A benzene sulfonamide derivative, and a preparation method and application thereof

Abstract

The application discloses a benzene sulfonamide derivative, and a structural formula of the derivative is as follows: The synthesis process of the application is simple, does not need anhydrous oxygen-free condition, and ingeniously combines microwave conditions in individual reactions, so that the yield of part of reactions is high and the time is short. Activity research shows that the compound has the activity of inhibiting tumor cell proliferation and migration, and can obviously inhibit the growth of tumors in tumor-bearing mice at a dose of 10 mg / kg; in addition, the compound also has the activities of reducing blood sugar and reducing blood fat. Therefore, the compound has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of compound synthesis and pharmaceutical application technology, and in particular to a benzenesulfonamide derivative, its preparation method and application. Background Technology

[0002] Benzenesulfonamides are a class of compounds containing a benzene ring and a sulfonamide functional group. In 1932, German scientists discovered that Prontosil had an inhibitory effect on Streptococcus pneumoniae, with sulfonamide being the active component, thus opening the door to sulfonamide antibacterial drugs. With the emergence of antibiotic overuse and the increasing prominence of drug resistance, antibiotic dosages have increased, efficacy has decreased, and other adverse reactions have occurred, limiting the development and application of antibiotics. Further research into the structure-activity relationship and mechanism of action of sulfonamide compounds has revealed that, in addition to antibacterial activity, these compounds also possess antitumor, antidepressant, anti-inflammatory, antidiabetic, and carbonic anhydrase-inhibiting biological activities. Among these, the antitumor activity of sulfonamide compounds has attracted considerable attention.

[0003] Studies have shown that sulfonamide compounds can reduce cancer cell proliferation and induce apoptosis. For example, Dubois et al. found that sulfonamide carbonic anhydrase IX inhibitors, when used in combination with radiotherapy, can effectively inhibit HT-29 cell activity. Hao Mingyue et al. synthesized a novel sulfonamide tumor inhibitor, investigated its effect on apoptosis in cervical cancer HeLa cells, and further studied its mechanism of action. Yin Luo et al. synthesized and evaluated the anti-tubulin polymerization activity and in vitro cell activity of novel sulfonamide derivatives with cinnamic acid acyl groups. This series of compounds showed effective microtubule polymerization inhibition and anti-proliferation activity against MCF-7 cells. Owa et al. reported a class of novel sulfonamide antitumor compounds with indigo as a lead compound, which can reversibly bind to the colchicine binding site on β-tubulin. Sun Hua et al. reported that 1,4-naphthoquinone derivatives have good inhibitory activity against human colon cancer cells, and their derivative structures contain sulfonamide groups.

[0004] Through searching, the following patent publications were found that are related to this invention's patent application:

[0005] 1. A 1,4-naphthoquinone derivative, its preparation method and application, application number 201711376664.8, authorized in January 2020. The main research content disclosed in this patent is a class of 1,4-naphthoquinone derivatives, some of which contain sulfonamide fragments. These compounds have anti-colon cancer and α-glucosidase inhibitory activity. The drawback of this patent is that the compound activity and solubility need to be further improved. This patent is based on our group's previous research, and its scope of protection does not cover the content of this patent. The content of this patent is a novel structure with more ideal activity and solubility, discovered after the synthesis and activity evaluation of a large number of compounds.

[0006] 2. A sulfonamide derivative, its preparation method and application, application number 202010688883.5. The main research content disclosed in this patent is a class of sulfonamide derivative compounds with anti-colon cancer and α-glucosidase inhibitory activity. This patent is also the basis for our preliminary research project. Based on patent 1 (201711376664.8), it modifies the structure of the 1,4-naphthoquinone core to obtain a series of novel derivative structures. The drawback of this patent is that the compound synthesis yield is low and the compound activity needs to be further improved. The content of this patent is a completely new structure discovered through extensive experimental exploration. In addition to retaining the benzenesulfonamide functional group, it also modifies multiple pharmacophores. The scope of protection of patent 2 does not cover the content of this patent.

[0007] By comparison, the present invention is fundamentally different from the aforementioned disclosed patents. First, there is a structural difference, and the present patent is not within the scope of protection of the aforementioned patents. Second, the aforementioned two patents have problems such as insufficient activity, low synthesis yield, and poor water solubility. Therefore, the aforementioned patent problems are the motivation for the technical solution of the present invention. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems existing in the prior art and to provide a benzenesulfonamide derivative, its preparation method and application.

[0009] The technical solution adopted by this invention to solve the technical problem is:

[0010] A benzenesulfonamide derivative, the structural formula of which is as follows:

[0011]

[0012] Among them, RNR' is and its hydrochloride salt, wherein n1 is equal to or not equal to n2, n1 = a natural number from 2 to 4, n2 = a natural number from 2 to 4, and R1 is an alkyl group;

[0013] Or, RNR' is and its hydrochloride salt, wherein n1 is equal to or not equal to n2, n1 = a natural number from 2 to 4, n2 = a natural number from 2 to 4, and m = a natural number from 1 to 3;

[0014] Or, RNR' is

[0015] Ar for Where R = H or halogen; or, Ar = Where R = H, methyl, trifluoromethyl, halogen; or, Ar = ... Where R = H, methyl, trifluoromethyl, halogen; or, Ar = ...

[0016] Further, R1 is methyl, ethyl, propyl or butyl.

[0017] The preparation method of the benzenesulfonamide derivatives described above follows the reaction route as follows:

[0018]

[0019] Among them, RNHR1 is

[0020] or, Among them, RNHR1 is

[0021] or, Among them, RNHR1 is

[0022] or,

[0023] or,

[0024] or,

[0025] or,

[0026] Furthermore, it includes the following steps:

[0027] The aliphatic amine raw material RNHR1 reacts with p-acetaminobenzenesulfonyl chloride to give VIII. Under alkaline conditions, the acetyl protecting group of the amino group is removed to give IX. IX reacts with a halogenated or trifluoromethanesulfonate (TfO)-substituted aryl group to give a benzenesulfonamide derivative. Microwave reaction conditions in some steps can increase the yield and shorten the reaction time. Then, product I is dissolved in methanol and hydrogen chloride gas is passed through it; the precipitated solid is the corresponding hydrochloride salt of product I.

[0028] The application of benzenesulfonamide derivatives as described above in the preparation of drugs for treating human colon cancer.

[0029] The application of benzenesulfonamide derivatives as described above in the preparation of antitumor drugs.

[0030] The application of benzenesulfonamide derivatives as described above in the preparation of drugs for treating disorders of glucose and lipid metabolism.

[0031] The application of benzenesulfonamide derivatives as described above in the preparation of lipid-lowering drugs.

[0032] The application of benzenesulfonamide derivatives as described above in the preparation of hypoglycemic drugs.

[0033] The beneficial effects achieved by this invention are:

[0034] 1. The benzenesulfonamide derivatives of this invention exhibit good anti-human colon cancer and liver cancer cell activity. These benzenesulfonamide derivatives also possess hypoglycemic and hypolipidemic activities, meaning they can alleviate comprehensive disorders of glucose and lipid metabolism.

[0035] 2. The reaction of the method of the present invention does not require anhydrous and oxygen-free operation, is simple to operate, and the raw materials and reagents are inexpensive and readily available, making it suitable for large-scale production and development.

[0036] 3. The key reaction steps in the method of this invention cleverly utilize microwave reaction conditions, which can improve the yield and shorten the reaction time.

[0037] 4. Some compounds of this invention are prepared as hydrochloride salts, which improves water solubility, improves lipid-water partition coefficient, and further enhances drug-like properties.

