Imidazoquinoline compounds with alkylthio or alkyldithio groups, their preparation methods and applications

Abstract

This application relates to the field of chemical pharmaceutical technology, and more particularly to an imidazoquinoline compound having an alkylthio or alkyldithio group, its preparation method, and its application. The imidazoquinoline compound of this application includes the imidazoquinoline compound shown in Formula I and its pharmaceutically acceptable salt. This imidazoquinoline compound can activate TLR7 and TLR8 receptors, and after local injection, the level of the inflammatory factor IL6 in the blood is further reduced compared to the prototype molecule 78A1. While achieving local immunostimulation, it minimizes the non-specific immunotoxicity caused by cytokine storms, thus significantly improving safety. Therefore, the imidazoquinoline compound provided in this application can be used more safely for local immune activation, such as for tumor treatment or as an immunostimulatory adjuvant for vaccines.

Description

Technical Field

[0001] This application belongs to the field of chemical pharmaceutical technology, and in particular relates to an imidazoquinoline compound having an alkylthio or alkyldithio group, its preparation method and application. Background Technology

[0002] Toll-like receptors (TLRs) are widely expressed across the immune cell spectrum, participating in both non-specific and specific immunity. TLRs are the innate immune system's surveillance and recognition of various disease-associated molecular patterns, forming the body's first line of defense against infectious diseases. Currently, 13 TLRs (TLR1-TLR13) have been identified in mammals. Because TLR activation can induce MyD88 or TRIF-dependent signaling pathways, thereby activating NF-κB, inducing cytokine and chemokine secretion, activating innate immune responses, and mediating adaptive immune responses, TLR agonists can be used as immune adjuvants in anti-tumor treatments and the treatment of infectious diseases.

[0003] TLR7 and TLR8 agonists are two important types of TLR agonists, especially dual TLR7 and TLR8 agonists (i.e., TLR7 / 8 agonists). Because they are generally small molecule compounds, they lack specific targeting when used as adjuvants, have short half-lives, and intravenous injection can lead to systemic immune system hyperactivity, resulting in cytokine release syndrome (CRS), making them difficult to tolerate in vivo. Therefore, TLR7 / 8 agonists are widely considered effective adjuvants for local antitumor therapy.

[0004] Although TLR7 / 8 agonists are effective adjuvants for local antitumor therapy, their severe toxic side effects remain a significant problem in current clinical applications. For example, the small molecule drug 78A1, after activating TLR7 / 8 receptors, can promote the release of tumor antigens and mediate tumor antigen-specific T-cell immunity, activating T cells to circulate throughout the body to kill distant metastatic tumors, achieving in situ immune activation and inducing systemic antitumor immune effects. However, due to its lack of specific targeting, even when 78A1 is administered via intratumoral injection, it is still difficult to overcome the systemic immunotoxicity caused by rapid absorption. Summary of the Invention

[0005] The purpose of this application is to provide an imidazoquinoline compound having an alkylthio or alkyldithio group, a method for its preparation, and its application, aiming to solve the technical problem of how to further reduce the side effects of 78A1.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides an imidazoquinoline compound having an alkylthio group or an alkyldithio group, including the imidazoquinoline compound shown in Formula I and its pharmaceutically acceptable salt;

[0008]

[0009] In Formula I, R1 is selected from hydrogen, C 1-10 Alkyl and C 1-10 Any of the alkoxy groups, where R2 is selected from hydrogen, C 1-10 Alkyl and C 1-10 Any of the alkoxy groups, R3 is selected from C 1-10 Alkyl or -S-R4, where R4 is a group represented by Formula II:

[0010]

[0011] In Formula II, R5 is selected from -(CH2). n -, n = 1-10.

[0012] Secondly, this application provides a method for preparing the above-mentioned imidazoquinoline compound, comprising:

[0013] Compound A and compound B were subjected to a condensation reaction to obtain the imidazoquinoline compound shown in Formula I; and R3 in the imidazoquinoline compound shown in Formula I was selected from C 1-10 alkyl;

[0014]

[0015] In compound B, X is -COOH, and Y is the same as R3.

[0016] Thirdly, this application provides a method for preparing the above-mentioned imidazoquinoline compound, comprising:

[0017] Compound A and compound C undergo a first reaction, and then compound B undergoes a second reaction to obtain intermediate compound D;

[0018] The intermediate compound D is condensed with the compound A to obtain the imidazoquinoline compound shown in Formula I; and R3 in the imidazoquinoline compound shown in Formula I is -S-R4.

[0019]

[0020] In compound B, X is -COOH and Y is hydrogen.

[0021] Fourthly, this application provides an application of the above-mentioned imidazoquinoline compound in the preparation of antitumor drugs and / or antiviral drugs.

[0022] Fifthly, this application also provides an application, namely, the application of the above-mentioned imidazoquinoline compound in the preparation of immunostimulatory adjuvants.

[0023] The imidazoquinoline compound provided in this application is a derivative of the TLR7 / 8 agonist 78A1 (CAS Registry No. 1258457-59-8). This imidazoquinoline compound can activate TLR7 and TLR8 receptors, and after local injection, the level of the inflammatory factor IL6 in the blood is further reduced compared to the original molecule 78A1. While achieving local immunostimulation, it minimizes the non-specific immunotoxicity caused by cytokine storms, thus significantly improving safety. Therefore, the imidazoquinoline compound of this application can be used more safely for local immune activation, such as for tumor treatment or as an adjuvant for vaccine immunostimulation.

[0024] The preparation method provided in this application allows for the use of different starting materials corresponding to the selectable groups in the imidazoquinoline compound. This method offers high yield and few byproducts. Furthermore, the prepared imidazoquinoline compound exhibits increased lipophilicity and reduced leakage into the bloodstream without significantly affecting the activation ability of compound 78A1 on TLR7 / 8 receptors, thereby ensuring local immunostimulatory effects while reducing systemic immunotoxicity.

[0025] Based on the aforementioned advantages of the imidazoquinoline compounds of this application, the imidazoquinoline compounds of this application can be used to prepare antitumor drugs and / or antiviral drugs and / or immunostimulatory adjuvants. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The mass spectrum of 78A1-SMe is shown.

[0028] Figure 2 The mass spectrum of 78A1-C(CH3)2SMe is shown.

[0029] Figure 3 The mass spectrum of 78A1-SS-C(CH3)2-78A1 is shown.

