A kind of six-membered sylvan ring compound and its preparation and application

Abstract

The application discloses a kind of six-membered semi-cucurbituril compounds and its preparation and application.Six-membered semi-cucurbituril compound of the application is prepared with compound F1 and BBr3 as raw material, with dry dichloromethane as solvent.It is a kind of brand-new compound, it has the effect of inhibiting CathepsinL, and has good application prospect in the prevention and treatment of novel coronavirus;And its preparation method is simple, easy to implement industrialization.

Description

TECHNICAL FIELD

[0001] The present application relates to compounds and preparation and application thereof, in particular to a kind of six-membered semi-cucurbituril compounds and preparation and application thereof BACKGROUND

[0002] Research has found that SARS-CoV-2 infects host cells by binding its own spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) of the receptor. After the virus binds to the target cell, the transmembrane serine protease (TMPRSS2) and cathepsin L of the host will cut the S protein at a specific site (S1 / S2), promoting the membrane fusion and endocytosis of the coronavirus, releasing the positive-strand RNA genetic material of the virus into the host cell, and using the host's translation mechanism, raw materials, etc. to propagate the virus. Therefore, to some extent, the expression of CathepsinL can be inhibited to block the entry of SARS-CoV-2 into host cells, thereby preventing the infection of the new coronavirus.

[0003] CathepsinL is a member of lysosomal cysteine proteases, and its spatial structure is mainly composed of an L domain composed of alpha helix and an R domain composed of beta fold. Its main function is to proteolyze protein antigens produced by pathogen endocytosis. A large number of studies have shown that CathepsinL is very important for the entry of SARS-CoV-2 into host cells, can hydrolyze viral S protein to change its conformation, thereby helping the virus to quickly enter host cells and grow, and is positively correlated with the course and severity of the disease in patients. Therefore, CathepsinL can be inhibited to block the invasion of the new coronavirus into the human body, and CathepsinL can be used as a potential drug target for the prevention and treatment of SARS-CoV-2 infection. SUMMARY

[0004] The present application aims to provide a kind of six-membered semi-cucurbituril compounds and preparation and application thereof. The six-membered semi-cucurbituril compound of the present application is a completely new compound, which has good inhibition effect on CathepsinL and good application prospect in the prevention and treatment of the new coronavirus. Moreover, its preparation method is simple and easy to implement industrially.

[0005] The technical scheme of the present application is a kind of six-membered semi-cucurbituril compounds, and its chemical structural formula is as follows:

[0006]

[0007] A kind of six-membered semi-cucurbituril compound preparation method, and the synthesis path is as follows:

[0008]

[0009] is prepared by using compound F1 and BBr3 as raw materials and dry dichloromethane as solvent. The name of compound F1 is: 1 4 ,1 6 -bis(4-methoxybenzyl)-1 1 ,1 2 ,1 3 ,1 3a ,1 4 ,1 5 ,1 6 ,1 6a -octahydro-1(1,3)-imidazo[4,5-d]imidazola-5,9(1,3)-diimidazolidina-3,7,11(1,3)-tribenzenacyclododecaphane-1 2 ,1 5 ,5 2 ,9 2 -tetraone.

[0010] Preferably, the preparation method of the aforementioned six-membered half-cucurbituril compound comprises the following steps:

[0011] (1) dissolving BBr3 in dry dichloromethane to obtain solution A;

[0012] (2) dissolving compound F1 in dry dichloromethane to obtain solution B;

[0013] (3) slowly adding solution A into solution B in a zero temperature environment to react, after the reaction is completed, the temperature is raised to room temperature, and then unreacted BBr3 and dichloromethane are removed to obtain solid C;

[0014] (4) after separating and purifying solid C, the compound 1, i.e. the six-membered half-cucurbituril compound, is obtained.

[0015] Preferably, the preparation method of the aforementioned six-membered half-cucurbituril compound, the molar concentration of BBr3 in solution A is 1M; the molar concentration of compound F1 in solution B is 0.035M.

[0016] Preferably, the preparation method of the aforementioned six-membered half-cucurbituril compound, in step (3), BBr3 and compound F1 are mixed in a molar ratio of 8:1-12:1 at-30℃, reacted for 12h, and then unreacted BBr3 and dichloromethane are removed by reduced pressure concentration.

