Thiourea derivative and application thereof

Abstract

The structure of the thiourea derivative is shown in the specification, and R1 is selected from any one of isopropyl, isobutyl, tertiary butyl, furyl, thienyl, phenyl and pyridyl; r2 is selected from any one of hydrogen, amino, hydroxyl, halogen and methyl; x is selected from any one of C and N; y is selected from any one of C and N; z is H or hydroxyl; n is 1 or 2. The thiourea derivative provided by the invention can activate TLR2, and has an immunostimulatory activity effect on human PBMC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a thiourea derivative and its uses. Background Technology

[0002] Toll-like receptors (TLRs) are an important class of pattern recognition receptors that play a crucial role in the innate immune system. Located on the cell membrane surface, their main function is to act as "forefront sentinels" of the immune system, continuously monitoring the extracellular environment and recognizing pathogen-associated molecular patterns (PAMPs) to initiate an immune response. TLR2, as an important member of the TLR family, has received widespread attention since its discovery due to its unique role in immune recognition and regulation.

[0003] The TLR2 gene is located on human chromosome 4q32 and encodes a transmembrane glycoprotein composed of extracellular, transmembrane, and intracellular regions. The extracellular region contains multiple leucine-rich repeats (LRRs), which are responsible for recognizing specific molecular structures on the surface of various pathogens. Compared to other TLRs, TLR2's most significant characteristic is its broad recognition spectrum and function-dependent nature. TLR2 cannot function independently; it must form heterodimers with TLR1 or TLR6 (forming TLR2 / 1 and TLR2 / 6 complexes, respectively) to effectively recognize and bind its ligands and initiate downstream signaling, thereby expanding its pathogen recognition range.

[0004] TLR2 plays a crucial role in the body's defense against pathogen infection. Studies have shown that TLR2-deficient mice are more susceptible to infections from various bacteria (such as Staphylococcus aureus and Streptococcus pneumoniae) and fungi (such as Candida albicans), suggesting that TLR2 plays a key role in fighting bacterial and fungal infections. Furthermore, TLR2 is involved in the immune response to certain viral infections, such as hepatitis C virus. Beyond anti-infective immunity, TLR2 is also closely related to the development and progression of various diseases. In autoimmune diseases, abnormal activation of TLR2 may lead to the immune system attacking its own tissues, such as in rheumatoid arthritis and systemic lupus erythematosus. In inflammatory diseases, TLR2-mediated excessive inflammatory responses may exacerbate tissue damage, such as in sepsis and atherosclerosis. In addition, TLR2 may also play a role in tumorigenesis, development, and immune escape; the specific mechanisms require further investigation.

[0005] In recent years, research on TLRs has made many new advances, and the clinical development of its targeted modulators (agonists and antagonists) has become a new direction for the treatment of infectious, oncological, and immune-related diseases. TLR agonists exert their effects by activating the immune response. For example, imiquimod, the first approved TLR7 / 8 agonist, is used topically for the treatment of condyloma acuminata and skin cancer; TLR9 agonists, such as CpG oligonucleotides, enhance the immune efficacy of hepatitis B vaccines as adjuvants and show synergistic anti-tumor potential in the treatment of lymphoma and other tumors; and TLR3 agonists, such as polyinosinic-polycytidylic acid, are used in antiviral and tumor immunotherapy clinical trials. Antagonists targeting TLR overactivation, such as the TLR4 antagonist Eritoran, have been attempted for the treatment of sepsis; the TLR2 antagonist TAK-042 is under clinical evaluation in rheumatoid arthritis; and TLR7 antagonists have shown the potential to suppress autoimmune responses in the treatment of systemic lupus erythematosus. Despite challenges in targeting specificity and delivery systems, TLR modulators have evolved from simple immune activation to precise intervention, providing innovative strategies for infection control, tumor immunology, and the treatment of autoimmune diseases. Their clinical value has been continuously expanded through randomized controlled trials. Summary of the Invention

[0006] In view of the above background technology, the purpose of this invention is to provide a thiourea derivative and its uses. The thiourea derivative provided by this invention can selectively activate TLR1-TLR2 and has an immune activation effect on human PBMCs, which has important research and development value and significance.

[0007] On the one hand, the present invention provides a thiourea derivative, the structure of which is shown in Formula I.

[0008]

[0009] In Formula I, R1 is selected from any one of isopropyl, isobutyl, tert-butyl, furanyl, thiophene, phenyl and pyridyl; R2 is selected from any one of hydrogen, amino, hydroxyl, halogen and methyl; X is selected from any one of C and N; Y is selected from any one of C and N; Z is H or hydroxyl; n is 1 or 2.

[0010] In the technical solution of this invention, the position of the R2 substituent is not particularly limited.

[0011] In some specific embodiments, the thiourea derivatives are listed below:

[0012]

[0013] In another aspect, the present invention provides a method for preparing the above-mentioned thiourea derivative, comprising the following steps:

[0014] 1) Precipitated sulfur and diethylamine undergo a reflux reaction to obtain

[0015] 2) It reacts with sulfur phosgene in the presence of an alkaline environment to obtain

[0016] 3) and The thiourea derivative was obtained by reflux reaction.

[0017] In a preferred embodiment, in step 1), the... The molar ratio of precipitated sulfur to diethylamine is 1:1~2:1~2:1~2;

[0018] Preferably, in step 1), the reflux reaction time is 4 to 6 hours;

[0019] Preferably, in step 1), the reflux reaction is carried out in a solvent; the solvent is selected from at least one of ethanol, isopropanol and methanol.