[0030] Figure 4This is a schematic diagram showing the detection results of the induction of TNF-α expression activity in mouse PBMC cells by 78A1, 78A1-SMe, 78A1-C(CH3)2SMe and 78A1-SS-C(CH3)2-78A1;

[0031] Figure 5 This is a schematic diagram showing the serum TNF-α and IL-6 levels after subcutaneous injection of 78A1, 78A1-SMe and 78A1-C(CH3)2SMe, where A represents the TNF-α detection result and B represents the IL-6 detection result.

[0032] Figure 6 This is a schematic diagram showing the serum TNF-α and IL-6 levels after intratumoral injection of 78A1, 78A1-SMe and 78A1-C(CH3)2SMe, where A represents the TNF-α detection result and B represents the IL-6 detection result.

[0033] Figure 7 The diagram shows the serum TNF-α and IL-6 levels after subcutaneous injection of 78A1 and 78A1-SS-C(CH3)2-78A1, where A represents the TNF-α detection results (data 1 hour after injection) and B represents the IL-6 detection results (data 4 hours after injection).

[0034] Figure 8 The results of the 78A1-SMe antitumor activity assay are shown in Figure A, where A is a schematic diagram of mouse weight change, B is a schematic diagram of tumor growth curve, C is a schematic diagram of tumor weight result, and D is a schematic diagram of tumor size at the experimental endpoint.

[0035] Figure 9 The results of the detection of antitumor activity of 78A1-C(CH3)2SMe are shown in Figure A, where A is a schematic diagram of mouse body weight change, B is a schematic diagram of tumor growth curve, C is a schematic diagram of tumor weight result, and D is a schematic diagram of tumor size at the experimental endpoint.

[0036] Figure 10 The results of the antitumor activity detection of 78A1-SS-C(CH3)2-78A1 are shown in Figure A, which is a schematic diagram of mouse body weight change, Figure B is a schematic diagram of tumor growth curve, Figure C is a schematic diagram of tumor weight result, and Figure D is a schematic diagram of tumor size at the experimental endpoint.

[0037] Figure 11 This is a schematic diagram comparing the detection results of 78A1-SH and 78A1-SMe on the induced expression of TNF-α in mouse PBMC cells. Detailed Implementation

[0038] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0039] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0040] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0041] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0042] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0043] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0044] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0045] 78A1, CAS number 1258457-59-8, is a small molecule TLR7 / 8 agonist with good antitumor activity. However, after administration, 78A1 not only activates the immune response within the tumor and increases cytokines such as TNF-α, mediating antigen-specific T-cell antitumor immunity, but also, because 78A1 is distributed outside the tumor, such as in plasma, it leads to an increase in the content of extratumor cytokines in the body, resulting in immunotoxicity. The compound shown in Formula I of this application, after local administration, reduces the secretion of extratumor cytokines such as TNF-α and chemokines, thereby reducing the non-specific immunotoxicity caused by the TNF-α cytokine storm.

[0046] In a first aspect, embodiments of this application provide an imidazoquinoline compound having an alkylthio group or an alkyldithio group, including the imidazoquinoline compound shown in Formula I and its pharmaceutically acceptable salt;

[0047]

[0048] In Formula I, R1 is selected from hydrogen, C 1-10 Alkyl and C 1-10 Any of the alkoxy groups, where R2 is selected from hydrogen, C 1-10 Alkyl and C 1-10 Any of the alkoxy groups, R3 is selected from C 1-10 Alkyl or -S-R4, where R4 is a group represented by Formula II:

[0049]

[0050] In Formula II, R5 is selected from -(CH2). n -, n = 1-10.

[0051] Specifically, R3 is selected from C 1-10 When R3 is alkyl, Formula I is an imidazoquinoline compound having an alkylthio group; when R3 is selected from -S-R4, Formula I is an imidazoquinoline compound having an alkyldithio group.

[0052] The imidazoquinoline compound provided in this application is a derivative of the TLR7 / 8 agonist 78A1, specifically a compound obtained by aminoacylation of the para-position of the benzene ring of 78A1, and also an alkylthiolated derivative. Upon administration, it releases 78A1 within the tumor, activating TLR7 and TLR8 receptors on tumor-associated immune cells, activating the expression of inflammatory cytokines, promoting the release of tumor antigens, and mediating tumor antigen-specific T-cell immunity, thereby achieving in situ immune activation and inducing anti-tumor immune effects. Simultaneously, after local injection, the levels of inflammatory factors in the blood of this imidazoquinoline compound are further reduced compared to the original molecule 78A1. While achieving local immune stimulation, it minimizes the non-specific immunotoxicity caused by cytokine storms, avoiding systemic immune system hyperactivity and cytokine release syndrome. Thus, while possessing anti-tumor activity, it significantly reduces systemic immunotoxicity and other side effects, thereby significantly improving safety.

[0053] C 1-10 Alkyl groups refer to straight-chain or branched alkyl groups containing 1-10 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.; C 1-10 Alkoxy groups are straight-chain or branched alkoxy groups containing 1-10 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.; n = 1-10, where n is an integer and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0054] In some embodiments, in the imidazoquinoline compound represented by Formula I, R1 is selected from hydrogen and C. 1-5 Any of the alkyl groups, R2 is selected from hydrogen and C. 1-5 Any of the alkyl groups, R3 is selected from C 1-5 Alkyl; or, R1 is selected from C 1-5 Alkyl group, R2 is selected from C 1-5 Alkyl group, R3 is selected from -S-R4, and R4 is a group represented by formula II.

[0055] In some embodiments, the imidazoquinoline compound is at least one of the following formulas (1)-(3):

[0056]

[0057]

[0058] Secondly, this application provides the above-mentioned imidazoquinoline compound (where R3 is C in this case). 1-10 The preparation method of alkyl groups includes the following steps:

[0059] Compound A (i.e., 78A1) and compound B (where X is -COOH and Y is the same as R3) were subjected to a condensation reaction to obtain the imidazoquinoline compound shown in Formula I.

[0060]

[0061] Compound A contains benzylamine, and compound B contains a carboxyl active group, allowing the benzylamine in compound A to undergo a dehydration condensation reaction with the carboxyl terminus of compound B to form the compound shown in Formula I. Furthermore, to improve synthesis efficiency, compound B can be first reacted with N-hydroxysuccinimide to generate an amine-reactive NHS ester, and then condensed with compound A.

