHz,CDCl₃)δ8.66(s,1H),7.63(dd,J＝8.0,12.0Hz,3H),7.53(d,J＝8.0Hz,2H),7.39(s, 1H),7.29(d,J＝12.0Hz,3H),7.07-7.01(m,3H),4.39-4.29(m,4H),4.03(s,3H),2.47-2.44(m,2H).

¹³C NMR(101MHz,CDCl₃)δ195.70,172.99,169.69,167.96,162.15,154.11,149.86,134.67,132.03, 128.60,124.31,123.42,121.81,116.97,116.23,115.52,100.68,65.34,64.69,56.38,29.70.

LC-MSm/z:611.8[M-H]^-.

Synthetic example 3: synthesis of QHS-3

Adding 4- (4-nitroanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7c) (0.389g, 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g, 2mmol), a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g, 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction solution into ice water, stirring for 30min, performing suction filtration, drying in vacuum, and separating dichloromethane by silica gel column chromatography: ethyl acetate (1:2) was added to 1mL of methanol to give 0.11g of an orange solid in 16% yield, Mp: 108-.

¹H NMR(400MHz,DMSO)δ10.05(s,1H),8.65(s,1H),8.30(d,J＝8.0Hz,2H),8.20(d,J＝12.0Hz,2H), 7.92-7.87(m,2H),7.81-7.76(m,1H),7.31(s,1H),7.13(dd,J＝4.0Hz,2H),4.34(dd,J＝4.0,20.0Hz,4H), 4.01(s,3H),2.33-2.30(m,2H).

¹³C NMR(101MHz,DMSO)δ215.26,174.23,162.39,162.00,156.16,154.47,152.59,149.92,142.12, 134.65,129.50,128.73,125.07,124.22,121.15,116.56,116.03,109.85,108.03,102.52,65.74,65.26,56.89, 28.71.

LC-MSm/z:579.8[M+H]⁺.

Synthetic example 4: synthesis of QHS-4

Adding 4- (3-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7d) (0.423g, 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g, 2mmol), a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g, 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, performing suction filtration, performing vacuum drying, and separating dichloromethane through silica gel column chromatography: ethyl acetate (2:1) was added to 1mL of methanol to give 0.12g of an orange solid in 20% yield, Mp: 104-.

¹H NMR(400MHz,DMSO)δ9.53(s,1H),8.53(s,1H),8.16(s,1H),7.90-7.75(m,5H),7.34(d,J＝8.0Hz, 1H),7.27(d,J＝16.0Hz,2H),7.12(d,J＝8.0Hz,2H),4.30(d,J＝20.0Hz,4H),3.98(s,3H),2.30(s,2H).

¹³C NMR(101MHz,DMSO)δ215.26,174.24,162.40,156.47,154.03,153.05,149.59,147.32,141.73, 134.64,130.83,129.50,128.73,126.10,124.49,124.22,121.65,121.06,116.03,115.84,109.44,108.34, 102.42,65.60,65.28,56.79,28.72.

LC-MSm/z:611.7[M-H]^-.

Synthesis example 5: synthesis of QHS-5

Adding the synthesized (7e) (0.411g and 1mmol) of 4- (3-trifluoromethylanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline, anhydrous DMF10mL, potassium carbonate (0.276g and 2mmol) and a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g and 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, performing suction filtration, drying in vacuum, and separating dichloromethane through silica gel column chromatography: ethyl acetate (2:1) was added to 1mL of methanol to give 0.12g of an orange solid in a yield of 20%, Mp: 109-.

¹H NMR(400MHz,DMSO)δ9.67(s,1H),8.54(s,1H),8.23(d,J＝8.0Hz,2H),7.87(d,J＝8.0Hz,3H), 7.75(s,1H),7.63(t,J＝8.0Hz,1H),7.44(d,J＝8.0Hz,1H),7.26(s,1H),7.13(d,J＝12.0Hz,2H), 4.35-4.28(m,4H),4.00(d,J＝5.0Hz,3H),2.32-2.29(m,2H).