[0020] In a preferred embodiment, in step 2), The molar ratio with sulfur phosgene is 1:2 to 3;

[0021] Preferably, in step 2), the reaction is carried out in an ice bath;

[0022] Preferably, in step 2), the reaction time is 0.5 to 1 hour;

[0023] Preferably, in step 2), the base is selected from at least one of triethylamine and N,N-diisopropylethylamine;

[0024] Preferably, in step 2), the reaction is carried out in a solvent; the solvent is selected from at least one of dichloromethane and tetrahydrofuran;

[0025] In a preferred embodiment, in step 3), and The molar ratio is 1:1.2 to 1.5;

[0026] Preferably, in step 3), the reflux reaction time is 6-8 hours;

[0027] Preferably, in step 3), the reflux reaction is carried out in a solvent; the solvent is selected from at least one of ethanol and methanol.

[0028] In some specific embodiments, the preparation method is as follows:

[0029]

[0030] Compounds A and B were dissolved in a solvent, and precipitated sulfur and diethylamine were added. Under reflux, the reaction was monitored by TLC until complete, and compound C was obtained by column chromatography. Compound C was dissolved in a solvent, and triethylamine was added. Under ice bath conditions, sulfur phosgene was added dropwise to carry out the reaction, and the reaction was monitored by TLC until completion. The solvent was evaporated under reduced pressure, and dichloromethane or tetrahydrofuran was added to the residue. The residue was washed, dried, and the solvent was removed under reduced pressure. Compound E and solvent were added, and the reaction was refluxed. The reaction was monitored by TLC until completion, and the thiourea derivative was obtained by column chromatography.

[0031] In another aspect, the present invention provides the use of the above-mentioned thiourea derivatives or pharmaceutically acceptable salts thereof in the preparation of TLR2 agonists.

[0032] In another aspect, the present invention provides the use of the above-mentioned thiourea derivatives or pharmaceutically acceptable salts thereof in the preparation of immunomodulators. In the technical solution of the present invention, the above-mentioned thiourea derivatives have an immune-activating effect on human PBMCs.

[0033] In another aspect, the present invention provides a pharmaceutical composition for activating TLR2, comprising the above-mentioned thiourea derivative or a pharmaceutically acceptable salt thereof as an active substance.

[0034] In another aspect, the present invention provides a pharmaceutical composition that can stimulate the activation of human PBMC cells, comprising the above-mentioned thiourea derivative or a pharmaceutically acceptable salt thereof as an active substance.

[0035] In the technical solution of the present invention, the pharmaceutical composition is an injectable preparation, an oral preparation, or a topical preparation.

[0036] In the technical solution of the present invention, the pharmaceutical composition is a tablet, capsule, powder, pill, granule, injection or emulsion.

[0037] The thiourea derivative provided by this invention can activate TLR2 and exhibits immunostimulatory activity against human PBMCs. Furthermore, the thiourea derivative provided in this application allows for in-depth research on TLR2, contributing not only to understanding the working mechanism of the innate immune system but also providing new targets for the diagnosis and treatment of related diseases. This thiourea derivative can be used to enhance the body's anti-infective immunity and for tumor immunotherapy. Attached Figure Description

[0038] Figure 1 This is the activation effect of compound 15 on TLR2.

[0039] Figure 2 This study investigated the immunostimulatory activity of compound 15 on human PBMCs. Detailed Implementation

[0040] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0042] Example 1

[0043] This embodiment provides a thiourea derivative 1-(4-fluoronaphthyl-1-yl)-3-(3-(furan-2-carbonyl)-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 1), the structure of which is The preparation process is as follows:

[0044] Will (1mmol) and Dissolve 1 mmol in 5 mL of ethanol, add 1 mmol of diethylamine and 1 mmol of precipitated sulfur, heat to reflux, monitor the reaction by thin-layer chromatography (TLC) until completion, evaporate the solvent under reduced pressure, and separate by column chromatography to obtain

[0045] Will Dissolve 1 mmol in 6 mL of dichloromethane, add 0.5 mL of triethylamine, and add 2 mmol of phosgene dropwise under ice bath conditions; monitor the reaction by TLC until completion, remove the solvent under reduced pressure, add an appropriate amount of dichloromethane, wash twice with water (2 × 5 mL), and dry with anhydrous sodium sulfate; remove the solvent under reduced pressure, add 5 mL of ethanol, and add... (1.2 mmol), heated to reflux, and the reaction was monitored by TLC until completion; the solvent was evaporated under reduced pressure, and compound 1 was separated by column chromatography.

[0046] The 1H NMR spectrum of compound 1 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 8.13 (d, J = 8.0 Hz, 1H), 7.64–7.49 (m, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.32–7.23 (m, 2H), 7.16 (t, J₁ = 9.2 Hz, J₂ = 18 Hz, 1H), 6.14 (s, 1H), 5.83 (s, 1H), 3.05–2.86 (m, 2H), 2.49–2.28 (m, 4H). The ¹H NMR spectrum of compound 1 is as follows: 13C NMR (101MHz, CDCl3) δ179.6,178.2,160.0,157.5,144.8,142.8,142.1,135.5,134.4,130.7,1 28.5,127.0,126.3,124.3,122.2,121.4,119.9,115.8,111.5,109.5,109.3,30.5,29.3,26.8.