[0038] 5. Activity studies have shown that these compounds inhibit tumor cell proliferation and migration, and significantly inhibit tumor growth in tumor-bearing mice at a dose of 10 mg / kg. Furthermore, these compounds also exhibit hypoglycemic and hypolipidemic activities. Therefore, this indicates that these compounds have promising application prospects. Attached Figure Description

[0039] Figure 1 This is the 1H NMR spectrum of compound 1 in deuterated methanol in this invention;

[0040] Figure 2 This is the 1H NMR spectrum of compound 2 in deuterated dichloromethane in this invention;

[0041] Figure 3 This is the 1H NMR spectrum of compound 3 in deuterated dimethyl sulfoxide in this invention;

[0042] Figure 4This is the 1H NMR spectrum of compound 4 in deuterated chloroform in this invention;

[0043] Figure 5 This is the 1H NMR spectrum of compound 5 in deuterated methanol in this invention;

[0044] Figure 6 This is the 1H NMR spectrum of compound 6 in deuterated chloroform in this invention;

[0045] Figure 7 This is the 1H NMR spectrum of compound 7 in deuterated acetone in this invention;

[0046] Figure 8 This is the 1H NMR spectrum of compound 8 in deuterated chloroform in this invention;

[0047] Figure 9 This is the 1H NMR spectrum of compound 9 in deuterated chloroform in this invention;

[0048] Figure 10 This is the 1H NMR spectrum of compound 10 in deuterated dimethyl sulfoxide in this invention;

[0049] Figure 11 This is the 1H NMR spectrum of compound 11 in deuterated chloroform in this invention;

[0050] Figure 12 This is the 1H NMR spectrum of compound 12 in deuterated dimethyl sulfoxide in this invention;

[0051] Figure 13 This is the 1H NMR spectrum of compound 13 in deuterated dimethyl sulfoxide in this invention;

[0052] Figure 14 This is the 1H NMR spectrum of compound 14 in deuterated acetone in this invention;

[0053] Figure 15 This is the 1H NMR spectrum of compound 15 in deuterated trichloromethane in this invention;

[0054] Figure 16 This is the 1H NMR spectrum of compound 16 in deuterated chloroform in this invention;

[0055] Figure 17 This is the 1H NMR spectrum of compound 17 in deuterated chloroform in this invention;

[0056] Figure 18 This is the 1H NMR spectrum of compound 18 in deuterated chloroform in this invention;

[0057] Figure 19 This is the 1H NMR spectrum of compound 19 in deuterated chloroform in this invention;

[0058] Figure 20 This is a statistical analysis of the effect of compound 3a in this invention on the migration of SW480 and HCT116 cells; among them, compared with the model group, ***P<0.01, **P<0.05, *P<0.01;

[0059] Figure 21 The effects of compounds 3a and 3 in this invention on cellular glucose consumption were shown; among them, compared with the model group, ***P<0.01;

[0060] Figure 22 The effects of compounds 3a and 3 in this invention on cellular lipid content are shown; among them, compared with the model group, **P<0.05, *P<0.01.

[0061] Figure 23 This is a graph showing the effect of compound 3a in this invention on tumor volume changes in tumor-bearing mice; where, compared with the model group, ***P<0.01;

[0062] Figure 24 The image shows the inhibitory effect of different doses of compound 3a on the appearance of tumors in tumor-bearing mice. Detailed Implementation

[0063] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments. However, the scope of protection of the present invention is not limited to the scope represented by the embodiments.

[0064] Unless otherwise specified, all raw materials used in this invention are conventional commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the field. All substances used in this invention are of conventional usage quality.

[0065] A benzenesulfonamide derivative, the structural formula of which is as follows:

[0066]

[0067] Among them, RNR' is and its hydrochloride salt, wherein n1 is equal to or not equal to n2, n1 = a natural number from 2 to 4, n2 = a natural number from 2 to 4, and R1 is an alkyl group;

[0068] Or, RNR' is and its hydrochloride salt, wherein n1 is equal to or not equal to n2, n1 = a natural number from 2 to 4, n2 = a natural number from 2 to 4, and m = a natural number from 1 to 3;

[0069] Or, RNR' is

[0070] Ar for Where R = H or halogen; or, Ar = Where R = H, methyl, trifluoromethyl, halogen; or, Ar = ... Where R = H, methyl, trifluoromethyl, halogen; or, Ar = ...

[0071] Preferably, R1 is methyl, ethyl, propyl or butyl.

[0072] The preparation method of the benzenesulfonamide derivatives described above follows the reaction route as follows:

[0073]

[0074] Among them, RNHR1 is

[0075] or, Among them, RNHR1 is

[0076] or, Among them, RNHR1 is

[0077] or,

[0078] or,

[0079] or,

[0080] or,

[0081] Preferably, the steps include:

[0082] The aliphatic amine raw material RNHR1 reacts with p-acetaminobenzenesulfonyl chloride to give VIII. Under alkaline conditions, the acetyl protecting group of the amino group is removed to give IX. IX reacts with a halogenated or trifluoromethanesulfonate (TfO)-substituted aryl group to give a benzenesulfonamide derivative. Microwave reaction conditions in some steps can increase the yield and shorten the reaction time. Then, product I is dissolved in methanol and hydrogen chloride gas is passed through it; the precipitated solid is the corresponding hydrochloride salt of product I.

[0083] The application of benzenesulfonamide derivatives as described above in the preparation of drugs for treating human colon cancer.

[0084] The application of benzenesulfonamide derivatives as described above in the preparation of antitumor drugs.

[0085] The application of benzenesulfonamide derivatives as described above in the preparation of drugs for treating disorders of glucose and lipid metabolism.

[0086] The application of benzenesulfonamide derivatives as described above in the preparation of lipid-lowering drugs.

[0087] The application of benzenesulfonamide derivatives as described above in the preparation of hypoglycemic drugs.

[0088] Specifically, the relevant preparation and testing methods are as follows:

[0089] Example 1: Synthesis of VIII side chain intermediate

[0090] The specific reaction route is as follows:

[0091]

[0092] General synthetic method for intermediates VIII-a to VIII-i:

[0093] Under ice bath conditions, acetaminobenzenesulfonyl chloride (1.50 mmol) was dissolved in 10 mL of anhydrous dichloromethane. Different amine groups (1.00 mmol) were added with stirring, followed by triethylamine (2.00 mmol). The reaction was carried out at room temperature for 5–10 h. After the reaction was monitored by thin-layer chromatography to ensure completion, 50 mL of water was added, followed by three extractions with 50 mL of dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated. The crude product was purified by silica gel column chromatography to obtain intermediates VIII-a to VIII-i, as detailed below:

[0094] Synthesis of (1) (S)-N-(4-((3-(dimethylamino)pyrrolidone-1-yl)sulfonyl)phenyl)acetamide (VIII-a)

[0095] The amine starting material was (S)-3-dimethylaminopyrrolidine dihydrochloride. Following the general synthesis method described above, a white solid VIII-a was obtained with a yield of 91.46%. 1 H-NMR (400MHz, CDCl3) δ7.83 (s, 1H), 7.76 (d, J = 8.4Hz, 2H), 7.70 (d, J = 8.8Hz, 2H), 3.48-3.52 (m, 1H), 3.34-3.40 (m, 1H), 3. 34-3.35(m,1H),2.95(t,J=9.0Hz,1H),2.59-2.67(m,1H),2.22(s,3H),2.17(s,6H),1.99-2.03(m,1H),1.60-1.67(m,1H).

[0096] (2) Synthesis of 4-(4-acetaminophenyl)sulfonyl)piperazine-1-carboxylic acid tert-butyl ester (VIII-b)

[0097] The amine starting material was mono-Boc piperazine. Following the general synthetic method described above, a white solid VIII-b was obtained with a yield of 95.64%. 1 H-NMR (400MHz, CDCl3) δ7.69 (s, 4H), 7.54 (s, 1H), 3.50 (t, J = 5.0Hz, 4H), 2.95 (t, J = 4.8Hz, 4H), 2.23 (s, 3H), 1.41 (s, 9H).