[0017] Preferably, in the aforementioned method for preparing the six-membered hemicyclic compound, the separation and purification in step (4) involves first adding a mixed solution of water and methanol in a volume ratio of 4:1 to solid C in a sub-zero temperature environment to quench the reaction, then adding dichloromethane for extraction three times, combining the organic phases, concentrating the organic phase under reduced pressure, and then separating and purifying it by silica gel column chromatography with a mesh size of 200-300; wherein the eluent during chromatography is dichloromethane / acetone in a volume ratio of 2:1.

[0018] Application of one of the aforementioned six-membered hemicyclic compounds in the preparation of drugs against the novel coronavirus.

[0019] An anti-novel coronavirus drug comprising the aforementioned hexacyclic cucurbitacinoid compound.

[0020] Application of one of the aforementioned six-membered hemicyclic compounds in the preparation of Cathepsin L inhibitors.

[0021] A Cathepsin L inhibitor comprising the aforementioned hexacucurbitacin compound.

[0022] Beneficial effects of the present invention

[0023] The six-membered hemicyclic compound of the present invention is a novel compound that has a good inhibitory effect on Cathepsin L and has good application prospects in the prevention and treatment of novel coronavirus; moreover, its preparation method is simple and easy to implement industrially.

[0024] The hexacyclic compound of this invention exhibited an inhibition rate of 58.09% against Cathepsin L at a concentration of 0.2 mg / mL, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The value was 20.63 μM. Attached Figure Description

[0025] Appendix Figure 1 This is a synthetic route diagram for the six-membered hemicyclic compound of the present invention.

[0026] Appendix Figure 2 For compound F1 1 HNMR image.

[0027] Appendix Figure 3 The six-membered hemicyclic compound of this invention 1 HNMR image.

[0028] Appendix Figure 4 The six-membered hemicyclic compound of this invention 13 CNMR image.

[0029] Appendix Figure 5 This is a high-resolution mass spectrum of the six-membered hemicyclic compound of the present invention.

[0030] attached Figure 6 The inhibition rate of the six-membered sylvan ring compound of the present application to Cathepsin L. DETAILED DESCRIPTION

[0031] The present application is further illustrated by the following examples, but is not limited to the following examples.

[0032] Embodiments of the present application

[0033] Example 1

[0034] Synthesis of six-membered sylvan ring compound

[0035] (1) Compound F1 (0.07 mmol) was weighed and dissolved in 2 mL of dry dichloromethane (Dry DCM).

[0036] (2) BBr3 (0.7 mmol) was measured and dissolved in 700 μL of dry dichloromethane (Dry DCM).

[0037] (3) The BBr3 solution was slowly added to the solution of compound F1 at -30°C, then gradually increased to room temperature within 12 h, and after the reaction was completed by TCL detection, the unreacted boron tribromide and dichloromethane were removed by vacuum concentration.

[0038] (4) The reaction was quenched by adding a mixed solution of water and methanol (v / v, 4:1) at 0°C, and extracted with dichloromethane (3 x 10 mL), the organic layers were combined and concentrated under reduced pressure, and the crude product was separated and purified by silica gel (200-300 mesh) column chromatography, eluent dichloromethane / acetone (v / v = 2:1), yield 45%, which 1 HNMR as Figure 3 shown, 13 CNMR as Figure 4 shown, HRMS as Figure 5 shown.

[0039] 1 HNMR (400 MHz, DMSO-d6):

[0040] δ 9.38 (s, 2H, Ar-OH), 7.33-7.28 (m, 4H, Ar-H), 7.16-7.10 (m, 6H, Ar-H), 6.89 (d, J = 8.0 Hz, 4H, Ar-H), 6.76 (br s, 2H, Ar-H), 6.65 (d, J = 8.0 Hz, 4H, Ar-H), 4.86 (s, 2H, CH2), 4.66 (d, J = 16.0 Hz, 2H, CH2), 4.53 (d, J = 15.0 Hz, 2H, CH2), 4.26 (d, J = 15.5 Hz, 6H, CH2), 4.15 (d, J = 15.4 Hz, 2H, C-H), 4.10 (d, J = 15.4 Hz, 2H, C-H), 3.97 (d, J = 15.4 Hz, 2H, C-H), 3.05-2.89 (m, 8H, CH2).

[0041] 13 CNMR (100 MHz, DMSO-d6):

[0042] 160.51, 158.93, 158.74, 156.76, 137.92, 137.89, 136.99, 129.09, 128.69, 128.58, 126.73, 126.51, 126.12, 124.73, 115.33, 66.81, 54.93, 47.56, 46.97, 45.99, 45.51, 41.84, 41.63.