[0047] Example 2

[0048] This embodiment provides a thiourea derivative 1-(4-fluoronaphthyl-1-yl)-3-(3-(thiophene-2-carbonyl)-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 2), the structure of which is

[0049] In this embodiment, the preparation method of compound 2 is the same as in Example 1, except that... Replace with

[0050]

[0051] The 1H NMR spectrum of compound 2 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 8.15–8.05 (m, 1H), 7.64–7.53 (m, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.28–7.16 (m, 2H), 7.14–7.02 (m, 1H), 6.89–6.59 (m, 2H), 2.94 (t, J₁ = 7.2 Hz, J₂ = 14.4 Hz, 2H), 2.37–2.23 (m, 2H), 2.22–1.97 (m, 2H). The ¹H NMR spectrum of compound 2 is as follows: 13 C NMR (101MHz, CDCl3) δ179.0,178.3,159.9,157.4,147.6,144.2,135.6,133.8,131.6,129.6,129. 5,128.5,127.1,127.0,126.0,125.9,124.1,122.3,121.5,121.1,109.4,109.2,30.4,28.4,26.7.

[0052] Preparation of Example 3:

[0053] This embodiment provides a thiourea derivative 1-(3-benzoyl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)-3-(4-fluoronaphthyl-1-yl)thiourea (compound 3), the structure of which is

[0054] In this embodiment, the preparation method of compound 3 is the same as in Example 1, except that... Replace with

[0055]

[0056] The 1H NMR spectrum of compound 3 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 12.64 (s, 1H), 8.28–7.98 (m, 2H), 7.69–7.49 (m, 3H), 7.44 (d, J = 7.2Hz, 1H), 7.41–7.32 (m, 1H), 7.28–7.15 (m, 2H), 7.09–6.62 (m, 2H), 3.05–2.64 (m, 2H), 2.37–2.12 (m, 2H), 2.09–1.97 (m, 2H). The ¹H NMR spectrum of compound 3 is as follows: 13 CNMR (101MHz, CDCl3) δ179.1,177.9,159.7,157.1,154.0,143.1,135.6,133.7,130.6,129.9,129.0,1 28.4,127.6,127.3,127.0,126.9,124.1,124.0,122.5,121.3,119.7,109.2,109.0,30.2,28.4,26.7.

[0057] Example 4

[0058] This embodiment provides a thiourea derivative 1-(4-fluoronaphthyl-1-yl)-3-(3-(pyridin-2-yl)formyl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)thiourea (compound 4), the structure of which is

[0059] In this embodiment, the preparation method of compound 4 is the same as in Example 1, except that... Replace with

[0060]

[0061] The 1H NMR spectrum of compound 4 is as follows: 1¹H NMR (400MHz, CDCl₃) δ 8.38 (s, 1H), 8.02 (d, J = 7.2Hz, 1H), 7.63–7.45 (m, 3H), 7.44–7.33 (m, 1H), 7.32–7.24 (m, 1H), 7.17–7.07 (m, 1H), 7.01 (t, J₁ = 8.8Hz, J₂ = 17.6Hz, 1H), 5.87 (s, 1H), 2.91 (t, J₁ = 7.2Hz, J₂ = 14.8Hz, 2H), 2.30–2.16 (m, 2H), 1.89 (s, 2H). The carbon NMR spectrum of compound 4 is as follows: 13 C NMR (101MHz, CDCl3) δ179.8,177.6,159.8,157.3,151.0,149.3,148.9,144.1,135.8,134.9,133. 3,130.5,128.4,127.5,126.9,124.3,124.0,123.8,122.8,121.2,119.2,109.2,30.3,28.2,26.7.

[0062] Example 5

[0063] This embodiment provides a thiourea derivative 1-(4-fluoronaphthyl-1-yl)-3-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 5), the structure of which is

[0064] In this embodiment, the preparation method of compound 5 is the same as in Example 1, except that... Replace with

[0065]

[0066] The 1H NMR spectrum of compound 5 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.19 (s, 1H), 8.65 (s, 1H), 8.17 (d, J = 8.0Hz, 1H), 7.96 (d, J = 8.0Hz, 1H), 7.76–7.41 (m, 3H), 7.33–7.15 (m, 1H), 3.08–3.00 (m, 2H), 2.92–2.79 (m, 4H), 2.42–2.34 (m, 2H), 0.91 (d, J = 6.8Hz, 6H). The ¹H NMR spectrum of compound 5 is as follows: 13C NMR (101MHz, CDCl3) δ202.2,178.1,160.0,157.5,155.9,138.9,132.9,131.5,131.4,128.1,127.8,126.9,126 .9,126.2,126.2,124.9,124.7,122.4,122.4,121.2,121.2,117.2,109.6,109.4,37.2,31.1,28.5,27.8,18.6.

[0067] Example 6

[0068] This embodiment provides a thiourea derivative 1-(4-fluoronaphthyl-1-yl)-3-(3-neopentyl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 6), the structure of which is

[0069] In this embodiment, the preparation method of compound 6 is the same as in Example 1, except that... Replace with

[0070]

[0071] The 1H NMR spectrum of compound 6 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 11.81 (s, 1H), 8.20 (d, J = 8.8Hz, 1H), 8.15–7.86 (m, 2H), 7.69–7.57 (m, 2H), 7.52 (s, 1H), 7.25 (s, 1H), 2.93–2.72 (m, 4H), 2.43–2.25 (m, 2H), 0.99 (s, 9H). The ¹H NMR spectrum of compound 6 is as follows: 13 C NMR (101MHz, CDCl3) δ206.9,160.2,157.7,138.0,134.8,131.6,128.3,127.6, 127.1,126.5,124.9,122.5,121.3,109.6,109.3,43.8,33.9,28.7,28.2,26.7.