[0098] (3) Synthesis of N-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)acetamide (VIII-c)

[0099] The amine starting material was N-methylpiperazine. Following the general synthetic method described above, a white solid VIII-c was obtained with a yield of 96.86%. 1 H-NMR (400MHz, CDCl3) δ7.71(d,J=9.2Hz,2H),7.68(d,J=9.2Hz,2H),7.35(s,1H),3.03(s,4H),2.47(t,J=5.0Hz,4H),2.64(s,3H),2.23(s,3H).

[0100] (4) Synthesis of N-(4-((3,5-dimethylpiperidin-1-yl)sulfonyl)phenyl)acetamide (VIII-d)

[0101] The amine starting material was 3,5-dimethylpiperidine. Following the general synthetic method described above, a white solid VIII-d was obtained with a yield of 92.84%. 1 H-NMR (400MHz, CDCl3) δ7.71(d,J=8.8Hz,2H),7.67(d,J=9.2Hz,2H),7.41(s,1H),3.71(d,J=8.8Hz, 2H), 2.23 (s, 3H), 1.65-1.78 (m, 4H), 0.98 (d, J = 6.8Hz, 1H), 0.84 (d, J = 6.0Hz, 6H), 0.41-0.50 (m, 1H).

[0102] (5) Synthesis of N-(4-(aza-1-ylsulfonyl)phenyl)acetamide (VIII-e)

[0103] The amine starting material was cycloheximine. Following the general synthetic method described above, a white solid VIII-e was obtained in 90.85% yield.

[0104] (6) Synthesis of N-(4-((4-methyl-1,4-diaza-1-yl)sulfonyl)phenyl)acetamide (VIII-f)

[0105] The amine starting material was N-methylperiperazine. Following the general synthetic method described above, a white solid VIII-f was obtained with a yield of 86.31%. 1 H-NMR (400MHz, MeOD) δ7.82 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 9.2 Hz, 2H), 3.58 (s, 2H), 3.37-3.43 (m, 6H), 2.91 (s, 3H), 2.16 (s, 3H), 2.12-2.15 (m, 2H).

[0106] (7) Synthesis of N-(4-(azo-1-ylsulfonyl)phenyl)acetamide (VIII-g)

[0107] The amine starting material was cyclooctylimine. Following the general synthetic method described above, a white solid VIII-g was obtained with a yield of 89.48%. 1 H-NMR (400MHz, CDCl3) δ7.73(d,J=8.4Hz,2H),7.65(d,J=8.4Hz,2H),7.46(s,1H),3.05(t,J=5.0Hz,4H),2.22(s,3H),1.72(s,8H),1.63(s,4H).

[0108] (8) Synthesis of N-(4-(N-(quinolin-8-yl)aminosulfonyl)phenyl)acetamide (VIII-h)

[0109] The amine starting material was 8-aminoquinoline. Following the general synthetic method described above, a white solid VIII-h was obtained with a yield of 79.82%. 1 H-NMR (400MHz, CDCl3) δ9.21(s,1H),8.76(dd,J=1.2,1.6Hz,1H),8.09(dd,J=1.6,1.2Hz,1H),7.85( d,J=8.8Hz,2H),7.81(dd,J=1.6,2.0Hz,1H),7.50(d,J=8.4Hz,2H),7.40-7.47(m,3H),2.14(s,3H).

[0110] (9) Synthesis of N-(4-((4-phenylpiperazin-1-yl)sulfonyl)phenyl)acetamide (VIII-i)

[0111] The amine starting material was N-phenylpiperazine. Following the general synthetic method described above, a white solid VIII-i was obtained with a yield of 91.36%. 1H-NMR (400MHz, DMSO-d6) δ10.39(s,1H),7.84(d,J=8.8Hz,2H),7.70(d,J=8.8Hz,2H),7.19(t,J=7.8Hz,2H ), 6.90 (d, J = 8.4Hz, 2H), 6.79 (t, J = 7.4Hz, 1H), 3.19 (t, J = 4.6Hz, 4H), 2.98 (t, J = 4.6Hz, 4H), 2.09 (s, 3H).

[0112] (10) Synthesis of N-(4-((4-benzylpiperazin-1-yl)sulfonyl)phenyl)acetamide (VIII-j)

[0113] Mono-Boc piperazine (1117 mg, 6 mmol) was dissolved in 15 mL of acetonitrile, and then anhydrous Na₂CO₃ (795 mg, 7.5 mmol) was added. Benzyl bromide (855 mg, 5 mmol) was added while stirring, and the reaction was carried out at room temperature for 5 h. After the reaction was complete, 50 mL of water was added, followed by extraction three times with 50 mL of dichloromethane. The organic phases were combined, dried with anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to dryness. The intermediate was purified by silica gel column chromatography. This intermediate was dissolved in 5 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise under ice bath, and the reaction was carried out for 5 h. After the reaction was completed, excess trifluoroacetic acid and solvent were evaporated under reduced pressure. The product was then dissolved in 5 mL of dichloromethane. While stirring in an ice bath, p-acetylbenzenesulfonyl chloride (882 mg, 3.78 mmol) and triethylamine (1.1 mL, 7.56 mmol) were added. The reaction was carried out at room temperature. After the reaction was complete, 50 mL of water was added, and the mixture was extracted three times with 50 mL of dichloromethane. The organic phases were combined, dried with anhydrous Na2SO4, filtered to remove the drying agent, and the solvent was evaporated. The crude product was purified by column chromatography to give compound VIII-j in 76.81% as a white solid. 1 H-NMR (400MHz, CDCl3) δ7.66-7.71(m,4H),7.38(s,1H),7.22-7.28(m,4H),3.48(s,2H),3.01(s,4H),2.52(t,J=4.8Hz,4H),2.23(s,3H).

[0114] (11) Synthesis of N-(4-((4-cinnamylpiperazin-1-yl)sulfonyl)phenyl)acetamide (VIII-k)

[0115] Using cinnamyl bromide as a raw material, the same synthesis method as that used for compound VIII-j was employed to obtain white solid VIII-k with a yield of 79.66%. 1H-NMR (400MHz, DMSO-d6) δ10.40(s,1H),7.83(d,J=8.8Hz,2H),7.66(d,J=8.8Hz,2H),7.40(d,J=7.2Hz,2H),7.30(t,J=7.4Hz,2 H),7.22(t,J=7.2Hz,1H),6.50(d,J=16Hz,1H),6.15-6.22(m,1H),3.08(d,J=5.4Hz,2H),2.88(s,4H),2.46(s,4H),2.10(s,3H).

[0116] (12) Synthesis of N-(4-((3-(dimethylamino)azacyclobut-1-yl)sulfonyl)phenyl)acetamide (VIII-l)

[0117] 1-tert-Butoxycarbonyl-3-azacyclobutanone (200 mg, 1.17 mmol) was dissolved in 5 mL of anhydrous dichloromethane. While stirring, dimethylamine hydrochloride (143 mg, 1.75 mmol) and triethylamine (0.4 mL, 2.95 mmol) were added. The mixture was stirred at room temperature for 3–5 h, followed by the addition of sodium cyanoborohydride (1.75 mmol). The reaction was allowed to proceed for 9.5 h at room temperature. After the reaction was complete, the reaction mixture was poured into 50 mL of water and extracted three times with 50 mL of dichloromethane:methanol = 10:1 (v / v). The combined organic phases were dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to dryness. The solution was then dissolved in 5 mL of anhydrous dichloromethane, and 1 mL of trifluoroacetic acid was added dropwise. The mixture was stirred for 3 h. After the reaction was complete, excess trifluoroacetic acid and solvent were removed by vacuum evaporation. The product was then dissolved in 5 mL of dichloromethane. Acetylbenzenesulfonyl chloride (409 mg, 1.75 mmol) and triethylamine (0.5 mL, 3.51 mmol) were added under stirring in an ice bath. The reaction was carried out at room temperature. After the reaction was complete, 50 mL of water was added, and the mixture was extracted three times with 50 mL of dichloromethane. The organic phases were combined and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the solvent was evaporated. The crude product was purified by silica gel column chromatography to give compound VIII-l in 64.26% yield as a white solid. 1 H-NMR (400MHz, CDCl3) δ7.78(d,J=8.6Hz,2H),7.73(d,J=8.4Hz,2H),7.47(s,1H),3.81( t, J=7.6Hz, 2H), 3.55 (d, J=7.2Hz, 2H), 3.03 (d, J=6.6Hz, 1H), 2.34 (s, 3H), 2.03 (s, 6H).