[0043] HRMS:

[0044] Calcd for C 48 H 48 N8O6 (833.3770), Found: 833.3783.

[0045] Example 2

[0046] Synthesis of six-membered cucurbit-like compounds

[0047] (1) Compound F1 (0.07 mmol) was dissolved in 2 mL of dry dichloromethane (Dry DCM).

[0048] (2) BBr3(0.56 mmol) was dissolved in 560 μL of dry dichloromethane (Dry DCM).

[0049] (3) The BBr3solution was slowly added to the solution of compound F1 at -30 °C, then gradually increased to room temperature within 12 h, and the reaction was complete by TCL detection. The unreacted boron tribromide and dichloromethane were removed by concentration under reduced pressure.

[0050] (4) The reaction was quenched by adding a mixed solution of water and methanol (v / v, 4:1) at 0℃, and extracted with dichloromethane (3x10 mL). The organic layers were combined and concentrated under reduced pressure. The crude product was separated and purified by silica gel (200-300 mesh) column chromatography with dichloromethane / acetone (v / v=2:1) as eluent.

[0051] Example 3

[0052] Synthesis of six-membered cucurbit-like compounds

[0053] (1) Compound F1 (0.07 mmol) was dissolved in 2 mL of dry dichloromethane (Dry DCM).

[0054] (2) BBr3 (0.84 mmol) was dissolved in 840 μL of dry dichloromethane (Dry DCM).

[0055] (3) The BBr3 solution was slowly added to the solution of compound F1 at -30℃, and then gradually increased to room temperature within 12 h. After the reaction was completed by TCL detection, the unreacted boron tribromide and dichloromethane were removed by reduced pressure concentration.

[0056] (4) The reaction was quenched by adding a mixed solution of water and methanol (v / v, 4:1) at 0℃, and extracted with dichloromethane (3x10 mL). The organic layers were combined and concentrated under reduced pressure. The crude product was separated and purified by silica gel (200-300 mesh) column chromatography with dichloromethane / acetone (v / v=2:1) as eluent.

[0057] Example 4

[0058] A new coronavirus drug, comprising the six-membered cucurbit-like compound prepared in Example 1, and according to the requirements of the dosage form, the corresponding conventional adjuvant or carrier can be added, and the production can be carried out according to the process of the corresponding dosage form. Common dosage forms can be selected from tablets, capsules, pills, granules, decoctions, oral liquids, dripping pills or syrup, etc.

[0059] Example 5

[0060] A Cathepsin L inhibitor, comprising the six-membered cucurbit-like compound prepared in Example 1, and according to the requirements of the dosage form, the corresponding conventional adjuvant or carrier can be added, and the production can be carried out according to the process of the corresponding dosage form. Common dosage forms can be selected from tablets, capsules, pills, granules, decoctions, oral liquids, dripping pills or syrup, etc.

[0061] Example 6:

[0062] Pharmacodynamic evaluation:

[0063] 1) Detection instrument: TECAN multifunctional enzyme label instrument (infinite M200 Pro); Shunyu Hengping analysis balance (FA1004); Aike ultrapure water machine (AKHL-III-08); Eppendorf pipette.

[0064] 2) Reagent kit:

[0065] Sensolyte @ 520 Cathepsin L kit Fluorimetric (Cat#: AS-72218, AnaSpec, San Jose, USA).

[0066] 3) Test drug: The six-membered cucurbituril compound prepared in Example 1.

[0067] 4) Positive control: Cathepsin L inhibitor (Cat#: AS-72218, AnaSpec, San Jose, USA).

[0068] 5) Cathepsin L protease experimental method and results:

[0069] 5.1. Preparation of test solution (according to the kit instruction manual):

[0070] Test compound:

[0071] Accurately weigh 2 mg of the test compound, and prepare a 50 mg / mL stock solution with DMSO, and then dilute the concentration to 5 mg / mL, 1 mg / mL, 0.2 mg / mL, 0.04 mg / mL, and 0.008 mg / mL with 10% DMSO, respectively.

[0072] Buffer solution: Dilute a certain amount of 1 M DTT (Component F) 125 times in the buffer solution (Component D), and place it in an ice bath for standby.

[0073] Cathepsin L substrate solution: Dilute 1 mM substrate (Component A) 100 times with the above prepared buffer solution containing DTT, and store it in the dark, and place it in an ice bath for standby.

[0074] Cathepsin L solution: Dilute 0.1 mg / mL Cathepsin L (Component C) 200 times with the above prepared buffer solution containing DTT, and place it in an ice bath for standby.