[0062] For example, in some embodiments, compound A is added to a solution containing N-hydroxysuccinimide, DMF, EDCI, HOBt, and DIPEA to react and obtain an intermediate product, which is then condensed with compound A.

[0063] In some embodiments, compound B is prepared by reacting 3,3-dialkylacrylic acid, as shown in compound E, with sodium alkylthiolate;

[0064]

[0065] Specifically, taking compound B where X is -COOH and R1, R2, and Y are all -CH3 as an example, the synthetic reaction of compound B is the reaction of 3,3-dimethacrylic acid with sodium methylthiolate. The synthetic route is as follows:

[0066]

[0067] Thirdly, this application provides a method for preparing the above-mentioned imidazoquinoline compound (where R3 is -S-R4), comprising the following steps:

[0068] Compound A (i.e., 78A1) and compound C undergo a first reaction, followed by a second reaction with compound B (where X is -COOH and Y is hydrogen) to obtain intermediate compound D; then intermediate compound D undergoes a condensation reaction with compound A to obtain the imidazoquinoline compound shown in Formula I.

[0069]

[0070] In some embodiments, the preparation method of compound B includes: reacting 3,3-dialkylacrylic acid (represented by compound E) with thioacetic acid, and then reacting it with concentrated sulfuric acid to obtain compound B;

[0071]

[0072] Specifically, taking compound B as an example where X is -COOH, R1 and R2 are -CH3, and Y is hydrogen, the synthetic reaction route of compound B is as follows:

[0073]

[0074] The aforementioned raw materials, namely compounds A, B, C, and E, can be obtained through commercial purchases or prepared using conventional synthetic methods.

[0075] The conventional synthetic route for compound A, 78A1, is as follows:

[0076]

[0077] The preparation method provided in this application can be used to prepare the compound from different starting materials, depending on the selection of the functional groups in the imidazoquinoline compound. The preparation method of this application has a high yield and few byproducts. Moreover, the prepared imidazoquinoline compound, without significantly affecting the activation ability of compound 78A1 on TLR7 / 8 receptors, exhibits increased lipophilicity and reduced leakage into the bloodstream, thereby ensuring local immunostimulatory effects while reducing systemic immunotoxicity.

[0078] Fourthly, this application provides an application of the imidazoquinoline compound represented by Formula I above in the preparation of antitumor drugs and / or antiviral drugs.

[0079] Fifthly, this application also provides an application, namely the use of the imidazoquinoline compound represented by Formula I above in the preparation of an immunostimulatory adjuvant.

[0080] Based on the embodiments of this application, after local (peritumoral and intratumoral) injection of the imidazoquinoline compound, it mainly acts on the immune cells within the tumor, mediates antigen-specific T cell anti-tumor immunity, and achieves in situ immune activation. At the same time, it effectively reduces the agonistic activity of TLR7 / 8 receptors on immune cells in the blood, thereby reducing systemic immunotoxicity. Thus, when the imidazoquinoline compound is used in anti-tumor drugs, it has the beneficial effect of low systemic immunotoxicity while possessing anti-tumor activity.

[0081] Based on the embodiments of this application, local injection of imidazoquinoline compounds can release TLR7 / 8 agonists within tumors, increase the content of inflammatory mediators within tumors, and effectively avoid the increase of pro-inflammatory cytokines such as TNF-α in plasma, making imidazoquinoline compounds have great application potential in the preparation of anti-tumor, antiviral and vaccine adjuvants.

[0082] In some embodiments, the tumor can be controlled as a solid tumor. In further embodiments, the solid tumor may include at least one of T-cell lymphoma, melanoma, breast cancer, rectal cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, bone cancer, and brain tumors. Solid tumors are easier to treat via intratumoral injection, which further increases the enrichment of TLR7 / 8 agonist molecules in the tumor and is beneficial to further improving the antitumor effect of the imidazoquinoline compound in the embodiments of this application.

[0083] Furthermore, this application also provides a pharmaceutical composition comprising the above-mentioned imidazoquinoline compound. After administration of the pharmaceutical composition of this application embodiment, the imidazoquinoline compound contained in the pharmaceutical composition, when applied topically, can release the TLR7 / 8 agonist A781 in the tumor area, activate immune cells within the tumor, and promote an increase in pro-inflammatory cytokines such as TNF-α, exhibiting good targeting.

[0084] In some embodiments, the administration method of the pharmaceutical composition can be controlled as injection, further, as subcutaneous injection and / or intratumoral injection, and further, it can include at least one of intratumoral rapid injection, intratumoral controlled injection, subcutaneous rapid injection, and subcutaneous controlled injection. Intratumoral rapid injection involves injecting the drug into the tumor tissue in a short time using a conventional syringe, while intratumoral controlled injection involves using a micro-infusion pump with an indwelling needle to control the flow rate, maintaining the drug concentration in the tumor tissue within the effective concentration range during continuous injection.

[0085] The following description is based on specific embodiments. In the embodiments of this application, room temperature refers to 25–30 degrees Celsius; various abbreviations are as follows: TLC stands for Thin-Layer Chromatography; HPLC stands for High-Performance Liquid Chromatography; NMR stands for Nuclear Magnetic Resonance; DMF stands for N,N-Dimethylformamide; NHS stands for N-hydroxysuccinimide; EDCI stands for 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt stands for 1-hydroxybenzotriazole; DIPEA stands for N,N-diisopropylethylamine; PE stands for petroleum ether; EA stands for ethyl acetate; DIEA stands for N,N-diisopropylethylamine; (methylsulfanyl)sodium stands for sodium methanethiol; PBMC stands for peripheral blood mononuclear cells.