¹³C NMR(101MHz,DMSO)δ215.24,174.22,170.79,162.40,156.51,154.07,153.01,149.62,147.42, 140.93,134.63,130.03,129.48,126.10,125.85,124.21,123.39,119.75,118.27,116.01,109.45,108.37, 102.40,65.58,65.28,56.79,28.72.

LC-MSm/z:602.8[M+H]⁺.

Synthetic example 6: synthesis of QHS-6

Adding synthesized (7f) (0.404g and 1mmol) of 4- (3, 4-dimethoxyanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline, anhydrous DMF10mL, potassium carbonate (0.276g and 2mmol) and catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ADT-OH (0.226g and 1mmol), reacting at 80 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, performing suction filtration, performing vacuum drying, and separating dichloromethane through silica gel column chromatography: ethyl acetate (2:1) was added to 1mL of methanol to give 0.1g of an orange solid in a yield of 17%, Mp: 116-.

¹H NMR(400MHz,DMSO)δ9.39(s,1H),8.42(s,1H),7.90-7.83(m,3H),7.75(s,1H),7.38(s,1H),7.32 (d,J＝8.0Hz,1H),7.21(s,1H),7.13(d,J＝8.0Hz,2H),6.99(d,J＝12.0Hz,1H),4.32-4.29(m,4H),3.97 (s,3H),3.79(d,J＝8.0Hz,6H),2.32-2.29(m,2H).

¹³C NMR(101MHz,DMSO)δ215.25,174.24,162.40,157.10,153.73,149.31,148.91,145.88,134.64, 133.02,129.50,128.72,116.54,116.02,115.83,115.62,112.26,109.24,108.57,108.14,102.66,65.49,65.29, 56.78,56.23,56.07,28.75.

LC-MSm/z:594.9[M+H]⁺.

Synthetic example 7: synthesis of QHS-13

Adding 4- (4-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7b) (0.423g and 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g and 2mmol), a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ACS81(0.356g and 2mmol), reacting at 70 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, extracting with ethyl acetate, combining organic layers, washing with saturated saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum, and separating petroleum ether by silica gel column chromatography: ethyl acetate (1:2) to give 0.09g of an off-white solid in a yield of 16%, Mp: 105-.

¹H NMR(400MHz,DMSO)δ9.53(s,1H),8.49(s,1H),7.84(d,J＝12.0Hz,3H),7.57(d,J＝12.0Hz,2H), 7.22(d,J＝8.0Hz,1H),5.84-5.69(m,1H),5.21-5.15(m,1H),5.11-5.05(m,1H),4.33-4.24(m,4H),3.98(s, 3H),3.16(d,J＝8.0Hz,2H),2.67-2.61(m,2H),2.51(s,2H),2.20-2.14(m,2H).

¹³C NMR(101MHz,DMSO)δ171.98,168.85,156.54,153.93,153.16,147.44,139.45,134.98,131.67, 128.77,124.43,117.57,115.32,109.43,102.46,65.69,61.67,56.77,34.49,34.07,28.31.

LC-MSm/z:563.8[M-H]^-.

Synthesis example 8: synthesis of QHS-15

Adding 4- (3-bromoanilino) -6-methoxy-7- (3-chloropropoxy) quinazoline (7d) (0.423g and 1mmol), anhydrous DMF10mL, potassium carbonate (0.276g and 2mmol), a catalytic amount of potassium iodide into a single-neck flask, stirring for 10min, adding ACS81(0.356g and 2mmol), reacting at 70 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, pouring the reaction liquid into ice water, stirring for 30min, extracting with ethyl acetate, combining organic layers, washing with saturated saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum, and separating petroleum ether by silica gel column chromatography: ethyl acetate (1:2) to give 0.11g of an off-white solid in a yield of 20%, Mp: 101-.

¹H NMR(400MHz,DMSO)δ9.54(s,1H),8.53(s,1H),8.16(s,1H),7.90(d,J＝8.0Hz,1H),7.84(s,1H), 7.36(t,J＝8.0Hz 1H),7.29(d,J＝8.0Hz,1H),7.24-7.22(m,1H),5.79-5.69(m,1H),5.14-5.05(m,2H), 4.33-4.23(m,4H),3.98(s,3H),3.16(d,J＝4.0Hz,2H),2.63(t,J＝4.0Hz,2H),2.52-2.51(m,2H), 2.17-2.11(m,2H).