[0118] Example 2 Synthesis of Compound 1

[0119] The specific synthesis route is as follows:

[0120]

[0121] The above-mentioned side-chain intermediate VIII-l (1.00 mmol) was dissolved in 10 mL of methanol, and 2 mL of sodium hydroxide aqueous solution (5 M) was added. The mixture was heated to reflux until the reaction was complete. The mixture was then extracted three times with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to obtain the crude product. The crude product (51 mg, 0.20 mmol) was dissolved in 2 mL of ethanol, and 2,3-dichloro-1,4-naphthoquinone (136 mg, 0.60 mmol) was added. The mixture was reacted in a microwave reactor at 70 °C for 4 h. The solvent was evaporated to dryness, and the product was purified by column chromatography to obtain product 1. Yield: 89%. The structural parameters of compound 1 are as follows: 1 H-NMR (400MHz, MeOD) δ8.15(t,J=7.8Hz,2H),7.85(s,2H),7.82(d,J=8.8Hz,2H),7.28(d,J=8.4Hz,2H),4.01-4.06(m,5H),2.82(s,6H); 13 C-NMR (100MHz, CDCl3) δ179.7,177.7,144.5,142.2,134.5,133.4,132.0,13 0.6,128.6,126.6,126.4,122.2,119.6,54.2,52.0,39.8.HRMS(ESI-TOF)m / z calcd.forC 21 H 21 ClN3O4S[M+H] + :446.0941,found446.0941.

[0122] Compound 1 is dissolved in methanol, and hydrogen chloride gas is passed through it. The solid that precipitates out of the system is the corresponding hydrochloride 1a.

[0123] Example 3 Synthesis of Compound 2

[0124]

[0125] Compound 2 was synthesized using the same method as compound 1, with side chain VIII-a (1.00 mmol) selected, yield: 88%; structural parameters of compound 2 are as follows: 1H-NMR (400MHz, CDCl3) δ8.21-8.23(m,1H),8.14-8.17(m,1H),7.80-7.83(m,3H),7.77(s,1H),7.71-7.76(m,1H),7.14(d,J=8.8Hz,2H),3.53-3 .57(m,1H),3.37-3.43(m,1H),3.26-3.32(m,1H),2.98(t,J=9.2Hz,1H) ,2.58-2.66(m,1H),2.17(s,6H),2.00-2.05(m,1H),1.64-1.72(m,1H); 13 C-NMR (100MHz, CDCl3) δ180.2,177.4,141.4,140.7,135.3,133.5,132.3,132.3,12 9.8,127.4,127.2,127.2,123.0,118.1,65.1,51.8,47.0,29.9.HRMS(ESI-TOF)m / z calcd.for C 22 H 23 ClN3O4S[M+H] + :446.1098,found446.1097.

[0126] Compound 2 was dissolved in methanol, and hydrogen chloride gas was passed through it. The solid that precipitated out of the system was the corresponding hydrochloride 2a.

[0127] Example 4 Synthesis of Compound 3

[0128]

[0129] The synthesis method of compound 3 is the same as that of compound 1, with side chain VIII-c selected, yield: 84%; the structural parameters of compound 3 are as follows: 1 H-NMR (400MHz, DMSO-d6) δ9.62 (s, 1H), 8.07 (dd, J = 1.2, 1.2Hz, 2H), 7.87-7.91 (m, 1H), 7.83-7. 87(m,1H),7.64(d,J=8.4Hz,2H),7.29(d,J=8.8Hz,2H),2.89(s,4H),2.37(s,4H),2.16(s,3H); 13C-NMR (100MHz, DMSO-d6) δ180.4,177.5,144.3,143.1,135.2,134.1,132.2,131.0,129.1,128.2,127.1,126.7,122.7,119.6,53.9,46.2,45.7.

[0130] Compound 3 was dissolved in methanol, and hydrogen chloride gas was passed through it. The solid that precipitated out of the system was the corresponding hydrochloride 3a.

[0131] Example 5: Synthesis of Compound 4

[0132]

[0133] The synthesis method of compound 4 is the same as that of compound 1, with side chain VIII-d selected, yield: 85%; the structural parameters of compound 4 are as follows: 1 H-NMR (400MHz, CDCl3) δ8.22(d,J=8.0Hz,1H),8.16(d,J=7.6Hz,1H),7.82(t,7.6Hz,1H),7.74(d,J=8.4Hz,2H),7.73(s,1H),7.69 (s,1H),7.14(d,J=8.8Hz,2H),3.75(d,J=7.6Hz,2H),1.75(m,4H),0.99(d,J=5.8Hz,1H),0.86(d,J=6.0Hz,6H),0.86-0.99(m,1H); 13 C-NMR (100MHz, CDCl3) δ180.2,177.4,141.3,140.8,135.3,132.4,132.3,129.8 ,128.1,127.4,127.2,123.0,118.1,52.9,41.4,30.9,19.0.HRMS(ESI-TOF)m / z calcd.for C 23 H 24 ClN2O4S[M+H] + :459.1145,found 4459.1142.

[0134] Example 6 Synthesis of Compound 5

[0135]

[0136] The synthesis method of compound 5 is the same as that of compound 1, with side chain VIII-f selected, yield: 95%; the structural parameters of compound 4 are as follows: 1H-NMR(400MHz,MeOD)δ8.12-8.16(m,2H),7.85-7.87(m,1H),7.81-7.83(m,1H),7.79(d,J=8 .4Hz,2H),7.24(d,J=8.8Hz,1H),3.61(s,2H),3.41-3.45(m,6H),2.94(s,3H),2.16(s,2H); 13 C-NMR (100MHz, MeOD) δ181.1,179.0,145.0,143.7,135.9,134.6,133.7,133.4,131.9,128. 6,127.9,127.7,124.0,120.0,59.1,56.3,48.3,47.6,45.0,44.6,25.9.HRMS(ESI-TOF)m / z calcd.for C 22 H 23 ClN3O4S[M+H] + :440.1098,found 440.1098.

[0137] Example 7 Synthesis of Compound 6

[0138]

[0139] The synthesis method of compound 6 is the same as that of compound 1, with side chain VIII-g selected, yield: 56%; the structural parameters of compound 6 are as follows: 1 H-NMR (400MHz, CDCl3) δ8.22(d,J=8.0Hz,1H),8.15(d,J=7.6Hz,1H),7.82(t,J=8.4Hz,2H),7.73(d,J= 7.6Hz,1H),7.68(s,1H),7.12(d,J=8.4Hz,2H),3.30(t,J=5.4Hz,4H),1.73-1.76(m,8H),1.62(s,4H); 13 C-NMR (100MHz, CDCl3) δ180.3,177.4,141.0,140.8,135.3,134.1,133.5,132.3,12 9.8,127.8,127.4,127.2,123.0,117.9,50.2,27.2,25.9,25.0.HRMS(ESI-TOF)m / z calcd.for C 24 H 26 ClN2O4S[M+H] + :473.1302,found473.1298.

[0140] Example 8: Synthesis of compound 7.