[0086] In addition, the significance analysis of the differences in this application specification uses one-way / two-way ANOVA (ns: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0087] Example 1

[0088] An imidazoquinoline compound of formula (1), abbreviated as 78A1-SMe, is prepared as follows:

[0089]

[0090] The product 78A1 can be prepared by direct condensation reaction with 3-thiomethylpropionic acid. However, to increase the efficiency of the synthesis reaction, the carboxyl group of 3-thiomethylpropionic acid is first converted to an amine-reactive NHS ester using NHS, and then reacted with 78A1 to obtain 78A1-SMe. Specifically, 3-thiomethylpropionic acid (99 mg, 1.05 eq.) was placed in a reaction tube, and anhydrous DMF (2 mL), NHS (90 mg, 1.0 eq.), EDCI (150 mg, 1.0 eq.), HOBt (106 mg, 1.0 eq.), and DIPEA (152 mg, 1.5 eq.) were added. The reaction was carried out at room temperature for 1 hour. Then, 78A1 (150 mg, 0.53 eq.) was added, and the reaction was carried out at room temperature for 1 hour. The molecular weight of the product was monitored by LCMS. After preparative purification, 94.69 mg of 78A1-SMe was obtained, and the NMR verification was as follows:

[0091] 1H NMR (400MHz, DMSO-d6) δ8.36(t,J=6.0Hz,1H),7.82-7.74(m,1H),7.57(dd,J=8.4,1.3Hz,1H),7.33(ddd,J=8.4 ,6.9,1.3Hz,1H),7.21(d,J=8.0Hz,2H),7.04(td,J=7.5,6.9,1.3Hz,1H),6.98(d,J=8.0Hz,2H),6.57(s,2H),5. The mass spectra of 78Al-SMe were as follows: 84 (s, 2H), 4.22 (d, J = 5.9 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 2.01 (s, 3H), 1.71 (p, J = 7.6 Hz, 2H), 1.39 (p, J = 7.4 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H). The purity was 99.8% (220 nm) as determined by HPLC. Figure 1 As shown.

[0092] Example 2

[0093] An imidazoquinoline compound of formula (2), abbreviated as 78A1-C(CH3)2SMe, is prepared as follows:

[0094]

[0095] At room temperature, 3,3-dimethacrylic acid (1 g, 9.988 mmol, 1.0 eq.) was added to a 100 mL single-necked flask, followed by sodium methanethiol aqueous solution (20%) (17.50 g, 49.943 mmol, 5.0 eq.), and the reaction was carried out overnight at 100 °C. TLC showed new spots. The mixture was extracted with water and EA, and the organic phase was concentrated to give 1.3 g of crude compound B (R1, R2, and Y are all -CH3). At room temperature, crude compound B (98.96 mg, 0.668 mmol, 1.2 eq.) was added to a 25 mL nitrogen-protected three-necked flask, followed by compound A (200 mg, 0.556 mmol, 1.0 eq.), EDCI (159.98 mg, 0.835 mmol, 1.5 eq.), HOBt (75.18 mg, 0.556 mmol, 1.0 eq.), and DIEA (107.86 mg, 0.835 mmol, 1.5 eq.). The reaction was allowed to proceed overnight at room temperature. The reaction was monitored by LCMS until complete, at which point the reaction was stopped, yielding 15.2 mg of the target product 78A1-C(CH3)2SMe. NMR verification is as follows:

[0096] 1H NMR (400MHz, DMSO-d6) δ8.34(t,J=5.9Hz,1H),7.77(dd,J=8.4,1.4Hz,1H),7.57(dd,J=8.4,1. 3Hz,1H),7.33(ddd,J=8.4,6.9,1.3Hz,1H),7.22(d,J=8.0Hz,2H),7.06–6.96(m,3H),6.65(s,2 The mass spectrum of 78Al-C(CH3)2SMe is as follows: 5.84(s,2H), 4.20(d,J=5.8Hz,2H), 2.94–2.86(m,2H), 2.35(s,2H), 1.97(s,3H), 1.71(p,J=7.6Hz,2H), 1.38(h,J=7.3Hz,2H), 1.28(s,6H), 0.87(t,J=7.3Hz,3H). Figure 2 As shown.

[0097] Example 3

[0098] The imidazoquinoline compound shown in formula (3), 78A1-SS-C(CH3)2-78A1, is prepared as follows:

[0099]

[0100] At room temperature, 100 g of 3,3-dimethacrylic acid (998.801 mmol, 1.0 eq.) and thioacetic acid (380.09 g, 4993.956 mmol, 5.0 eq.) were added to a 1000 mL single-necked flask, and the mixture was reacted overnight at 98 °C. The reaction was monitored by TLC until it was complete, and the reaction was stopped. The reaction solution was concentrated and filtered through a column with a volume ratio of PE:EA = 30:1 to 2:1 to obtain 38 g of crude intermediate S1. At room temperature, intermediate S1 (19 g, 107.814 mmol, 1.0 eq.) was added to a 1000 mL single-necked flask, followed by water (200 mL) and concentrated sulfuric acid (42.29 g, 431.223 mmol, 4.0 eq.). The reaction was carried out overnight at 100 °C. The reaction was monitored by TLC until it was complete. The reaction was then stopped, and water and ethyl acetate were added. The mixture was stirred and separated to obtain the organic phase. The organic phase was concentrated to obtain 13 g of crude compound B (R1 and R2 are both -CH3, and Y is hydrogen).

[0101] At room temperature, compound A (2 g, 5.564 mmol, 1.0 eq.), DMF (20 mL), N-hydroxysuccinimide 3-(2-pyridinedithio)propionic acid (2.09 g, 6.691 mmol, 1.2 eq.), and DIEA (1.08 g, 8.372 mmol, 1.5 eq.) were added to a 100 mL single-necked flask. The reaction was carried out for three hours, and the reaction was stopped after LCMS monitoring until complete. The reaction solution was directly used for the next step. At room temperature, compound B (0.48 g, 3.577 mmol, 1.0 eq.), methanol (20 mL) were added to the reaction solution from the previous step, and the reaction was carried out for three hours. The reaction was stopped after LCMS monitoring until complete, and 1.2 g of intermediate S2 was prepared.

[0102] Under nitrogen protection at room temperature, intermediate S2 (50 mg, 0.086 mmol, 1.0 eq.), compound A (37.20 mg, 0.103 mmol, 1.2 eq.), EDCI (24.73 mg, 0.129 mmol, 1.5 eq.), HOBt (11.62 mg, 0.086 mmol, 1.0 eq.), and DIEA (16.67 mg, 0.129 mmol, 1.5 eq.) were added to a 10 mL three-necked flask. The reaction was carried out at room temperature for 3 hours. LC-MS showed the presence of the product, yielding 11.9 mg of the target product 78A1-SS-C(CH3)2-78A1. NMR verification is as follows:

[0103] 1H NMR(400MHz,Chloroform-d)δ7.80(ddd,J=8.5,5.3,1.2Hz,2H),7.66(d,J=8.3Hz,2H),7.41(dddd,J=8.3,7 .0,2.9,1.3Hz,2H),7.24(dd,J=10.4,8.0Hz,4H),7.14(dddd,J=8.4,7.1,2.9,1.2Hz,2H),6.97(dd,J=8.0, The mass spectra of 78A1-SS-C(CH3)2-78A1 are as follows: 5.3Hz, 4H), 6.44(dt, J = 23.1, 5.8Hz, 2H), 5.70(s, 4H), 4.36(t, J = 5.9Hz, 4H), 2.97–2.83(m, 6H), 2.45(t, J = 7.1Hz, 4H), 1.82(p, J = 7.6Hz, 4H), 1.55–1.20(m, 14H), 0.95(t, J = 7.4Hz, 6H). Figure 3 As shown.