¹³C NMR(101MHz,DMSO)δ171.98,165.97,156.44,153.99,153.11,149.55,147.51,134.97,130.82, 128.77,126.04,124.44,121.65,121.01,117.56,109.43,102.37,65.71,61.50,56.77,34.48,34.07,28.14.

LC-MSm/z:565.1[M+H]⁺.

Integral synthesis route and steps of LHS series compound

2-amino-5-nitrobenzoic acid is taken as a raw material, and an intermediate (4a,4b,4c) is synthesized by ring closure, chlorination, nitration and reduction reaction and then connected with H₂S donor to obtain 6 LHS series compounds, and the specific synthetic route is as follows.

Synthesis (1) of 6-nitro-4 (3H) -quinazolinone

2-amino-5-nitrobenzoic acid (3.64g, 20mmol), formamidine acetate (4.16g, 40mmol) and 50mL of ethylene glycol monomethyl ether were added to a 100mL three-necked flask and reacted under reflux. TLC monitoring, after the reaction, the reaction solution was poured into a beaker, placed in a freezer at-20 ℃ overnight, filtered to obtain 3.0g of yellow solid with yield of 78.5% and Mp of 280 plus 283 ℃.

Synthesis of 4- (3-trifluoromethylanilino) -6-nitroquinazoline (3a)

6-Nitro-4 (3H) -quinazolinone (1) (3.8g, 20mmol) was added to a 250ml three-necked flask, SOCl was added₂40mL, slowly dripping 4mL of DMF, heating to 80 ℃, carrying out reflux reaction for 1h, and then supplementing 20mL of SOCl₂And 2ml of DMF, and the reaction was continued under reflux. TLC monitoring, after the reaction, the reaction solution was transferred to a 250mL eggplant-shaped bottle and SOCl was removed by reduced pressure rotary evaporation₂Then, petroleum ether was added thereto, the solid was washed, and the obtained solid was put into a 250mL single-neck eggplant-shaped bottle containing 3-trifluoromethylaniline (6.64g, 40mmol) and 60mL of isopropyl alcohol, and the mixture was refluxed at an elevated temperature. TLC monitoring, after the reaction is finished, suction filtration is carried out, and the filter cake is washed by isopropanol to obtain yellow solid with the yield of 60%.

Synthesis of 4- (3-bromoanilino) -6-nitroquinazoline (3b)

6-Nitro-4 (3H) -quinazolinone (1) (3.8g, 20mmol) was added to a 250mL three-necked flask, and thionyl chloride (SOCl) was added₂) 40mL, slowly adding 4mL of N, N-Dimethylformamide (DMF) dropwise, heating to 80 ℃, carrying out reflux reaction for 1h, and supplementing 20mL of SOCl₂And 2mL of DMF was added to the mixture,the reaction was continued under reflux. TLC monitoring, after the reaction, the reaction solution was transferred to a 250mL eggplant-shaped bottle and SOCl was distilled off under reduced pressure₂Adding petroleum ether, washing the solid, adding the obtained solid into a 250ml single-opening eggplant-shaped bottle weighed to contain 3-bromoaniline (6.88g, 40mmol) and 60ml of isopropanol, heating, carrying out reflux reaction, monitoring by TLC, after the reaction is finished, carrying out suction filtration, and washing a filter cake by using the isopropanol to obtain a yellow solid with the yield of 90%.

Synthesis of 4- (3-chloro-4-fluoroanilino) -6-nitroquinazoline (3c)

6-Nitro-4 (3H) -quinazolinone (1) (3.8g, 20mmol) was added to a 250mL three-necked flask, and thionyl chloride (SOCl) was added₂) 40mL, slowly adding 4mL of N, N-Dimethylformamide (DMF) dropwise, heating to 80 ℃, carrying out reflux reaction for 1h, and supplementing 20mL of SOCl₂And 2mL of DMF, and the reaction was continued at reflux. TLC monitoring, after the reaction, the reaction solution was transferred to a 250mL eggplant-shaped bottle and SOCl was removed by reduced pressure rotary evaporation₂Adding petroleum ether, washing the solid, adding the obtained solid into a 250mL single-mouth eggplant-shaped bottle which is called to contain 3-chloro-4-fluoroaniline (5.87g, 40mmol) and 60mL of isopropanol, heating, carrying out reflux reaction, monitoring by TLC, after the reaction is finished, carrying out suction filtration, and washing a filter cake by using the isopropanol to obtain a yellow solid with the yield of 50%.