[0141]

[0142] The synthesis method of compound 7 is the same as that of compound 1, with side chain VIII-i selected, yield: 56%; the structural parameters of compound 7 are as follows: 1 H-NMR (400MHz, Acetone-d6) δ8.80(s,1H),8.13(t,J=7.6Hz,2H),7.89-7.93(m,1H),7.84-7.87(m,1H),7.79(d,J=8.8Hz,2H),7 .41(d,J=8.8Hz,2H),7.20-7.24(m,2H),6.95(d,J=8.0Hz,2H),6.81-6.84(m,1H),3.28(t,J=4.8Hz,4H),3.15(t,J=5.0Hz,4H); 13 C-NMR (100MHz, CDCl3) δ180.3,177.4,150.7,141.8,140.7,135.3,133.6,132.3,131.1,129 .9,129.3,128.5,127.5,127.3,122.8,121.0,118.6,117.0,48.2,46.2.HRMS(ESI-TOF)m / z calcd.for C 26 H 23 ClN3O4S[M+H] + :508.1098,found 508.1092.

[0143] Example 9: Synthesis of Compound 8

[0144]

[0145] The synthesis method of compound 8 is the same as that of compound 1, with side chain VIII-j selected, yield: 53%; the structural parameters of compound 8 are as follows: 1H-NMR (400MHz, CDCl3) δ8.23(d,J=7.6Hz,1H),8.16(d,J=7.6Hz,1H),7.80-7.84(m,1H),7.75-7.77(m,1H),7.72(d,J=8.4Hz ,2H),7.69(s,1H),7.28-7.29(m,2H),7.24-7.26(m,1H),7.12(d,J=8.4Hz,2H,.350(s,2H),3.07(s,4H),2.52-2.55(m,4H); 13 C-NMR (100MHz, CDCl3) δ141.5,140.7,137.4,135.3,133.5,132.3,131.4,129.8,12 9.1,128.4,127.4,127.4,127.2,122.8,118.4,62.6,52.1,46.1.HRMS(ESI-TOF)m / z calcd.for C 27 H 25 ClN3O4S[M+H] + :522.1254,found 522.1250.

[0146] Example 10 Synthesis of Compound 9

[0147]

[0148] The synthesis method of compound 9 is the same as that of compound 1, with side chain VIII-k selected, yield: 66%; the structural parameters of compound 9 are as follows: 1 H-NMR (400MHz, CDCl3) δ8.22(d,J=7.6Hz,1H),8.16(d,J=7.6Hz,1H),7.81(t,J=7.4Hz,1H),7.75(t,J=8.2Hz,3H),7.69(s,1H),732-7.36(m, 2H),7.26-7.30(m,2H),7.23(s,1H),7.12(d,J=8.4Hz,2H),6.52(d,J= 16.0Hz,1H),6.15-6.19(m,1H),3.16(s,2H),3.12(m,4H),2.61(m,4H); 13C-NMR (100MHz, CDCl3) δ180.2,177.4,141.6,140.6,136.5,135.3,133.5,132.3,131.1,129.8 ,128.6,128.4,127.8,127.4,127.2,126.4,122.7,118.5,60.6,52.1,46.0.HRMS(ESI-TOF)m / z calcd.for C 29 H 27 ClN3O4S[M+H] + :548.1411,found 548.1408.

[0149] Example 11 Synthesis of Compound 10

[0150] The specific preparation method is as follows:

[0151]

[0152] The side chain VIII-e (1.00 mmol) was dissolved in 10 mL of methanol, and 2 mL of sodium hydroxide aqueous solution (5 M) was added. The mixture was heated to reflux until complete. After extraction three times with 50 mL of dichloromethane, the organic phases were combined and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the solvent was evaporated to obtain the crude product. The crude product (0.20 mmol) was dissolved in 2 mL of THF / H₂O (3:1 v / v). Under ice bath conditions, 2,4-dichloroquinazoline (0.30 mmol) and sodium acetate (0.60 mmol) were added. The mixture was reacted in a sealed tube at 100 °C until complete. After cooling to room temperature, 20 mL of water was added, and the mixture was extracted three times with 50 mL of dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated to dryness. The product was purified by column chromatography to obtain final product 10. Yield: 78%; Structural parameters of product 10: 1 H-NMR (400MHz, DMSO-d6) δ10.43(s,1H),8.63(d,J=8.4Hz,1H),8.11(d,J=8.4Hz,2H),7.94(t,J=7.6Hz,1H),7.8 5(d,J=8.8Hz,2H),7.78(d,J=8.4Hz,2H),7.71(t,J=7.4Hz,1H),3.24(t,J=5.8Hz,4H),1.65(s,4H),1.52(s,4H); 13C-NMR(100MHz,DMSO-d6)δ159.6,156.2,151.5,142.6,134.9,134.4,128.0,127.5,127.4,124.1,122.5,114.3,48.2,29.0,26.8.HRMS(ESI-TOF)m / z calcd.for C 20 H 22 ClN4O2S[M+H] + :417.1152,found 417.1151.

[0153] Example 12 Synthesis of Compound 11

[0154]

[0155] The synthesis method of Example 11 was the same as that of Compound 10 described above, using side chain VIII-g (1.00 mmol). Yield: 45%; Structural parameters of Compound 11: 1 H-NMR (400MHz, CDCl3) δ8.01(d,J=8.4Hz,1H),7.97(s,1H),7.92(d,J=8.8Hz,2H),7.88-7.89( m,2H),7.85(d,J=8.4Hz,2H),7.59-7.63(m.1H),3.10-3.13(m,4H),1.75(s,8H),1.67(s,4H); 13 C-NMR (100MHz, CDCl3) δ158.3,156.7,151.6,141.3,134.3,133.5,128.6,12 8.5,127.3,121.2,120.7,113.5,50.3,27.3,26.0,25.1.HRMS(ESI-TOF)m / z calcd.for C 22 H 26 ClN4O2S[M+H] + :445.1465,found445.1463.

[0156] Example 13 Synthesis of Compound 12

[0157] The synthesis route is as follows:

[0158]

[0159] The side chain VIII-e (1.00 mmol) was dissolved in 10 mL of methanol, and 2 mL of sodium hydroxide solution (5 M) was added. The mixture was heated to reflux until the reaction was complete. The mixture was then extracted three times with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to obtain the crude product. The crude product (0.50 mmol) was dissolved in 2 mL of LDMF, and 2-chloroquinazoline-4(3H)-one (1.0 mmol) was added. The mixture was reacted in a microwave reactor at 150 °C for 3 h. After cooling to room temperature, the solid was poured into 10 mL of ice water, collected, and purified by column chromatography to obtain product 12. Yield: 90%; Structural parameters of product 12: 1 H-NMR (400MHz, DMSO-d6) δ10.95(s,1H),9.15(s,1H),8.00(d,J=8.0Hz,1H),7.96(d,J=8.8Hz,2H),7.69-7. 71(m,1H),7.47(d,J=8.0Hz,1H),7.29(t,J=7.4Hz,1H),3.19-3.35(m,4H),1.63(s,4H),1.49-1.51(m,4H); 13 C-NMR(100MHz,DMSO-d6)δ162.0,149.9,147.3,143.3,135.1,132.2,128.4,126.4,126.0,124.2,119.2,48.2,29.0,26.8.HRMS(ESI-TOF)m / z calcd.for C 20 H 23 N4O3S[M+H] + :399.1491,found 399.1486.

[0160] Example 14 Synthesis of Compound 13

[0161]

[0162] The synthesis method of Example 14 is the same as that of Compound 12 described above, using side chain VIII-g (1.00 mmol). Yield: 50%; Structural parameters of Compound 13: 1H-NMR (400MHz, DMSO-d6) δ10.95(s,1H),9.15(s,1H),7.99(d,J=8.4Hz,2H),7.98(d,J=8.8Hz,2H),7.75(d,J=8.8Hz,2H ),7.70(d,J=7.2Hz,1H),7.47(d,J=8.0Hz,1H),7.29(t,J=7.4Hz,1H),3.02(d,J=4.8Hz,4H),1.66(s,4H),1.59(s,8H); 13 C-NMR(100MHz,DMSO-d6)δ179.8,162.0,149.9,147.3,143.4,135.1,130.8, 128.7,126.4,126.0,124.2,119.2,5.0,27.2,26.0,25.0.HRMS(ESI-TOF)m / z calcd.for C 22 H 27 N4O3S[M+H] + :427.1804,found427.1802.