[0072] Example 7

[0073] This embodiment provides a thiourea derivative, 1-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl)-3-(4-fluoronaphthyl-1-yl)thiourea (compound 7), with the following structure:

[0074] In this embodiment, the preparation method of compound 7 is the same as in Example 1, except that... Replace with Will Replace with

[0075] The 1H NMR spectrum of compound 7 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 8.01 (d, J = 8.8 Hz, 1H), 7.61–7.42 (m, 3H), 7.26–7.08 (m, 4H), 7.05–6.92 (m, 1H), 6.83 (t, J₁ = 7.6 Hz, J₂ = 15.2 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 5.72 (s, 1H), 2.72 (t, J₁ = 6.0 Hz, J₂ = 12.0 Hz, 2H), 1.79–1.64 (m, 2H), 1.61–1.42 (m, 4H). The carbon NMR spectrum of compound 7 is as follows: 13 C NMR (101MHz, CDCl3) δ177.9,174.2,159.6,157.0,154.1,138.1,133.7,130.8,129.9,129.2,128.3,1 27.6,127.5,127.2,127.0,126.8,126.7,124.1,122.5,121.3,109.1,108.9,26.0,24.7,22.1,21.9.

[0076] Example 8

[0077] This embodiment provides a thiourea derivative, 1-(4-fluoronaphthyl-1-yl)-3-(3-(furan-2-carbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl)thiourea (compound 8), with the following structure:

[0078] In this embodiment, the preparation method of compound 8 is the same as in Example 1, except that... Replace with

[0079] The 1H NMR spectrum of compound 8 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 8.16–8.07 (m, 1H), 7.61–7.52 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.27–7.16 (m, 2H), 7.15–7.05 (m, 1H), 6.13–6.03 (m, 1H), 5.87 (d, J = 3.6 Hz, 1H), 2.77 (t, J₁ = 6.4 Hz, J₂ = 12.4 Hz, 2H), 1.92–1.77 (m, 4H), 1.69–1.58 (m, 2H). The ¹H NMR spectrum of compound 8 is as follows: 13C NMR (101MHz, CDCl3) δ178.6,174.9,159.9,157.3,143.8,142.7,141.2,139.7,134.1,130.7,128. 4,126.9,126.5,126.2,124.5,122.3,121.3,114.5,111.0,109.3,109.1,26.2,23.6,22.3,21.9.

[0080] Example 9

[0081] This embodiment provides a thiourea derivative 1-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)-3-(naphthyl-1-yl)thiourea (compound 9), the structure of which is as follows:

[0082] In this embodiment, the preparation method of compound 9 is the same as in Example 1, except that... Replace with Will Replace with

[0083] The 1H NMR spectrum of compound 9 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.24 (s, 1H), 8.06 (s, 1H), 7.96–7.80 (m, 3H), 7.63 (s, 2H), 7.56–7.53 (m, 2H), 3.04–3.01 (m, 1H), 2.92–2.83 (m, 4H), 2.43–2.38 (m, 2H), 0.90 (d, J = 6.8 Hz, 6H). The ¹H NMR spectrum of compound 9 is as follows: 13 C NMR (101MHz, CDCl3) δ202.0,178.0,155.9,138.9,134.8,132.9,131.7,129.8,129 .1,128.5,127.1,126.6,125.8,125.5,122.1,117.2,37.2,31.1,28.5,27.8,18.6.

[0084] Example 10

[0085] This embodiment provides a thiourea derivative 1-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)-3-(2-methylnaphthyl-1-yl)thiourea (compound 10), the structure of which is as follows:

[0086] In this embodiment, the preparation method of compound 10 is the same as in Example 1, except that... Replace with Will Replace with

[0087] The 1H NMR spectrum of compound 10 is as follows: 1 ¹H NMR (400MHz, CDCl₃). δ 12.80 (s, 1H), 7.96–7.89 (m, 4H), 7.53–7.46 (m, 3H), 3.00–2.80 (m, 5H), 2.51 (s, 3H), 2.40–2.35 (m, 2H), 0.83 (d, J = 6.4 Hz, 6H). The ¹H NMR spectrum of compound 10 is as follows: 13 C NMR (101MHz, CDCl3) δ201.8,178.0,155.5,138.8,135.2,133.3,132.8,130.8,129.3, 129.0,128.8,128.2,127.2,125.7,122.1,117.3,37.2,31.1,28.5,27.8,18.6,18.3.

[0088] Example 11

[0089] This embodiment provides a thiourea derivative 1-(7-hydroxynaphthyl-1-yl)-3-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 11), the structure of which is as follows:

[0090] In this embodiment, the preparation method of compound 11 is the same as in Example 1, except that... Replace with Will Replace with

[0091] The 1H NMR spectrum of compound 11 is as follows: 1 ¹H NMR (400MHz, DMSO-d6). δ 12.98 (s, 1H), 10.97 (s, 1H), 9.88 (s, 1H), 7.85 (t, J1 = 8.8Hz, J2 = 14.8Hz, 2H), 7.42–7.32 (m, 2H), 7.10 (d, J = 7.2Hz, 2H), 3.15 (s, 1H), 2.94–2.77 (m, 4H), 2.38–2.31 (m, 2H), 0.96 (s, 6H). The ¹H NMR spectrum of compound 11 is as follows: 13CNMR(101MHz,DMSO-d6)δ202.0,178.1,156.6,156.4,139.1,132.0,132.0,130.4,129.3, 128.2,126.4,122.8,119.4,116.3,104.4,56.5,37.1,31.1,28.5,27.8,19.4,19.2,19.0.