Synthesis of 4- (3-trifluoromethylanilino) -6-aminoquinazoline (4a)

4- (3-trifluoromethylanilino) -6-nitroquinazoline (3a) (4.3g), 150mL of ethanol and 50mL of acetic acid are added into a 500mL three-necked flask, mechanically stirred, heated, and then iron powder (4.5g) activated by hydrochloric acid is added into the flask in portions and refluxed. And monitoring by TLC, filtering while the solution is hot after the reaction is finished, carrying out reduced pressure spin-drying on the filtrate, adding distilled water, stirring, pouring into a 500mL beaker, and carrying out suction filtration to obtain a light green solid with the yield of 60%.

Synthesis of 4- (3-bromoanilino) -6-aminoquinazoline (4b)

4- (3-bromoanilino) -6-nitroquinazoline (3b) (5.0g), 150mL of ethanol and 50mL of acetic acid are added into a 500mL three-necked flask, mechanically stirred, heated, added with iron powder activated by hydrochloric acid (5.0g) in portions into the flask, and refluxed for reaction. And monitoring by TLC, filtering while the solution is hot after the reaction is finished, carrying out reduced pressure spin-drying on the filtrate, adding distilled water, stirring, pouring into a 500mL beaker, and carrying out suction filtration to obtain a light green solid with the yield of 40%. Synthesis of 4- (3-chloro-4-fluoroanilino) -6-aminoquinazoline (4c)

4- (3-chloro-4-fluoroanilino) -6-nitroquinazoline (3c) (3.8g), 150mL of ethanol and 50mL of acetic acid were placed in a 500mL three-necked flask, mechanically stirred, warmed, and iron powder (4.5g) activated with hydrochloric acid was added in portions to the flask, and the reaction was refluxed. Monitoring by TLC, filtering while the solution is hot after the reaction is finished, carrying out rotary evaporation on the filtrate under reduced pressure until the filtrate is dry, adding distilled water, stirring, pouring into a 500mL beaker, and carrying out suction filtration to obtain a light green solid with the yield of 40%.

Synthesis of 5-p-carboxyethoxyphenyl-3H-1, 2-dithiole-3-thione (ACS-60)

Adding ADT-OH (0.45g, 2mmol), potassium carbonate (0.55g, 4mmol) and ethyl bromoacetate (0.66mL, 6mmol) into a three-neck flask, adding anhydrous DMF30mL, fully stirring for dissolving, reacting at 50 ℃ for 3h, monitoring by TLC, cooling to room temperature after the reaction is finished, extracting with ethyl acetate, washing with distilled water for 3 times, drying the ethyl acetate layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a tan oily substance. The oily substance (0.31g, 1mmol) obtained in the previous step is directly used for the next step of reaction, 50% sulfuric acid (2.2mL, 20mmol) and 20mL acetic acid are added, the reaction is carried out at 100 ℃ for 2h, the reaction is monitored by TLC, the reaction is cooled to room temperature after the reaction is finished, distilled water is added, and the deep green solid is obtained by suction filtration, and the yield is 49%.

Synthetic example 9: synthesis of FHS-1

Adding ACS-60(0.312g, 1.1mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.418g, 1.1mmol) and triethylamine (0.122g, 1.2mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-trifluoromethylanilino) -6-aminoquinazoline (0.30g, 1.1mmol) (4a), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water after the reaction is finished, stirring and filtering to obtain a gray solid, and performing silica gel column chromatography to separate a final light orange solid product, wherein the yield is 15%, and the Mp:108 and 109 ℃.