[0163] Example 15 Synthesis of Compound 14

[0164] The specific synthesis route is as follows:

[0165]

[0166] The side chain VIII-e (1.00 mmol) was dissolved in 10 mL of methanol, and 2 mL of sodium hydroxide aqueous solution (5 M) was added. The mixture was heated to reflux until the reaction was complete. The mixture was then extracted three times with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to obtain the crude product. The crude product (0.5 mmol) was dissolved in 2 mL of DMF, and 2,4-dichloro-5,6,7,8-tetrahydroquinazoline (1.0 mmol), palladium acetate (0.1 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (0.1 mmol), and cesium carbonate (1.0 mmol) were added. The mixture was reacted in a microwave reactor at 100 °C for 3 h. After cooling to room temperature, the solid was poured into 10 mL of ice water, collected, and purified by column chromatography to obtain product 14. Yield: 45%; Structural parameters: 1H-NMR (400MHz, Acetone-d6) δ8.28(s,1H),8.00(d,J=8.8Hz,2H),7.77(d,J=8.8Hz,2H),3.28(t,J=5.8Hz,4H ),2.70(t,J=6.0Hz,2H),2.65(t,J=6.2Hz,2H),1.84-1.90(m,4H),1.70(d,J=3.6Hz,4H),1.57-1.60(m,4H); 13 C-NMR (100MHz, Acetone-d6) δ167.3,160.6,157.1,143.9,134.8,128.6,121.6,114.1,48.9,32.7,27.6,22.6,22.5,18.9.

[0167] Example 16 Synthesis of Compound 15

[0168]

[0169] The synthesis method of Example 16 was the same as that of Compound 14 above, using side chain VIII-g (1.00 mmol). Yield: 45%; Structural parameters of Compound 15: 1 H-NMR (400MHz, CDCl3) δ7.78 (s, 4H), 6.63 (s, 1H), 3.09 (t, J = 5.0Hz, 4H), 2.80 (t, J = 6.0Hz, 2H),2.52(t,J=6.0Hz,2H),1.93-1.96(m,2H),1.85-1.89(m,2H),1.74(s,8H),1.65(s,4H); 13 C-NMR(100MHz, CDCl3)δ166.8,158.9,157.0,141.9,132.7,128.6,120.1,112.0,50.2,27.3,26.0,25.0,22.2,21.9,21.7.HRMS(ESI-TOF)m / zcalcd.for C 22 H 30 ClN4O2S[M+H] + :449.1778,found 449.1778.

[0170] Example 17 Synthesis of Compound 16

[0171]

[0172] The side chain VIII-e (1.00 mmol) was dissolved in 10 mL of methanol, and 2 mL of sodium hydroxide aqueous solution (5 M) was added. The mixture was heated to reflux until the reaction was complete. The mixture was then extracted three times with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered to remove the drying agent, and the solvent was evaporated to obtain the crude product. The crude product (0.5 mmol) was dissolved in 2 mL of DMF, and 1.0 mmol of 2-phenyl-7,8-dihydro-6H-thiopyran[3,2-d]pyrimidin-4-yltrifluoromethanesulfonate was added. The mixture was reacted at 100 °C for 3 h, cooled to room temperature, poured into 10 mL of ice water, and the precipitated solid was collected. The solid was purified by column chromatography to obtain the final product 16. Yield: 40%; Structural parameters of product 16: 1 H-NMR (400MHz, CDCl3) δ8.34(d,J=5.6Hz,2H),7.90(d,J=8.8Hz,2H),7.81(d,J=9.6Hz,2H),7.46(d,J=5.6Hz,3H),6.81(s ,1H),3.30(t,J=5.8Hz,4H),3.16(t,J=6.4Hz,2H),3.00(t,J=6.2Hz,2H),2.30(t,J=5.8Hz,2H),1.73(s,4H),1.59(s,4H); 13 C-NMR (100MHz, CDCl3) δ160.1,156.8,155.1,142.9,137.8,133.1,130.2,128.5,1 28.2,127.8,199.6,110.1,48.3,32.2,29.2,27.8,27.0,23.2.HRMS(ESI-TOF)m / z calcd.forC 25 H 29 N4O2S2[M+H] + :481.1732,found 481.1732.

[0173] Example 18 Synthesis of Compound 17

[0174]

[0175] The synthesis method of Example 18 was the same as that of Compound 16 described above, using side chain VIII-g (1.00 mmol). Yield: 40%; Structural parameters of Compound 17: 1H-NMR (400MHz, CDCl3) δ8.34-8.36(m,2H),7.91(d,J=8.4Hz,2H),7.82(d,J=8.8Hz,2H),7.47(d,J=6.0Hz,2H),7.46(s,1H),6. 81(s,1H),3.18(t,J=5.8Hz,2H),3.11(t,J=5.0Hz,2H),3.01(t,J=6.4Hz,2H),2.32(t,J=6.0Hz,2H),1.75(s,8H),1.66(s,4H); 13 C-NMR (100MHz, CDCl3) δ160.1,158.8,155.1,143.0,137.8,131.6,130.2,128.5, 127.8,119.5,110.1,50.2,32.2,27.8,27.3,26.0,25.0,23.2.HRMS(ESI-TOF)m / z calcd.for C 27 H 33 N4O2S2[M+H] + :509.2045,found 509.2045.

[0176] Example 19 Synthesis of Compound 18

[0177]

[0178] Example 19 was synthesized using the same method as compound 16 described above, but with side chain VIII-e (1.00 mmol) reacted with 2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-6H-thiopyran[3,2-d]pyrimidin-4-yltrifluoromethanesulfonate to give compound 18. Yield: 44%; Structural parameters of compound 18: 1 H-NMR (400MHz, CDCl3) δ8.45(d,J=8.0Hz,2H),7.81-7.88(m,4H),7.72(d,J=8.4Hz,2H),3.31(t,J=5.8Hz,2H),3.3 1(t,J=5.8Hz,4H),3.20(t,J=5.8Hz,2H),3.02(t,J=6.4Hz,2H),2.30-2.36(m,2H),1.74(s,4H),1.59-1.62(m,4H); 13C-NMR (100MHz, CDCl3) δ160.0,157.2,155.1,142.6,141.1,133.5,131.8,131.5,128.2,128.0,1 25.6,125.4,125.4,122.9,119.8,111.2,48.3,32.2,29.2,27.8,26.9,23.0.HRMS(ESI-TOF)m / z calcd.for C 26 H 28 F3N4O2S2[M+H] + :549.1606,found549.1606.

[0179] Example 20 Synthesis of Compound 19

[0180]

[0181] Example 20 was synthesized using the same method as compound 16 described above, but with side chain VIII-e (1.00 mmol) reacted with 2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyran[4,3-d]pyrimidin-4-yltrifluoromethanesulfonate to give compound 19. Yield: 47%; Structural parameters of compound 19: 1 H-NMR (400MHz, CDCl3) δ8.47(d,J=8.0Hz,2H),7.80-7.85(m,4H),7.72(d,J=8.4Hz,2H),6.63(s,1H),3.70( s,2H),3.32(t,J=6.0Hz,4H),3.25(t,J=5.8Hz,2H),3.01(t,J=6.0Hz,2H),1.75(s,4H),1.60-1.62(m,4H); 13 C-NMR (100MHz, CDCl3) δ163.8,159.7,157.0,142.4,133.9,132.2,128.3,128.2,1 25.5,125.4,120.3,110.4,48.3,34.2,29.2,26.9,25.0,23.5.HRMS(ESI-TOF)m / z calcd.forC 26 H 28 F3N4O2S2[M+H] + :549.1606,found 549.1606.