[0075] Cathepsin L inhibitor: Dilute 100 μL Cathepsin L inhibitor (Component E) 100 times with the above prepared buffer solution containing DTT, and place it in an ice bath for standby.

[0076] 5.2. Assay method (operation according to kit instruction manual):

[0077] Add 10 μL of DTT-containing buffer solution (Substrate Control, SC), 2 μL of 10% DMSO blank solvent (Vehicle Control, VC), 2 μL of Cathepsin L positive control (Positive Control, PC) and 2 μL of test compound solution (Test Compound Control, TCC) into a 384-well black enzyme plate (square well flat bottom, REF3573, Corning, USA, Lot#. 36221009), then add 8 μL of Cathepsin L (no addition for the Substrate Control group) and 10 μL of Cathepsin L substrate solution in turn. The final volume of each well is 20 μL, and each solution is added to 3 wells in parallel. After slight shaking and mixing, incubate at 37°C for 30 min, and then test the fluorescence intensity using a TECAN multifunctional enzyme plate reader (infinite M200Pro). The fluorescence excitation wavelength is 485 nm, the emission wavelength is 525 nm, and the gain is 91.

[0078] 5.3. Data processing and analysis:

[0079] Inhibition rate calculation formula: Inhibition rate % = (F VC -F TCC ) / (F VC -F SC ) x 100%; In the formula: F VC is the fluorescence intensity of the solvent blank, F TCC is the fluorescence intensity of the test compound, and F SC is the fluorescence intensity of the substrate blank solution.

[0080] IC 50 is the concentration corresponding to B / B0=50%, which can be calculated by non-linear fitting using GraphPad Prism 9.3.0 software. In the formula: B is the fluorescence intensity value of the well with added inhibitor, and B0 is the fluorescence intensity value of the control well without added inhibitor.

[0081] 5.4. Screening results of anti-Cathepsin L activity of hexameric cucurbituril compounds:

[0082] Table 1. Inhibition rate of hexameric cucurbituril compound 1 on Cathepsin L at different concentrations

[0083]

[0084]

[0085] The above merely describes preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, within the technical scope disclosed by the present application, according to the technical scheme and inventive concept of the present application, makes equivalent replacement or change, should be covered within the protection scope of the present application.

Claims

1. A six-membered sylvanoid compound, characterized in that, The chemical structural formula is as follows:

2. A method for preparing a six-membered sylvanoid compound according to claim 1, characterized by, The synthesis path is as follows: The compound 1 is prepared by taking compound F1 and BBr3 as raw materials and dry dichloromethane as a solvent.

3. The method of claim 2, wherein the method is characterized by, The specific steps are as follows: (1) BBr3 is dissolved in dry dichloromethane to obtain solution A; (2) compound F1 is dissolved in dry dichloromethane to obtain solution B; (3) solution A is slowly added to solution B in a zero-temperature environment to react, after the reaction is completed, the temperature is raised to room temperature, and then unreacted BBr3 and dichloromethane are removed to obtain solid C; (4) after solid C is separated and purified, the compound 1, i.e. a six-membered semi-cucurbituril compound, is obtained.

4. The method of claim 3, wherein the method comprises: The molar concentration of BBr3 in the solution A is 1M; the molar concentration of compound F1 in the solution B is 0.035M.

5. The method of claim 3, wherein the method is characterized by: In the step (3), BBr3 and compound F1 are mixed in a molar ratio of 8:1-12:1 at-30℃ for 12h, and then unreacted BBr3 and dichloromethane are removed by reduced pressure concentration.

6. The method of claim 3, wherein the method is characterized by: The separation and purification in step (4) is as follows: first, a mixed solution of water and methanol in a volume ratio of 4:1 is added to solid C to quench the reaction in a zero-temperature environment, then dichloromethane is added for extraction 3 times, the organic phase is combined, the organic phase is concentrated under reduced pressure, and then separated and purified by silica gel column chromatography with a pore size of 200-300; wherein the eluent is dichloromethane / acetone in a volume ratio of 2:

1.

7. Use of the six-membered semi-cucurbituril compound of claim 1 in the preparation of an anti-new coronavirus drug.

8. An anti-SARS-CoV-2 drug, characterized in that: The six-membered semi-cucurbituril compound of claim 1.

9. Use of the six-membered semi-cucurbituril compound of claim 1 in the preparation of a Cathepsin L inhibitor.

10. A Cathepsin L inhibitor, characterized by: The six-membered semi-cucurbituril compound of claim 1.

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