[0104] Performance verification experiment

[0105] (1) Detection of in vitro TNF-α expression induction activity in mouse PBMC cells

[0106] 1.1 SPF-grade BALB / c mice, 5-6 weeks old, 18-20 grams, were used. Whole blood was extracted from these mice via enucleation for the extraction of peripheral blood mononuclear cells (PBMCs). An equal volume of whole blood diluent was added to the whole blood and mixed thoroughly. This mixture was then added to an equal volume of mouse peripheral blood lymphocyte separation medium. The mixture was centrifuged at 1000g for 20 min, and the lymphocyte layer was collected. The lymphocytes were washed with 10 mL of phosphate-buffered saline (PBS) and centrifuged at 250g for 10 min. The supernatant was discarded, and the lymphocytes were resuspended in complete RPMI-1640 cell culture medium. The mouse whole blood diluent and lymphocyte separation medium used in the experiment were purchased from Beijing Solarbio Science & Technology Co., Ltd. The mouse peripheral blood lymphocyte separation medium kit was catalog number P8620.

[0107] Eight concentrations of RPMI-1640 cell culture medium were prepared for each of the following compounds: 78A1 (control), 78A1-SMe, and 78A1-C(CH3)2SMe. 78A1 served as the positive control, 78A1-SMe and 78A1-C(CH3)2SMe as the experimental groups, and RPMI-1640 cell culture medium as the blank control. 40 μL of each group's RPMI-1640 cell culture medium was added to a final volume of 160 μL of 1.6*10⁻⁶ ppm RPMI-1640 cell culture medium. 5Peripheral lymphocyte culture solutions were prepared in each well of normal PBMC cells, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 1.

[0108] Table 1

[0109]

[0110] After incubating the peripheral lymphocyte culture solutions of each group at 37℃ and 5% CO2 for 24 hours, centrifuging at 1000 rpm for 5 minutes, the cell supernatant was collected, and the TNF-α concentration in the peripheral blood mononuclear cell culture supernatant was detected according to the ELISA instructions. The mouse TNF-α double antibody sandwich ELISA detection kit used for the detection was manufactured by Wuhan Sanying, and the product code was KE10002.

[0111] Experimental results are as follows Figure 4 As shown in A, within the tested dose range, relative to the blank control group, the TNF-α concentration in the cell supernatant of the 78A1 group increased with increasing 78A1 concentration in the range of 0.0006μM-0.1563μM, but the TNF-α level decreased with further increases in drug concentration; the TNF-α content of the 78A1-SMe and 78A1-C(CH3)2SMe groups showed that their stimulation levels and trends were basically consistent with the positive control 78A1.

[0112] 2.1 SPF-grade BALB / c mice, 5-6 weeks old, 18-20 grams, were used. Whole blood was extracted from these mice via enucleation for the extraction of peripheral blood mononuclear cells (PBMCs). An equal volume of whole blood diluent was added to the whole blood and mixed thoroughly. This mixture was then added to an equal volume of mouse peripheral blood lymphocyte separation medium. The mixture was centrifuged at 1000g for 20 min, and the lymphocyte layer was carefully collected. The cells were washed with 10 mL of phosphate-buffered saline (PBS) and centrifuged at 250g for 10 min. The supernatant was discarded, and the lymphocytes were resuspended in complete RPMI-1640 cell culture medium. The mouse whole blood diluent and lymphocyte separation medium used in this experiment were purchased from Beijing Solarbio Science & Technology Co., Ltd. The mouse peripheral blood lymphocyte separation medium kit was catalog number P8620.

[0113] Seven concentrations of RPMI-1640 cell culture medium were prepared for each compound, namely 78A1 and 78A1-SS-C(CH3)2-78A1. 78A1 served as the positive control, 78A1-SS-C(CH3)2-78A1 as the experimental group, and RPMI-1640 cell culture medium as the blank control. 40 μL of each group's RPMI-1640 cell culture medium was added to a final volume of 160 μL of 1.6*10⁻⁶ ppm RPMI-1640 cell culture medium. 5Peripheral lymphocyte culture solutions were prepared in each well of normal PBMC cells, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 2.

[0114] Table 2

[0115]

[0116] After incubating the peripheral lymphocyte culture solutions of each group at 37℃ and 5% CO2 for 24 hours, centrifuging at 1000 rpm for 5 minutes, the cell supernatant was collected, and the TNF-α concentration in the peripheral blood mononuclear cell culture supernatant was detected according to the ELISA instructions. The mouse TNF-α double antibody sandwich ELISA detection kit used for the detection was manufactured by Wuhan Sanying, and the product code was KE10002.

[0117] Experimental results are as follows Figure 4 As shown in B, within the tested dose range, compared to the blank control group, the TNF-α concentration in the cell supernatant of the 78A1 group increased with increasing 78A1 concentration in the range of 0.0024μM-2.5μM, and then decreased slightly. The TNF-α content in the 78A1-SS-C(CH3)2-78A1 group showed a lower stimulation level at low concentrations compared to the positive control 78A1 group. It only showed an activating effect at concentrations greater than 0.1563uM. Subsequently, the stimulation level increased with increasing drug concentration, reaching the same level as the positive control at the highest concentration of 10uM.