[0092] Example 12

[0093] This embodiment provides a thiourea derivative 1-(4-hydroxynaphthyl-1-yl)-3-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 12), the structure of which is

[0094] In this embodiment, the preparation method of compound 12 is the same as in Example 1, except that... Replace with Will Replace with

[0095] The 1H NMR spectrum of compound 12 is as follows: 1 ¹H NMR (400MHz, DMSO-d6). δ 12.70 (s, 1H), 10.74 (s, 1H), 10.44 (s, 1H), 8.21 (d, J = 8Hz, 1H), 7.71 (d, J = 7.6Hz, 1H), 7.54–7.45 (m, 2H), 7.33 (d, J = 7.6Hz, 1H), 6.95 (d, J = 7.6Hz, 1H), 3.19 (s, 1H), 2.89–2.74 (m, 4H), 2.31 (s, 2H), 0.90 (s, 6H). The carbon NMR spectrum of compound 12 is as follows: 13 C NMR(101MHz,DMSO-d6)δ201.9,139.0,132.0,131.3,127.2,127.0,125.7,125. 3,123.0,122.7,116.3,108.1,55.3,49.0,37.0,31.1,28.5,27.7,19.4,19.2.

[0096] Example 13

[0097] This embodiment provides a thiourea derivative, 1-(4-aminonaphthyl-1-yl)-3-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 13), with the following structure:

[0098] In this embodiment, the preparation method of compound 13 is the same as in Example 1, except that... Replace with Will Replace with

[0099] The 1H NMR spectrum of compound 13 is as follows: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.14 (d, J = 8.4Hz, 1H), 7.63 (d, J = 8.4Hz, 1H), 7.57–7.31 (m, 2H), 7.20 (d, J = 8.0Hz, 1H), 6.72 (d, J = 8.0Hz, 1H), 6.01 (s, 1H), 2.89 (s, 2H), 2.77 (t, J₁ = 7.2Hz, J₂ = 14.4Hz, 2H), 2.34–2.30 (m, 2H), 0.82 (s, 6H). The ¹H NMR spectrum of compound 13 is as follows: 13 C NMR (101MHz, DMSO-d6) δ139.0,131.9,131.1,127.3,126.7,124.4,123.6,123.3,122.8,107.2,55.3,37.0,31.1,28.5,27.7,19.2.

[0100] Example 14

[0101] This embodiment provides a thiourea derivative 1-(4-bromonaphth-1-yl)-3-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)thiourea (compound 14), the structure of which is

[0102] In this embodiment, the preparation method of compound 14 is the same as in Example 1, except that... Replace with Will Replace with

[0103] The 1H NMR spectrum of compound 14 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.36 (s, 1H), 8.33 (d, J = 9.2Hz, 1H), 8.10 (s, 1H), 8.00 (d, J = 9.2Hz, 1H), 7.92 (d, J = 8.0Hz, 1H), 7.73–7.55 (m, 2H), 7.49 (d, J = 8.0Hz, 1H), 3.08–3.01 (m, 1H), 2.94–2.81 (m, 4H), 2.44–2.36 (m, 2H), 0.93 (d, J = 6.8Hz, 6H). The ¹H NMR spectrum of compound 14 is as follows: 13C NMR (101MHz, CDCl3) δ202.4,177.8,155.9,139.0,133.2,133.0,131.8,131.0,129 .8,128.0,128.0,127.9,125.9,123.6,122.7,117.2,37.2,31.1,28.5,27.8,18.7.

[0104] Example 15

[0105] This embodiment provides a thiourea derivative 1-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)-3-(quinolin-5-yl)thiourea (compound 15), the structure of which is as follows:

[0106] In this embodiment, the preparation method of compound 15 is the same as in Example 1, except that... Replace with Will Replace with

[0107] The 1H NMR spectrum of compound 15 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.41 (s, 1H), 9.00 (s, 1H), 8.53–8.26 (m, 2H), 8.05 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.52 (s, 1H), 3.05 (d, J = 6.8 Hz, 1H), 2.94–2.83 (m, 4H), 2.56–2.31 (m, 2H), 0.92 (d, J = 6.4 Hz, 6H). The ¹H NMR spectrum of compound 15 is as follows: 13 C NMR (101MHz, DMSO-d6) δ202.4,178.5,156.4,151.0,148.5,139.2,132.3,129.8,128.8,126.3,125.4,122.1,116.4,37.2,31.1,28.5,27.8,19.2.

[0108] Example 16

[0109] This embodiment provides a thiourea derivative 1-(3-isobutyryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)-3-(isoquinoline-5-yl)thiourea (compound 16), the structure of which is

[0110] In this embodiment, the preparation method of compound 16 is the same as in Example 1, except that... Replace with Will Replace with

[0111] The 1H NMR spectrum of compound 16 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.36 (s, 1H), 9.38 (s, 1H), 8.57 (d, J = 5.6 Hz, 1H), 8.30 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.80–7.75 (m, 2H), 3.09–3.02 (m, 1H), 2.94–2.83 (m, 4H), 2.44–2.37 (m, 2H), 0.91 (d, J = 6.8 Hz, 6H). The ¹H NMR spectrum of compound 16 is as follows: 13 C NMR (101MHz, CDCl3) δ202.6,177.8,155.9,152.7,143.8,139.0,133.1,132 .9,131.5,129.8,128.5,127.5,117.2,115.4,37.2,31.1,28.5,27.8,18.6.