¹H NMR(400MHz,DMSO)δ10.52(s,1H),10.05(s,1H),8.79(s,1H),8.60(s,1H),8.27(s,1H),8.20(d,J ＝8.0Hz,1H),7.96-7.91(m,3H),7.83(d,J＝8.0Hz,1H),7.76(d,J＝4.0Hz,1H),7.60(t,J＝8.0Hz,1H), 7.43(s,1H),7.20(d,J＝8.0Hz,2H),4.95(s,2H).

¹³C NMR(101MHz,DMSO)δ215.38,176.88,166.77,161.68,157.86,153.67,147.43,140.73,136.50, 134.86,129.92,129.87,129.55,129.45,129.02,128.71,128.10,128.07,126.16,124.84,124.55,118.66, 116.28,115.86,67.54.

LC-MSm/z:571.1[M+H]⁺.

Synthetic example 10: synthesis of FHS-2

Adding ACS81(0.267g, 1.5mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.57g, 1.5mmol) and triethylamine (0.167g, 1.6mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-trifluoromethylanilino) -6-aminoquinazoline (0.252g, 0.8mmol) (4a), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum, and separating by silica gel column chromatography to obtain a final beige solid product 0.06g, with the yield of 16%, Mp:107 and 108 ℃.

¹H NMR(400MHz,DMSO)δ10.37(s,1H),10.02(s,1H),8.76(s,1H),8.57 (s,1H),8.27-8.20(m,2H),7.82(dd,J＝8.0Hz,2H),7.60(d,J＝8.0Hz,1H),7.41(s,1H),5.90-5.80(m, 1H),5.19(dd,J＝16.0Hz,2H),3.43(d,J＝8.0Hz,2H),3.05(m,2H),2.51-2.50(m,2H).

¹³C NMR(101MHz,DMSO)δ169.88,157.82,149.53,147.12,140.82,137.33,134.03,129.91,128.96, 128.04,126.13,126.06,119.15,118.42,117.29,115.95,100.00,41.52,36.41,33.93.

LC-MSm/z:465.1[M+H]⁺.

Synthetic example 11: synthesis of XHS-1

Adding ACS-60(0.43g, 1.5mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.57g, 1.5mmol) and triethylamine (0.182g, 1.6mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-bromoanilino) -6-aminoquinazoline (0.472g, 1.5mmol) (4b), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water after the reaction is finished, stirring, performing suction filtration to obtain a gray solid, and performing silica gel column chromatography to separate a final light orange solid product, wherein the yield is 10%, and the Mp is 112 ion 113 ℃.

¹H NMR(400MHz,DMSO)δ10.55(s,1H),9.90(s,1H),8.77(s,1H),8.57(s,1H),8.17(s,1H),7.97(d,J ＝12.0Hz,1H),7.87(d,J＝8.0Hz,2H),7.79(d,J＝8.0Hz,2H),7.72(s,1H),7.30(d,J＝8.0Hz,1H),7.25 (s,1H),7.19(d,J＝8.0Hz,2H),4.93(s,2H).

¹³C NMR(101MHz,DMSO)δ215.34,173.94,166.71,161.66,157.80,153.69,147.40,141.55,136.42, 134.83,130.63,129.39,128.68,126.34,125.56,124.84,121.59,121.33,116.26,116.07,115.88,67.54.

LC-MSm/z:583.0[M+H]⁺.

Synthetic example 12: synthesis of XHS-2

Adding ACS81(0.267g, 1.5mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.57g, 1.5mmol) and triethylamine (0.167g, 1.6mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-bromoanilino) -6-aminoquinazoline (0.252g, 0.8mmol) (4b), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum, and separating by silica gel column chromatography to obtain a final beige solid product 0.06g, with the yield of 16%, Mp:108 and 109 ℃.

¹H NMR(400MHz,DMSO)δ10.35(s,1H),9.88(s,1H),8.71(s,1H),8.57(d,J＝4.0Hz,H),8.17(t,J＝ 4.0Hz,1H),7.87(d,J＝8.0Hz,2H),7.78(dd,J＝4.0Hz,1H),7.32-7.25(m,2H),5.90-5.80(m,1H),5.18 (dd,J＝16.0Hz,2H),3.43(d,J＝8.0Hz,2H),3.04(t,J＝8.0Hz,2H),2.51-2.50(m,2H).