[0118] Figure 4 Data A and B demonstrate that in in vitro cell experiments, 78A1-SMe and 78A1-C(CH3)2SMe exhibit comparable stimulatory activity to 78A1, but 78A1-SS-C(CH3)2-78A1 has a higher minimum effective concentration in PBMCs. This is primarily because 78A1-SS-C(CH3)2-78A1 has a large molecular weight, requiring metabolism to open disulfide bonds and generate the active molecules 78A1-SMe and 78A1-C(CH3)2SMe to exert its activity. However, in in vitro experiments, the amount of these active molecules generated by 78A1-SS-C(CH3)2-78A1 is relatively small, thus requiring a higher dose of 78A1-SS-C(CH3)2-78A1 to achieve its active effect. This indirectly suggests that the two molecules 78A1-SMe and MeS-C(CH3)2-78A1 can be directly applied externally in vitro to exert their effects as drug molecules, while 78A1-SS-C(CH3)2-78A1, as a prodrug molecule, is not suitable for direct in vitro use.

[0119] (2) Detection of the induction of TNF-α and IL-6 expression in mouse serum by subcutaneous injection

[0120] 78A1, 78A1-SMe, and 78A1-C(CH3)2SMe were used to prepare PBS solutions of appropriate concentrations containing the three different components, using phosphate-buffered saline (PBS) as the solvent. SPF-grade BALB / c female mice, 5-6 weeks old and weighing 18-20 grams, were purchased from the Henan Provincial Experimental Animal Center. These mice were divided into four groups: Group 1, Group 2, Group 3, and Group 4, with 9 mice in each of Groups 1, 2, and 3, and 3 mice in Group 4.

[0121] Mice in group 1 were subcutaneously injected with 100 μL of the above-mentioned 78A1 solution; mice in group 2 were subcutaneously injected with 100 μL of the above-mentioned 78A1-SMe solution; mice in group 3 were subcutaneously injected with 100 μL of the above-mentioned 78A1-C(CH3)2SMe solution; and mice in group 4 were subcutaneously injected with 100 μL of PBS solution. The injection dosages for each group of mice are shown in Table 3.

[0122] Table 3

[0123] Grouping Injected compound Dosage Group 1 78A1 57μg Group 2 78A1-SMe The molar amount is consistent with that of 57 μg 78A1. Group 3 <![CDATA[78A1-C(CH3)2SMe]]> The molar amount is consistent with that of 57 μg 78A1. Group 4 PBS ——

[0124] In groups 1, 2, and 3, orbital blood was collected from three mice at 30, 60, and 240 minutes after subcutaneous injection. In group 4, orbital blood was collected from three mice 1 hour after subcutaneous injection. After orbital blood collection, the mouse blood was centrifuged at 3800 rpm for 10 minutes to separate serum. The concentration of TNF-α in the serum was detected using the same method as in Example 21.

[0125] Experimental results are as follows Figure 5 As shown in Figure A, the results indicated that in the first group of mice receiving subcutaneous rapid injection of 78A1, the serum TNF-α level significantly increased, and the serum TNF-α level reached its peak 1 hour after injection, with an increase of 10-15 times. In the subcutaneous rapid injection groups of 78A1-SMe and 78A1-C(CH3)2SMe, the serum TNF-α level in the 78A1-SMe group was relatively low 1 hour after injection.

[0126] Mice in the same experimental group as described above were used to detect the concentration of IL-6 in their serum. The mouse IL-6 double antibody sandwich ELISA kit used for the test was manufactured by Wuhan Sanying, and the product code was KE10007.

[0127] Experimental results are as follows Figure 5As shown in Figure B, 4 hours after subcutaneous injection of 78A1, the serum IL-6 level in mice was close to 400 pg / mL, approximately 25 times higher than that in the PBS control group. However, 1 hour after subcutaneous injection of 78A1-SMe and 78A1-C(CH3)2SMe, the serum IL-6 level was highest, and then began to decline, with peak levels ranging from 80 to 150 pg / mL, representing a 5-8 fold increase compared to the PBS group. The IL-6 levels in the 78A1-SMe and 78A1-C(CH3)2SMe groups were significantly different from those in the 78A1 control group (p<0.0001), indicating that the safety of 78A1-SMe and 78A1-C(CH3)2SMe was improved at the tested doses.

[0128] (3) Detection of the induction of TNF-α and IL-6 expression in mouse serum by intratumoral injection

[0129] The same mice (SPF grade BALB / C, 5-6 weeks old, 18-20 grams female mice) were divided into four groups: group 5, group 6, group 7 and group 8. Groups 5, 6 and 7 each had 9 mice, and group 8 had 3 mice.

[0130] CT-26 colon cancer cells purchased from ATCC were passaged in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. The specific method was as follows: the culture supernatant was discarded, and the cells were washed 1-2 times with calcium- and magnesium-free PBS. 3-5 ml of digestion buffer (0.25% Trypsin-0.53 mM EDTA) was added to the culture flask, and the flask was incubated at 37°C for 1-2 minutes. The digestion was then observed under a microscope. If most cells became rounded and detached, the flask was quickly returned to a clean workbench, and 5 ml of complete culture medium containing 10% serum was added to stop the digestion. The cells were gently pipetted until completely detached, then aspirated and centrifuged at 250g for 8-10 minutes. The supernatant was discarded, and 1-2 ml of culture medium was added and resuspended. Culture medium was added at a ratio of 5-6 ml / flask, and the cell suspension was aliquoted into fresh T75cm culture flasks containing 5-6 ml of culture medium at a ratio of 1:2 to 1:4. 2 Cells in the logarithmic growth phase were selected and grafted into the left axilla of mice in groups 5 to 8 to establish a mouse tumor model. On the day of inoculation, the tumor cell solution was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the final cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 5 One cell / 100 μL was injected subcutaneously into the left axilla to graft tumors in mice.

[0131] 78A1, 78A1-SMe, and 78A1-C(CH3)2SMe were used to prepare PBS solutions of appropriate concentrations containing the three different components, using phosphate-buffered saline (PBS) as the solvent. Approximately one week after inoculation, the tumors grew to 100 mm. 3 The mice were injected with 50 μL of each of the above-mentioned PBS, 78A1 solution, 78A1-SMe, and 78A1-C(CH3)2SMe solutions into their respective tumors using a 1 mL syringe. The dosages for each group of mice are shown in Table 4.