[0182] Example 21: Experiment on the inhibition of tumor cell proliferation by compounds 1-19 and their hydrochlorides.

[0183] Tumor cells (SW480, HCT116, HepG2) were cultured in IMDM medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum under constant temperature conditions of 37°C and 5% CO2. Tumor cells in the logarithmic growth phase were used, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at 100 μL / mL in 96-well plates, with blank and control wells included. After 24 h of culture, 0.5 μL of the compound was added to each well at final concentrations of 0.001, 0.01, 0.1, 1, and 10 μM, with three replicates for each drug concentration. Blank wells contained only culture medium and no cells, DMSO, or the compound. Control wells contained only complete culture medium with the same concentration of DMSO to act on the cells. Place the cells in a 37°C, 5% CO2 incubator. After 6 h, 12 h, 24 h, and 48 h, add 20 μL of 5 mg / mL MTT solution (prepared with PBS and sterilized by filtration through a 0.22 μm filter) to each well. Continue incubation at 37°C, 5% CO2 for another 4 h. Terminate the culture, carefully remove the culture supernatant from the wells, add 100 μL of DMSO to each well, mix thoroughly by pipetting, and incubate at 37°C for 10 min to allow the purple crystals to dissolve completely. Measure the absorbance (OD) of each well using a microplate reader (490 nm, 630 nm). Calculate the cell inhibition rate using the following formula.

[0184] Cell viability (%) = (Experimental group OD - Blank group OD) / (Control group OD - Blank group OD) × 100%

[0185] Based on the MTT results, derive the linear regression equation and calculate the IC50 for each compound. 50 Value (half-maximal inhibitory concentration).

[0186] The in vitro antitumor activity of compounds 1-19 as their hydrochloride salts is shown in Table 1.

[0187] Table 1. In vitro antitumor activity of compounds 1-19 with their hydrochloride salts

[0188]

[0189] Note: a Camptothecin was used as a positive control.

[0190] Table 1 shows that compounds 1-19 all exhibited inhibitory activity against human colon cancer cells. During the cell inhibitory activity test, it was found that the solubility of hydrochloride salts 1a, 2a, and 3a was superior to that of their corresponding unsalted precursor compounds, and their inhibitory activity remained relatively stable.

[0191] Example 22: Experiment on the inhibition of tumor cell proliferation by compound 3a

[0192] A representative compound, 3a, was selected for further evaluation of its antitumor activity. To observe whether compound 3a could effectively inhibit cancer cell migration, an in vitro scratch assay was used for evaluation. Figure 20 As shown, compound 3a has the ability to inhibit cancer cell migration. At 12 h, 2 μM of compound 3a showed preliminary inhibitory activity against both HCT-116 and SW480 cells. After 24 h of administration, the migration of HCT-116 and SW480 cells in the control group was approximately 40% and 30%, respectively, and 2 μM of compound 3a effectively inhibited cell migration. At 48 h, without drug intervention, the migration distance of the compound reached more than 50%, and 0.5 μM of compound 3a could effectively inhibit HCT-116 and SW480 cells, with 2 μM showing the most significant inhibitory effect, which was concentration-dependent.

[0193] Example 23 Evaluation of the cellular glucose consumption promoting activity of the compound

[0194] The glucose consumption assay was used to evaluate the amount of glucose-promoting activity of different concentrations of compounds, thereby reflecting their hypoglycemic activity at the cellular level. Metformin was used as a positive control. The specific method was as follows: HepG2 cells in logarithmic growth phase were routinely digested, cultured in DMEM low-glucose medium, and seeded at a cell density of 5 × 10⁶ cells / year. 4 Cells were cultured in 96-well plates at a concentration of 100 μL per well. The plates were incubated overnight. After cell attachment, the supernatant was discarded, and the cells were washed once with 1×PBS. The cells were then starved for 24 h in serum-free DMEM high-glucose medium, washed once with 1×PBS, and cultured in DMEM high-glucose medium. A drug control group (medium medium + test substance + cells), a negative control group (medium medium + DMSO + cells), a blank control group (medium medium only + test substance), and a positive control group (medium medium + metformin + cells) were established. Each concentration was used in triplicate, with 0.5 μL of diluent containing different concentrations of the test substance added to each well. Cells were then incubated in a CO2 incubator. Relative glucose consumption was measured using a glucose assay kit after 24 h.

[0195] First, representative compounds 3 and 3a were selected to evaluate the cytotoxicity of the compounds after 24 hours of treatment. At a concentration of 1 μM, cell viability was approximately 80%. Therefore, 1 μM and 0.2 μM concentrations were chosen to test the effect on glucose consumption by cells. Figure 21 As shown, compound 3a significantly promoted glucose consumption at both 1 μM and 0.2 μM, while compound 3 significantly promoted glucose consumption at 1 μM, and its activity was comparable to that of the positive control metformin at 500 μM, indicating that both compounds have good activity in promoting glucose consumption.

[0196] Further, the activity of compounds 1-19 and their hydrochloride in promoting glucose consumption was evaluated using a 1 μM concentration, as shown in Table 2.

[0197] Table 2. Glucose consumption-promoting activity of compounds 1-19 via their hydrochloride salts

[0198]

[0199] Note: Metformin was used as a positive control, and the test concentration was 500 μM.

[0200] As shown in Table 2, all tested compounds exhibited some degree of promoting activity against cellular glucose consumption at a concentration of 1 μM, indicating that these compounds have the potential to lower blood glucose. Furthermore, tumor cell proliferation requires a large amount of energy, affecting glucose consumption, which may be related to inhibiting tumor proliferation.

[0201] Example 24: Study on the cellular-level lipid-lowering activity of the compound

[0202] Oil Red O staining was used to evaluate the cellular-level lipid-lowering activity of representative compounds at different concentrations. Lovastatin was used as a positive control. The specific method was as follows: HepG2 cells in logarithmic growth phase were harvested and cultured in DMEM low-glucose medium at a cell density of 1×10⁻⁶ cells / year. 5 HepG2 cells were seeded at 2 mL / mL in 6-well plates and incubated overnight at 37°C. After cell attachment, the supernatant was discarded, and the cells were washed once with 1×PBS. The cells were then starved in serum-free DMEM high-glucose medium for 24 h, washed once with 1×PBS, and induced for 24 h in DMEM low-glucose complete medium (containing 1% BSA) containing inducers (0.5% sodium oleate and 0.25% sodium palmitate) to establish a HepG2 cell lipid accumulation model. A drug control group (inducer + test substance + cells), a negative control group (inducer + DMSO + cells), a blank control group (DMEM low-glucose medium solution containing only 1% BSA), and a positive control group (inducer + lovastatin + cells) were set up. Each concentration was used in triplicate. 10 μL of diluent containing different concentrations of the test substance was added to each well. The cells were incubated in a CO2 incubator for 24 h, then washed three times with 1×PBS. Cells were fixed with 2 mL of 4% paraformaldehyde per well for 30 min, washed three times with 1×PBS, and treated with 2 mL of 60% isopropanol per well for 5 min to increase cell permeability. The cells were stained with 2 mL of Oil Red O per well for 1 h in the dark at room temperature. After washing the cells four times with distilled water, 1 mL of isopropanol was added to each well for 10 min. The cells were then shaken and washed out. The absorbance was measured at 492 nm using a microplate reader.

[0203] Since the compound had little effect on cell viability at concentrations of 1 μM and 0.2 μM over 24 hours, its lipid-lowering activity was still tested at these two concentrations. Figure 22 As shown, compounds 3a and 3 at a concentration of 1 μM significantly reduced intracellular lipid content.

[0204] Further, the lipid-lowering activity of compound 1-19 and its hydrochloride was evaluated at the cellular level using a 1 μM concentration, as shown in Table 3.

[0205] Table 3. Glucose consumption-promoting activity of compounds 1-19 via their hydrochloride.