¹³C NMR(101MHz,DMSO)δ169.91,157.78,153.47,147.06,141.61,137.26,134.02,130.73,128.90, 126.37,124.82,121.59,121.37,119.17,115.92,100.00,41.50,36.39,33.93.

LC-MSm/z:476.0[M+H]⁺.

Synthetic example 13: synthesis of LHS-1

Adding ACS-60(0.313g, 1.1mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.418g, 1.1mmol) and triethylamine (0.122g, 1.2mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-chloro-4-fluoroanilino) -6-aminoquinazoline (0.317g, 1.1mmol) (4c), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water after the reaction is finished, stirring, performing suction filtration to obtain a gray solid, and performing silica gel column chromatography to separate a final light orange solid product 0.095g, wherein the yield is 15%, and the Mp:115-116 ℃.

¹H NMR(400MHz,CDCl₃)δ8.83(s,1H),8.76(s,1H),8.53(s,1H),7.98(d,J＝12.0Hz,2H),7.75-7.70 (m,3H),7.63(d,J＝8.0Hz,1H),7.57(d,J＝8.0Hz,1H),7.43(s,1H),7.22(d,J＝8.0Hz,1H),7.19-7.16 (m,2H),4.79(s,2H).

¹³C NMR(101MHz,DMSO)δ215.28,172.40,166.73,161.61,136.80,134.79,129.29,128.60,128.19, 124.85,124.74,119.53,119.35,116.88,116.66,116.22,116.02,113.08,100.00,67.49.

LC-MSm/z:556.0[M+H]⁺.

Synthesis example 14: synthesis of LHS-2

Adding ACS81(0.196g, 1.1mmol) and DMF10mL into a single-neck flask, stirring to dissolve, adding HATU (0.418g, 1.1mmol) and triethylamine (0.122g, 1.2mmol), stirring and activating at room temperature for 20min, slowly adding 4- (3-chloro-4-fluoroanilino) -6-aminoquinazoline (0.317g, 1.1mmol) (4c), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction liquid into 100mL of ice water, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum to obtain a beige solid, separating by silica gel column chromatography to obtain the final beige solid product of 0.084g, yield of 17%, Mp of 121 ℃.

¹H NMR(400MHz,DMSO)δ10.35(s,1H),9.90(s,1H),8.71(s,1H),8.54(s,1H),8.13(dd,J＝4.0Hz, 1H),7.86-7.75(m,3H),7.38(s,1H),5.90-5.80(m,1H),5.18(dd,J＝16.0Hz,2H),3.43(d,J＝8.0Hz,2H), 3.04(t,J＝8.0Hz,2H),2.51-2.50(m,2H).

¹³C NMR(101MHz,DMSO)δ169.86,157.79,153.43,152.59,147.03,137.27,137.13,134.02,128.91, 124.26,123.12,119.32,119.14,116.95,115.82,112.01,41.53,36.40,33.95.

LC-MSm/z:450.1[M+H]⁺.

Study of biological Activity

Preliminary screening for in vitro Activity of Compounds

Proliferation inhibition experiment of adherent cells-SRB method: to examine the possible biological activity of the synthesized compounds, cells in logarithmic growth phase were plated in 96-well plates (200. mu.L/well) in certain numbers, incubated for 24h to adhere to the walls and then dosed. Each drug concentration is provided with 2 multiple wells, and corresponding zero setting wells and blank control are arranged. After 72h of drug action, the adherent cells were added with 50% TCA (50. mu.L/well), fixed at 4 ℃ for 1h, the fixative was poured off, washed 5 times with distilled water, and naturally dried. Add 100. mu.L of 4mg/mL SRB to each well, stain at room temperature for 15min, discard, wash with 1% glacial acetic acid for 5 times, and dry naturally. Finally, 150. mu.L of 10mM Tris solution was added to each well, shaken well, and OD was measured at 565nm using a variable wavelength microplate reader (VERSAmaxTM, Molecular Device). The cell growth inhibition rate was calculated by the formula.

Inhibition ratio (%) - (OD value)_{Control well}OD value_{Medicine feeding hole}) OD value_{Control well}×100％

Calculating half inhibitory concentration IC by LOGIT method according to each concentration inhibition rate₅₀. The experiment was repeated 3 more times and the data are expressed as mean ± SD.