[0112] Example 17: Preparation of compound 17:

[0113] This embodiment provides a thiourea derivative 1-(3-isobutyryl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl)-3-(quinoline-5-yl)thiourea (compound 17), the structure of which is as follows:

[0114] In this embodiment, the preparation method of compound 17 is the same as in Example 1, except that... Replace with Will Replace with Will Replace with

[0115] The 1H NMR spectrum of compound 17 is as follows: 1¹H NMR (400MHz, CDCl₃) δ 13.28 (s, 1H), 8.98 (s, 1H), 8.36 (d, J = 8.4Hz, 1H), 8.29 (d, J = 8.4Hz, 1H), 8.22 (s, 1H), 7.87 (t, J₁ = 7.6Hz, J₂ = 16Hz, 1H), 7.68 (d, J = 7.2Hz, 1H), 7.48–7.45 (m, 1H), 3.14–3.04 (m, 1H), 2.70–2.66 (q, 4H), 1.80 (s, 4H), 0.85 (d, J = 6.8Hz, 6H). The ¹H NMR spectrum of compound 17 is as follows: 13 C NMR (101MHz, CDCl3) δ203.9,178.4,151.0,150.6,131.9,131.4,130.3,129.6, 128.8,127.5,126.3,125.6,121.8,121.6,37.6,26.8,24.5,23.0,22.6,18.9.

[0116] Example 18

[0117] This embodiment provides a thiourea derivative, 1-(3-isobutyryl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl)-3-(naphthyl-1-yl)thiourea (compound 18), with the following structure:

[0118] In this embodiment, the preparation method of compound 18 is the same as in Example 1, except that... Replace with Will Replace with Will Replace with

[0119] The 1H NMR spectrum of compound 18 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.05 (s, 1H), 8.02–7.95 (m, 4H), 7.64–7.59 (m, 2H), 7.57–7.51 (m, 2H), 3.09–3.03 (m, 1H), 2.68 (t, J₁ = 5.2 Hz, J₂ = 10.8 Hz, 4H), 1.90–1.72 (m, 4H), 0.83 (d, J = 6.4 Hz, 6H). The ¹H NMR spectrum of compound 18 is as follows: 13C NMR (101MHz, CDCl3) δ203.6,178.3,150.8,134.8,131.6,129.9,129.3,128.6,128.5, 127.3,127.2,126.6,125.8,125.6,122.1,121.8,37.6,26.8,24.5,23.1,22.6,18.8.

[0120] Example 19

[0121] This embodiment provides a thiourea derivative 1-(3-neovaleryl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)-3-(quinolin-5-yl)thiourea (compound 19), the structure of which is as follows:

[0122] In this embodiment, the preparation method of compound 19 is the same as in Example 1, except that... Replace with Will Replace with

[0123] The 1H NMR spectrum of compound 19 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 11.78 (d, J = 90.8 Hz, 1H), 8.98 (s, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.28–8.24 (m, 2H), 7.87–7.83 (m, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.48–7.45 (m, 1H), 2.90–2.81 (m, 4H), 2.38–2.31 (m, 2H), 1.04 (s, 9H). The ¹H NMR spectrum of compound 19 is as follows: 13 C NMR (101MHz, CDCl3) δ207.2,178.7,150.8,149.0,138.2,132.0,131.3,130.5,129.4,126.4,125.7,121.9,43.8,33.8,28.7,28.1,27.1,26.8.

[0124] Example 20

[0125] This embodiment provides a thiourea derivative 1-(quinolin-5-yl)-3-(3-(thiophene-2-carbonyl)-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 20), the structure of which is as follows:

[0126] In this embodiment, the preparation method of compound 20 is the same as in Example 1, except that... Replace with Will Replace with

[0127] The 1H NMR spectrum of compound 20 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 12.06 (s, 1H), 8.96 (s, 1H), 8.47–8.30 (m, 2H), 8.26 (d, J = 8.8 Hz, 1H), 7.94–7.81 (m, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.46–7.43 (m, 1H), 7.33 (s, 1H), 7.03 (t, J₁ = 4 Hz, J₂ = 8.4 Hz, 1H), 2.85 (t, J₁ = 7.2 Hz, J₂ = 14 Hz, 2H), 2.50 (t, J₁ = 6.8 Hz, J₂ = 14 Hz, 2H), 2.35–2.17 (m, 2H). The ¹H NMR spectrum of compound 20 is as follows: 13 C NMR (101MHz, CDCl3) δ184.9,177.8,154.3,150.9,149.0,143.0,139.8,134.6,132.5,1 32.4,131.7,131.0,130.5,129.4,127.0,126.1,125.4,121.9,119.0,31.3,28.9,28.4.

[0128] Example 21

[0129] This embodiment provides a thiourea derivative 1-(3-(furan-2-carbonyl)-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)-3-(naphthyl-1-yl)thiourea (compound 21), the structure of which is

[0130] In this embodiment, the preparation method of compound 21 is the same as in Example 1, except that... Replace with

[0131]

[0132] The 1H NMR spectrum of compound 21 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 7.92–7.89 (m, 2H), 7.59–7.42 (m, 4H), 7.34 (d, J = 7.2 Hz, 1H), 7.31–7.19 (m, 1H), 6.11–6.09 (m, 1H), 5.73 (d, J = 3.6 Hz, 1H), 3.00–2.95 (m, 2H), 2.66–2.17 (m, 4H). The ¹H NMR spectrum of compound 21 is as follows: 13C NMR (101MHz, CDCl3) δ179.5,178.1,144.6,144.5,142.8,142.3,138.4,135.6,134.1,129. 6,129.4,128.6,127.6,126.6,126.1,125.4,122.0,119.8,115.7,111.5,30.5,29.3,26.8.