[0206]

[0207] Note: Lovastatin was used as a positive control, and the test concentration was 10 μM.

[0208] As shown in Table 3, all tested compounds at a concentration of 1 μM had a certain effect on reducing the increase in intracellular lipids after induction by cell inducers, indicating that these compounds also have certain lipid-lowering activity.

[0209] Example 25: Study on the in vivo antitumor activity of compound 3a

[0210] (1) Establishment of Balb / C nude mouse colon cancer model

[0211] HCT116 cells in logarithmic growth phase and in good condition (viable cell count >95%) were selected and centrifuged at 1000 rpm for 5 min to obtain cell pellet. After washing twice with pre-chilled phosphate buffer, the cells were counted. The cell number was adjusted to 5 × 10⁶ cells using pre-chilled physiological saline according to the desired cell density. 6 Prepare a suspension of 100 cells / mL, place it in a tube, and keep it on ice for later use.

[0212] Balb / C nude mice (5 weeks old) that had been acclimatized for one week were stabilized, and the right axilla of each mouse was disinfected with a 75% medical cotton ball. Using a 1 mL syringe, the HCT116 cell suspension was drawn up, excess air bubbles were removed, and 0.2 mL was injected subcutaneously into the right axilla of each mouse. The needle was then removed, and the puncture site was gently pressed with a sterile cotton swab to prevent cell fluid from leaking out with the needle.

[0213] After HCT116 cell inoculation, the mice were observed for any abnormal changes in their diet, water intake, and body weight. They were observed daily to check for nodule growth at the inoculation site. The successful establishment of the subcutaneous tumor model was indicated by the palpable presence of a solid mass. Once the tumor in the nude mice reached approximately 1 cm in diameter, preparations were made for a second transplantation.

[0214] Before tumor transplantation, afodin solution was prepared using tert-amyl alcohol, prepared fresh each time, and stored at 4°C. The solution was preheated to 37°C before use and administered via intraperitoneal injection. The anesthetic dose of afodin was 0.4 mL per nude mouse body weight.

[0215] Tumors were removed from nude mice in a laminar flow hood and washed in a culture dish containing physiological saline. Well-grown tumor fragments were selected, their capsules and connective tissues were removed, and they were cut into tissue blocks approximately 2 mm in diameter using sterile instruments for later use.

[0216] The anesthetized mouse was placed flat in a laminar flow hood, and the right axilla of the mouse was disinfected with a 75% medical cotton ball. An incision of approximately 0.4 cm was made using sterile scissors. A tumor graft was picked up using a tumor grafting needle, placed into a tumor grafting sleeve, and then inserted subcutaneously. Absorbable surgical sutures were used for suturing.

[0217] (2) Grouping and administration of tumor-bearing nude mice

[0218] The tumor volume of nude mice was measured daily. When the tumor volume of the nude mice grew to 200 mm, the tumor was recorded. 3 The mice were grouped according to their weight and tumor size. The test compounds were dissolved in glucose injection containing 5% Tween-80 and administered via intraperitoneal injection. Mice were divided into five groups based on the type and concentration of the drug: normal group, model group, low-dose group (5 mg / kg), high-dose group (10 mg / kg), and positive control fluorouracil group (20 mg / kg). The dosing cycle was as follows: once every other day for three weeks in the first week; no drug administration in the second week; once every other day for two weeks in the second week; no drug administration in the third week; and sacrificed on day 30.

[0219] (3) Evaluation of pharmacological and physicochemical indicators

[0220] The short and long axes of the tumors were measured and recorded every two days, and a curve showing the overall change in tumor volume in each group of nude mice was plotted. The mice were weighed before sacrifice. Blood was collected from the femoral artery, allowed to stand for 30 minutes, and then centrifuged at 3500 rpm for 10 minutes at 4°C to collect serum. The mice were dissected, the tumors were removed, washed with physiological saline, and excess water was blotted with filter paper before being weighed, recorded, and photographed on a precision balance.

[0221] Relative tumor weight: Organ index = tumor weight / nude mouse body weight;

[0222] Relative tumor inhibition rate: The relative tumor volume (RTV) and relative tumor proliferation T / C (%) were calculated according to the following formulas: RTV = Vt / V0, where Vt: the tumor volume obtained from each measurement; V0: the initial tumor volume (first administration); T / C (%) = average RTV of the administration group / average RTV of the control group × 100%;

[0223] Tumor volume inhibition rate: IRTV (%) = 100% - T / C;

[0224] Tumor volume in mice was measured during drug administration, and tumor growth curves were plotted (see...). Figure 23 In the control group, nude mice injected with the same volume of 5% Tween-80 glucose solution showed rapid tumor growth. On day 30, there was no significant difference in tumor volume between the 5 mg / kg treatment group and the control group (P>0.05), indicating no significant inhibitory activity. At a dose of 10 mg / kg, tumor volume was significantly inhibited (P<0.001), and tumor growth was very slow overall, with significant suppression of tumor growth. Nude mice in the fluorouracil group experienced mortality, with all mice dying after 12 days.

[0225] After the tumor was removed, photos were taken, and the results were as follows: Figure 24 As shown, direct observation revealed that the tumors in the nude mice in the 10 mg / kg group were significantly smaller and more regularly shaped than those in the control group. Tumor weight in nude mice is another intuitive indicator of drug efficacy. Weighing the tumors showed that the tumor weight in the 10 mg / kg group was significantly reduced, only 41% of that in the control group (Table 3). To rule out the influence of decreased mouse body weight on tumor weight, calculations of relative tumor weight showed that the compound remained effective at 10 mg / kg. Table 3 shows that the relative tumor inhibition rate of compound 3a reached 80% at 10 mg / kg, which is six times the inhibition rate at a dose of 5 mg / kg. Therefore, compound 3a exhibits good tumor-inhibiting activity at a dose of 10 mg / kg and has the potential to be developed into a novel antitumor drug.

[0226] Table 3. Tumor weight and relative tumor inhibition rate (IRTV) of compound 3a at different doses.

[0227] Group Dosage (mg / kg) Tumor weight (g) IRTV (%) Model group - 3.50±0.51 - Group 1 10 1.44±0.27*** 80.17 Group 2 5 3.11±0.85 12.14

[0228] In summary, this invention synthesized a series of benzenesulfonamide derivatives, optimized the synthetic process, and evaluated the in vitro antitumor, hypoglycemic, and hypolipidemic activities of the compounds. The synthetic process of this invention is simple, requiring no anhydrous or oxygen-free conditions, and some reactions cleverly incorporate microwave conditions, resulting in high yields and short reaction times for certain reactions. Subsequent activity studies showed that these compounds inhibit tumor cell proliferation and migration, significantly inhibiting tumor growth at an in vivo dose of 10 mg / kg; furthermore, they also exhibit hypoglycemic and hypolipidemic activities. Therefore, this demonstrates the promising application prospects of these compounds.

[0229] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A benzenesulfonamide derivative, characterized in that: The specific structural formula of the derivative is as follows: 、 、 、 、 、 。 2. The method for preparing benzenesulfonamide derivatives as described in claim 1, characterized in that: Its reaction route is as follows: ； Among them, RNHR1 is , ; or, Among them, RNHR1 is , ; or, Among them, RNHR1 is , .

3. The use of the benzenesulfonamide derivatives as described in claim 1 in the preparation of drugs for treating human colon cancer.

4. The use of the benzenesulfonamide derivatives as described in claim 1 in the preparation of drugs for treating human liver cancer.

5. The use of the benzenesulfonamide derivatives as described in claim 1 in the preparation of drugs for treating disorders of glucose and lipid metabolism.

6. The use of the benzenesulfonamide derivatives as described in claim 1 in the preparation of lipid-lowering drugs.

7. The use of the benzenesulfonamide derivatives as described in claim 1 in the preparation of hypoglycemic drugs.

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