Preliminary screening of the compounds

Cells in logarithmic growth phase were plated in 96-well plates (200. mu.L/well) in a certain number, and cultured for 24h to adhere to the walls and then dosed with drugs. After 72 hours of drug action, the OD values were measured and the average value was calculated to calculate the cell proliferation inhibition rate (Table 3).

TABLE 3 growth inhibition ratio of each compound

a. The final concentration of the detection target compound was 20. mu.M. The positive control drugs MHS-1 and MHS-3 have concentrations of 45. mu.M and 55. mu.M, respectively.

b. The compound concentration was 10. mu.M. c. The concentration of the compound was 50. mu.M.

TABLE 4 IC of the Compounds on HepG2 cell line₅₀Value of

(mean±SD，n＝3)

Target compound release H₂Determination of S level

By means of H₂H released by fluorescent probe technology of S detection on target compound₂And S, measuring.

Preparing a probe solution: NIR-HS was dissolved in acetone to prepare a 1mM stock solution, which was diluted to 200. mu.M solution for use, and the probe solution was stored in low temperature and protected from light.

Na₂Preparing an S stock solution: 5mg EDTA was dissolved in 10mL double distilled water, and nitrogen was introduced into the solution for 15 min. Under the protection of nitrogen, 48mg of Na₂S·9H₂O dissolved in the solution to give 20mM Na₂S stock solution, which is diluted into 100 mu M solution for standby and needs to be prepared for use.

Preparation of PBS: potassium dihydrogen phosphate (KH)₂PO₄)0.24g of disodium hydrogen phosphate (Na)₂HPO₄)1.44g, sodium chloride (NaCl)8.0g, potassium chloride (KCl)0.2g, and water to 1000 mL. pH 7.4, requires low temperature storage.

Preparation of QHS-6 Compound: QHS-6 was dissolved in acetone to prepare a 2.5mM stock solution, which was diluted to 1.25mM, 625. mu.M, 100. mu.M, and 50. mu.M solutions for future use, and stored at low temperature.

Formulation of QHS-13 Donor Compounds: QHS-13 was dissolved in acetone to prepare a 5mM stock solution, which was diluted to 1mM, 500. mu.M, 100. mu.M, and 50. mu.M solutions for future use, and stored at low temperature.

Preparation of LHS-1 donor Compound: LHS-1 was dissolved in acetone to prepare a 2.5mM stock solution, which was diluted to 1.25mM, 625. mu.M, 100. mu.M, 50. mu.M solutions for future use and stored at low temperature.

3.1.2.3 qualitative determination of H released by target Compound₂S

H is to be₂The S fluorescent probe NIR-HS (final concentration of 10 μ M) was incubated with the target compound (0 μ M,5 μ M, 10 μ M,50 μ M, 100 μ M and 200 μ M) in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃). Thereafter, the fluorescence emission spectrum (. lamda.) was measured with an F4600 fluorescence spectrophotometer_ex＝670nm，λ_em723nm), at least three replicates per concentration were tested at least 3 times.

Quantitative determination of H released by target Compounds₂S

(1) Establishment of a standard curve:

h is to be₂NIR-HS (final concentration of 10. mu.M) and Na as S fluorescent probes₂S (0 μ M,2 μ M,4 μ M,6 μ M,8 μ M) was incubated in PBS buffer (PBS: acetone ═ 2:3) for 3h (37 ℃). Thereafter, the fluorescence emission spectrum (. lamda.) was measured with an F4600 fluorescence spectrophotometer_ex＝670nm，λ_em723nm) and based on the fluorescence intensity, a standard curve was made. There were at least three replicates per concentration, measured at least 3 times.

(2)H₂Quantitative determination of S release:

h is to be₂The S fluorescent probe NIR-HS (final concentration of 10 μ M) and the target compound (20 μ M) were incubated in PBS buffer (PBS: acetone ═ 2:3) for 0min, 5min, 30min, 60min, 90min, 120min, 150min, 180min (37 ℃) respectively. Thereafter, the fluorescence emission spectrum (. lamda.) was measured_ex＝670nm，λ_em＝723nm)，

According to Na₂S Standard Curve, converted to H Release from each Donor Compound₂The amount of S. At least three replicates were tested at each time point for at least 3 times.