[0133] Example 22

[0134] This embodiment provides a thiourea derivative 1-(naphth-1-yl)-3-(3-(pyridin-2-yl)formyl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)thiourea (compound 22), the structure of which is

[0135] In this embodiment, the preparation method of compound 22 is the same as in Example 1, except that... Replace with Will Replace with

[0136] The 1H NMR spectrum of compound 22 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 8.38 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.73 (d, J = 4.4Hz, 1H), 7.58 (d, J = 8.0Hz, 1H), 7.52–7.45 (m, 2H), 7.33 (d, J = 6.4Hz, 2H), 7.06 (t, J₁ = 5.2Hz, J₂ = 12.4Hz, 1H), 2.91 (t, J₁ = 7.2Hz, J₂ = 14.4Hz, 2H), 2.29–2.20 (m, 2H), 1.84 (m, 3H). The ¹H NMR spectrum of compound 22 is as follows: 13 C NMR (101MHz, CDCl3) δ179.7,177.6,151.0,149.4,148.8,143.8,137.4,135.5,135.0,133. 9,129.5,129.2,128.4,127.5,127.3,126.6,125.0,124.1,122.6,119.1,30.3,28.2,26.7.

[0137] Example 23: Preparation of compound 23:

[0138] This embodiment provides a thiourea derivative 1-(3-benzoyl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)-3-(naphthyl-1-yl)thiourea (compound 23), the structure of which is as follows:

[0139] In this embodiment, the preparation method of compound 23 is the same as in Example 1, except that... Replace with Will Replace with

[0140] The 1H NMR spectrum of compound 23 is as follows: 1 H NMR(400MHz, CDCl3)δ7.78(d,J=7.6Hz,1H),7.72(d,J=8.0Hz,1H),7.53-7.45(m ,3H),7.34(t,J1=7.6Hz,J2=15.6Hz,1H),7.29-7.20(m,3H),7.14(t,J1=6.8Hz,J 2=14.0Hz,1H),6.84(t,J1=7.6Hz,J2=15.2Hz,1H),6.74(d,J=7.6Hz,1H),2.90( t, J1=7.2Hz, J2=14.4Hz, 2H), 2.26-2.19(m,2H), 1.99-1.80(m,2H), 1.69(s,1H). The carbon NMR spectrum of compound 23 is as follows: 13 C NMR (101MHz, CDCl3) δ179.0,177.9,153.9,142.9,137.8,135.6,134.0,130.7,129.7,129. 3,129.3,129.0,128.5,127.4,127.2,127.1,126.5,125.0,122.3,119.5,30.2,28.4,26.7.

[0141] Example 24

[0142] This embodiment provides a thiourea derivative, 1-(naphthyl-1-yl)-3-(3-neopentyl-5,6-dihydro-4H-cyclopentano[b]thiophene-2-yl)thiourea (compound 24), with the following structure:

[0143] In this embodiment, the preparation method of compound 24 is the same as in Example 1, except that... Replace with Will Replace with

[0144] The 1H NMR spectrum of compound 24 is as follows: 1¹H NMR (400MHz, CDCl₃) δ 11.69 (s, 1H), 8.12 (s, 1H), 8.00–7.94 (m, 3H), 7.63–7.54 (m, 4H), 2.89–2.80 (m, 4H), 2.36–2.32 (m, 2H), 1.00 (s, 9H). The ¹H NMR spectrum of compound 24 is as follows: 13 C NMR (101MHz, CDCl3) δ207.1,178.3,138.0,134.8,131.7,130.1,129.3,128 .5,127.2,126.7,125.9,125.9,125.8,122.3,43.8,33.8,28.7,28.1,26.7.

[0145] Example 25

[0146] This embodiment provides a thiourea derivative 1-(3-isovaleryl-5,6-dihydro-4H-cyclopentano[b]thiophen-2-yl)-3-(naphthyl-1-yl)thiourea (compound 25), the structure of which is

[0147] In this embodiment, the preparation method of compound 25 is the same as in Example 1, except that... Replace with Will Replace with

[0148] The 1H NMR spectrum of compound 25 is as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 13.11 (s, 1H), 8.14 (s, 1H), 8.06–7.83 (m, 3H), 7.72–7.43 (m, 4H), 2.92–2.72 (m, 4H), 2.45–2.25 (m, 4H), 1.92–1.74 (m, 1H), 0.72 (d, J = 6.0 Hz, 6H). The ¹H NMR spectrum of compound 25 is as follows: 13 C NMR (101MHz, CDCl3) δ197.8,178.0,155.3,139.4,134.8,132.8,131.6,130.0,129.4, 128.5,127.3,126.7,125.8,125.7,122.1,118.2,49.7,31.5,28.5,27.8,24.9,22.3.

[0149] Example 1:

[0150] This invention investigated the activation effect of the thiourea derivatives prepared in Examples 1-25 on TLR2. The HEK BLUE TLR2 cells used in the experiment were owned by the applicant; fetal bovine serum was purchased from GIBICO, USA; cell culture plates were purchased from Corning Incorporated, USA; DMEM medium was purchased from GIBICO, USA; and Quanti-blue was purchased from InvivoGen, USA.