[0132] Table 4

[0133] mice Injection Dosage Group 5 78A1 57μg Group 6 78A1-SMe The molar amount is consistent with that of 57 μg 78A1. Group 7 <![CDATA[78A1-C(CH3)2SMe]]> The molar amount is consistent with that of 57 μg 78A1. Group 8 PBS ——

[0134] In groups 5, 6, and 7, orbital blood was collected from three mice 30 min, 60 min, and 240 min after intratumoral injection, respectively. In the PBS group, orbital blood was collected from three mice 1 h after intratumoral injection. After collecting orbital blood from each group of mice, the blood was centrifuged at 3800 rpm for 10 min to separate the serum. The concentrations of TNF-α and IL-6 in the serum were then measured using the same methods as in the previous experiments.

[0135] Experimental results are as follows Figure 6 As shown in Figure A, the results indicated that intratumoral rapid injection of 78A1 significantly increased the serum TNF-α level in mice. One hour after injection, the serum TNF-α level reached its peak, with an increase of 25-30 times, which was significantly different from the PBS group. Within 4 hours of intratumoral rapid injection of 78A1-SMe, the serum TNF-α level only increased by 10-15 times, which was weaker compared to the 25-30 times increase in serum TNF-α level in mice injected with 78A1. Figure 6As shown in Figure B, the results indicated that 4 hours after intratumoral injection of 78A1, the serum IL-6 level in mice was close to 900 pg / mL, representing a 30-fold increase compared to the PBS group, with a significant difference. However, within 4 hours after intratumoral injection of 78A1-SMe and 78A1-C(CH3)2SMe, the serum IL-6 levels were only 3-7 times higher than those in the PBS group, with no significant difference, and were significantly lower than those in mice injected with 78A1. IL-6, as a core marker of cytokine storms, is associated with multiple aspects of vascular homeostasis and cellular inflammation. It stimulates energy flow in muscle and adipose tissue, raises body temperature, and can cross the blood-brain barrier, altering the body's temperature set point by affecting PGE2 synthesis. The terrifying side effects of excessive IL-6 production have been reported in CAR-T cell therapy, and it is also a fatal factor in severe cases of COVID-19 infection, as well as a key marker of the side effects of immune agonist drugs. These results indicate that intratumoral injection of 78A1-SMe and 78A1-C(CH3)2SMe at the tested doses significantly reduced blood IL-6 levels compared to the original molecule 78A1, potentially representing better clinical application potential.

[0136] Figure 5 subcutaneous injection and Figure 6 Intratumoral injection data show that, compared to the 78A1 molecule, the 78A1-SMe of this application reduces TNF-α concentration, and both 78A1-SMe and 78A1-C(CH3)2SMe significantly reduce IL6 concentration. Because 78A1-SMe and 78A1-C(CH3)2SMe have higher lipid solubility than 78A1, they can release molecules more slowly in the blood to exert their effects. While ensuring drug activity, they can further reduce side effects. This indirectly indicates that 78A1-SMe and MeS-C(CH3)2-78A1 are safer and more suitable for in vivo injection as active drugs than 78A1.

[0137] (4) Detection of the induction activity of subcutaneous injection of 78A1-SS-C(CH3)2-78A1 on the expression of TNF-α and IL-6 in mouse serum.

[0138] Take 78A1 and 78A1-SS-C(CH3)2-78A1, and use phosphate buffered saline (PBS) as solvent to prepare PBS solutions of appropriate concentrations containing the two different components.

[0139] Identical mice were divided into three groups: group 9, group 10, and group 11, with 9 mice in each of groups 9 and 10, and 3 mice in group 11. Mice in group 9 were subcutaneously injected with 100 μL of the aforementioned 78A1 solution, mice in group 10 were subcutaneously injected with 100 μL of the aforementioned 78A1-SS-C(CH3)2-78A1 solution, and mice in group 11 were subcutaneously injected with 100 μL of PBS solution. The injection dosages for each group are shown in Table 5.

[0140] Table 5

[0141]

[0142]

[0143] In groups 9 and 10, orbital blood was collected from three mice at 30, 60, and 240 minutes after subcutaneous injection. In group 11, orbital blood was collected from three mice 1 hour after subcutaneous injection. After orbital blood collection, the mouse blood was centrifuged at 3800 rpm for 10 minutes to separate serum. The concentrations of TNF-α and IL-6 in the serum were detected using the same method described above.

[0144] Experimental results are as follows Figure 7 As shown, according to Figure 7 The results of the study showed that mice injected subcutaneously with 78A1 had a significantly increased serum TNF-α level, with the peak level reaching 1 hour after injection, representing an 8-fold increase. In the subcutaneous 78A1-SS-C(CH3)2-78A1 group, the serum TNF-α level remained essentially at the baseline of the PBS control group within 4 hours of injection, showing no significant increase. Compared to the increase in the 78A1-injected group, the increase in TNF-α in the 78A1-SS-C(CH3)2-78A1-injected group was significantly reduced. Figure 7 The results of the study showed that mice injected subcutaneously with 78A1 experienced a significant increase in serum IL-6 levels, with peak levels occurring 4 hours after injection, representing a 20-24 fold increase. In contrast, mice injected subcutaneously with 78A1-SS-C(CH3)2-78A1 showed only a 1-2 fold increase in serum IL-6 levels within 4 hours of injection. Compared to the increase observed in the 78A1-injected group, the increase in IL-6 levels in the 78A1-SS-C(CH3)2-78A1-injected group was significantly weaker.

[0145] Figure 7 Data Explanation: In vivo experiments showed that, compared to the 78A1 molecule, 78A1-SS-C(CH3)2-78A1 could better reduce the concentrations of TNF-α and IL-6, indicating that the precursor molecule 78A1-SS-C(CH3)2-78A1 had fewer side effects and significantly improved safety in vivo, making it more suitable for in vivo injection.

[0146] (5) Antitumor activity detection

[0147] 5.1 Using the same mice as described above (SPF-grade BALB / C, 5-6 weeks old, 18-20 grams female mice), divided into 3 groups (groups 12 to 14), with 6 mice in each group, tumor models were established in mice using the same method described above. Approximately one week after inoculation, the tumors grew to 100 mm. 3 Using PBS as a solvent, solutions of 78A1 and 78A1-SMe at appropriate concentrations were prepared and administered to mice via intratumoral injection. Groups 12 and 13 received rapid intratumoral injection using a 1 mL syringe, with an injection volume of 50 μL. Group 14 received an intratumoral injection of 50 μL of PBS solution. The dosages of the drugs and compounds administered to mice in groups 12 through 14 are shown in Table 6.