H₂Measurement of S level

1. Target compound release H₂Qualitative determination of S

Selecting compounds with relatively good activity (QHS-6, QHS-13 and L) from QHS-a series, QHS-b series and LHS-a series of compoundsHS-1), determination of its H₂And (4) releasing S.

The experimental results show that: after incubation of compound QHS-6(10,50,100, 200. mu.M) with fluorescent probe NIR-HS (10. mu.M), a significant fluorescence response was produced with a significant increase in fluorescence intensity (10. mu.M, 33.77 + -1.55; 50. mu.M, 50.15 + -2.10; 100. mu.M, 66.08 + -3.59; 200. mu.M, 97.27 + -3.27) compared to no donor (0. mu.M, 29.65 + -1.42) ((0. mu.M, 29.65 + -1.42))^**P<0.01) (fig. 1), and the fluorescence intensity gradually increased with increasing compound concentration. This indicates that the compound QHS-6 is capable of releasing H in PBS buffer₂And S. The results are shown in FIG. 1.

The experimental results show that: after incubation of compound QHS-13(10,50,100, 200. mu.M) with fluorescent probe NIR-HS (10. mu.M), a significant fluorescence response was generated with a significant increase in fluorescence intensity (10. mu.M, 48.41 + -5.95; 50. mu.M, 66.58 + -6.34; 100. mu.M, 84.95 + -5.48; 200. mu.M, 120.39 + -7.21) compared to the absence of donor (0. mu.M, 36.78 + -1.29) ((0. mu.M, 36.78 + -1.29))^**P<0.01) (fig. 2), and the fluorescence intensity gradually increased with increasing compound concentration. This indicates that the compound QHS-13 is capable of releasing H in PBS buffer₂S。

The experimental results show that: after incubation of compound LHS-1(10,50,100, 200. mu.M) with fluorescent probe NIR-HS (10. mu.M), a significant fluorescence response was produced with a significant increase in fluorescence intensity (10. mu.M, 41.10 + -2.65; 50. mu.M, 83.35 + -2.83; 100. mu.M, 116.11 + -12.59; 200. mu.M, 204.02 + -14.38) compared to no donor (0. mu.M, 29.04 + -1.08) ((0. mu.M, 29.04 + -1.08))^*P<0.05,^**P<0.01) (fig. 3), and the fluorescence intensity gradually increased with increasing compound concentration. This indicates that LHS-1, a compound capable of releasing H in PBS buffer₂S。

2. Target compound release H₂Quantitative determination of S

Fluorescence intensity and Na of probe NIR-HS₂The standard curve of S concentration (0,2,4,6, 8. mu.M) is shown in FIG. 4, and shows a good linear relationship (0. mu.M, 1.0. + -. 0.0; 2. mu.M, 2.05. + -. 0.045; 4. mu.M, 3.45. + -. 0.24; 6. mu.M, 4.78. + -. 0.39; 8. mu.M, 5.78. + -. 0.23).

Incubating the compounds (QHS-6, QHS-13, LHS-1, 20 μ M) with fluorescent probe NIR-HS, detecting the fluorescence intensity every 30min, and then, according to a standard curve, measuring the fluorescence intensityConversion of the value into H₂Concentration of S, i.e. H₂S concentration-time curve.

The experimental results show that: the compounds QHS-6, QHS-13 and LHS-1 can release H₂And S. H of compound QHS-13₂The S release is obviously more than that of the compounds QHS-6 and LHS-1. H of compound QHS-13 within 1H₂S release speed is high, and H is obtained after 2H₂The release of S tends to be smooth; within 2H, H of the compound LHS-1₂S is released slowly, and H is released after 2H₂S, accelerating the speed; the compound QHS-6 releases H in 0-3H₂The S speed was slower (table 5, fig. 5).

TABLE 5 liberated H of the test compounds₂Change in S concentration

(mean±SD，n＝3)。

Claims (3)

1. A compound or a pharmaceutically acceptable salt thereof,

2. a pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, together with pharmaceutically acceptable excipients.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating liver cancer.

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