[0151] The experimental procedure is as follows:

[0152] First, adherent HEK-Blue hTLR2 cells were pipetted off to form a cell suspension, and then counted three times, with the average value taken. Next, the cells were plated by adding 30 μL of cell suspension (containing 2 × 10⁶ cells / mL) to each well of a 384-well plate. 4 (cells). Third, add 10 μL of the drug solution to each well and incubate the cells with the specific concentration of the test compound in an incubator containing 5% CO2 at 37°C for 24 hours. Finally, add 40 μL of the pre-prepared Quanti-Blue solution to each well of the 384-well plate. Wrap the plate in aluminum foil and incubate in the dark for 10-60 minutes, measuring the absorbance at 620 nm every 10 minutes. EC 50 Nonlinear fitting was performed using Hill1 in Growth / Sigmoidal. This experiment was independently repeated three times, and the standard deviation (SD) value was calculated.

[0153] The experimental results of this embodiment demonstrate that compounds 1-25 at different concentrations all have an activating effect on TLR2. The results of their TLR2 activation are shown in Table 1. Among them, the activation effect of compound 15 at different concentrations on TLR2 is as follows: Figure 1 As shown.

[0154] Table 1. Activation activity of compounds on TLR2

[0155]

[0156] a EC 50 The data were obtained from three independent, repeated experiments.

[0157] Example 2

[0158] The present invention tested the immunostimulatory activity of the thiourea derivative prepared in Example 15 on human peripheral blood mononuclear cells (PBMCs). Fetal bovine serum was purchased from GIBICO, USA; cell culture plates were purchased from Corning Incorporated, USA; DMEM and RPMI media were purchased from GIBICO, USA; and flow cytometry antibodies were purchased from Biolegend, USA.

[0159] The experimental steps are as follows:

[0160] First, on the first day, SK-OV-3 cells labeled with the red fluorescent protein dye mCherry were introduced at a density of 5 × 10⁶ cells per well. 4 The tumor cells were seeded in 6-well plates. After the tumor cells adhered, PBMC cells were seeded into the same 6-well plates, and a specified concentration of compound 15 was added. This process used RPMI 1640 complete medium (containing 10% FBS and 1% penicillin / streptomycin). The effector-to-target ratio of PBMC cells to tumor cells was 50:1. After co-incubation for 48 h, PBMC cells were collected and washed with PBS. They were then mixed with antibodies against human CD3-FITC, CD8a-PE, CD69-PECy5.5, CD14-ER780, CD19-PE / TR, and CD56-EV450, and incubated for 30 min in the dark. After the incubation time, the cells were washed several times with PBS, and then analyzed using flow cytometry. The results were analyzed using Flowjo software.

[0161] Experimental results prove CD3 + T lymphocytes, CD8 + The activation rates of T lymphocytes, monocytes, B cells, and NK cells all increased to varying degrees with increasing concentrations of compound 15. Figure 2 Therefore, compound 15 has an immune-activating effect.

[0162] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thiourea derivative, characterized in that, The structure of the thiourea derivative is shown in Formula I. In Formula I, R1 is selected from any one of isopropyl, isobutyl, tert-butyl, furanyl, thiophene, phenyl and pyridyl; R2 is selected from any one of hydrogen, amino, hydroxyl, halogen and methyl; X is selected from any one of C and N; Y is selected from any one of C and N; Z is H or hydroxyl; n is 1 or 2.

2. The thiourea derivative according to claim 1, characterized in that, The thiourea derivatives include:

3. The method for preparing the thiourea derivative according to any one of claims 1-2, characterized in that, Includes the following steps: 1) Precipitated sulfur and diethylamine undergo a reflux reaction to obtain 2) It reacts with sulfur phosgene in the presence of an alkaline environment to obtain 3) and The thiourea derivative was obtained by reflux reaction.

4. The preparation method according to claim 3, characterized in that, In step 1), the The molar ratio of precipitated sulfur to diethylamine is 1:1~2:1~2:1~2; Preferably, in step 1), the reflux reaction time is 4 to 6 hours; Preferably, in step 1), the reflux reaction is carried out in a solvent; the solvent is selected from at least one of ethanol, isopropanol and methanol.

5. The preparation method according to claim 3, characterized in that, In step 2), The molar ratio with sulfur phosgene is 1:2 to 3; Preferably, in step 2), the reaction is carried out in an ice bath; Preferably, in step 2), the reaction time is 0.5 to 1 hour; Preferably, in step 2), the base is selected from at least one of triethylamine and N,N-diisopropylethylamine; Preferably, in step 2), the reaction is carried out in a solvent; the solvent is selected from at least one of dichloromethane and tetrahydrofuran.

6. The preparation method according to claim 3, characterized in that, In step 3), and The molar ratio is 1:1.2 to 1.5; Preferably, in step 3), the reflux reaction time is 6-8 hours; Preferably, in step 3), the reflux reaction is carried out in a solvent; the solvent is selected from at least one of ethanol and methanol.

7. Use of the thiourea derivative or pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the preparation of a TLR2 agonist.

8. Use of the thiourea derivative or pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the preparation of an immunomodulator.

9. A pharmaceutical composition for activating TLR2, characterized in that, The active ingredient is a thiourea derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-2.

10. A pharmaceutical composition that can stimulate the activation of human PBMC cells, characterized in that, The active ingredient is a thiourea derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-2.

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