[0148] Table 6

[0149] mice Injection Dosage Group 12 78A1 solution 11μg Group 13 78A1-SMe The molar amount is consistent with that of 11 μg 78A1. Group 14 PBS solution ——

[0150] Mice in groups 12 to 14 of this embodiment were administered the drug twice, once every 3 days. During the drug administration period, changes in mouse body weight and tumor volume were measured every 2 days until day 19 post-administration. Tumor volume was measured using calipers to determine the major axis (l) and minor axis (w) of the tumor, and the volume was calculated using the formula: v = 0.5 × l × w. 2 The tumor volume was calculated. After 20 days, the treatment ended, the mice were euthanized, and the tumors were dissected, photographed, and the results analyzed.

[0151] Experimental results are as follows Figure 8 As shown: Figure 8 The results showed that, compared to the PBS injection group, the mice in the intratumoral rapid injection 78A1 group exhibited a significant decrease in body weight in the early stages of treatment, with the most pronounced decrease occurring on day 7 after the first administration. However, the body weight of mice in the intratumoral rapid injection 78A1-SMe group did not show a significant change compared to the PBS injection group. Figure 8 As shown in Figures B to D, both intratumoral rapid injection of 11 μg of 78A1 and 78A1-SMe (equimolar amount of 78A1) effectively inhibited tumor growth, and there was no significant difference between the 78A1-SMe group and the 78A1-injected group. This indicates that the antitumor effect of 78A1-SMe is basically equivalent to that of 78A1. However, because the 78A1 group experienced a more significant reduction in body weight, 78A1-SMe has fewer side effects and therefore better clinical application value than 78A1.

[0152] 5.2 Replace 78A1-SMe in Table 6 with 78A1-C(CH3)2SMe, and perform the same experimental procedure as described above. The experimental results are as follows: Figure 9As shown.

[0153] Figure 9 As shown in Figure A, compared to the PBS injection group, mice in both the intratumoral rapid injection of 78A1 and 78A1-C(CH3)2SMe groups exhibited a transient weight loss trend in the early stages of treatment, with the 78A1 group experiencing a smaller average weight loss. However, near the end of the experiment, the 78A1-C(CH3)2SMe group showed a greater weight recovery, more closely resembling the PBS group. Figure 9 As shown in B, C, and D, both 11 μg of 78A1 and 78A1-C(CH3)2SMe (equimolar amount of 78A1) administered intratumorally effectively inhibited tumor growth. The antitumor effect of the 78A1-C(CH3)2SMe group was weaker than that of the 78A1 group, but the tumor inhibition rate was still greater than 60%. Because 78A1-C(CH3)2SMe contains two methyl groups, its molecular steric hindrance is greater, affecting its binding to the receptor and thus its molecular activity, but it still has some antitumor effect. These results indicate that 78A1-C(CH3)2SMe showed a significant decrease in antitumor effect compared to the original molecule, with a slight improvement in animal weight, failing to demonstrate ideal application value. This suggests that modifications based on the 78A1 structure have significant uncertainties, and in vitro experimental results must be verified using animal models.

[0154] 5.3 Replace 78A1-SMe in Table 6 with 78A1-SS-C(CH3)2-78A1, and perform the same experimental procedure as described above. The experimental results are as follows: Figure 10 As shown.

[0155] Figure 10 The results showed that, compared to the PBS injection group, the mice in the intratumoral rapid injection 78A1 group exhibited a significant decrease in body weight in the early stages of treatment. However, the body weight of mice injected with intratumoral rapid injection 78A1-SS-C(CH3)2-78A1 showed no significant change compared to the PBS injection group. Figure 10 As shown in B, C, and D, an intratumoral injection of 11 μg of 78A1-SS-C(CH3)2-78A1 effectively inhibited tumor growth, showing no significant difference compared to the results of 78A1 injection. Although the addition of two methyl groups to 78A1-SS-C(CH3)2-78A1 increases its steric hindrance and affects its binding to receptors, as a precursor molecule, it can be enzymatically hydrolyzed in vivo to produce 78A1-C(CH3)2SMe and 78A1-SMe to exert its active effect. Therefore, compared to using 78A1-C(CH3)2SMe alone as a drug, 78A1-SS-C(CH3)2-78A1 exhibits better drug activity and fewer side effects, making it more suitable for in vivo injection therapy for tumors.

[0156] (6) Comparison experiment with 78A1-SH

[0157] Mouse PBMCs were prepared using the same method described above. Six concentrations of RPMI-1640 cell culture medium were prepared for each of the two compounds, 78A1-SH and 78A1-SMe. 78A1-SH served as the positive control, 78A1-SMe as the experimental group, and RPMI-1640 cell culture medium as the blank control. 40 μL of RPMI-1640 cell culture medium from each group was added to a final volume of 160 μL (1.6 x 10⁻⁶). 5 Peripheral lymphocyte culture solutions were prepared in each well of normal PBMC cells, and the concentrations of different compounds in each peripheral lymphocyte culture solution were as shown in Table 7.

[0158] Table 7

[0159]

[0160]

[0161] After incubating the peripheral lymphocyte culture solutions of each group at 37℃ and 5% CO2 for 24 hours, centrifuging at 1000 rpm for 5 minutes, the cell supernatant was collected, and the TNF-α concentration in the peripheral blood mononuclear cell culture supernatant was detected according to the ELISA instructions. The mouse TNF-α double antibody sandwich ELISA detection kit used for the detection was manufactured by Wuhan Sanying, and the product code was KE10002.

[0162] The structural formula of the 78A1-SH is:

[0163] Experimental results are as follows Figure 11 As shown, within the tested dose range, the TNF-α concentrations produced by 78A1-SH and 78A1-SMe exhibited a dose-dependent relationship. However, the minimum effective concentration of 78A1-SMe was lower than that of 78A1-SH. This is because the 78A1-SH molecule contains a thiol group, making it unstable and prone to dimerization. This reduces its ability to directly target the infection site. In contrast, the 78A1-SMe molecule of this application is stable and can directly target the infection site, resulting in a lower effective concentration. Therefore, 78A1-SMe of this application exhibits better pharmacological activity than 78A1-SH.

[0164] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An imidazoquinoline compound having an alkylthio group, characterized in that, Having the structure shown in formula (1) or a medicinal salt thereof; 。 2. The use of the imidazoquinoline compound as described in claim 1 in the preparation of an anti-colon cancer drug.

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