A Cas protein activity inhibitor and its application

Abstract

The present invention discloses a Cas protein activity inhibitor and its application. The present invention provides a Cas protein activity inhibitor, which can inhibit the activity of SpCas9 protein both in vitro and in cells, reduce the off-target cleavage activity of SpCas9 protein, increase the safety of SpCas9 protein in clinical use, and can be used to regulate CRISPR / SpCas9 system, reduce the off-target cleavage activity of SpCas9 protein, and also help to broaden the application scope of SpCas9 in clinical practice. In addition, the Cas protein activity inhibitor of the present invention has a simple structure and is worth further development and transformation.

Description

Technical Field

[0001] The present invention belongs to the field of CRISPR-Cas9 gene editing technology. More specifically, it relates to a Cas protein activity inhibitor and its application. Background Art

[0002] The CRISPR / Cas9 system is an adaptive immune system of bacteria and archaea that protects them from invasion by viruses or plasmids. The CRISPR / Cas system can cut foreign DNA or RNA under the guidance of crRNA (CRISPR-RNA).

[0003] Cas9, derived from S. pyogenes, has been used in clinical treatment. Cas9 can recognize PAM sequences, identify specific DNA sites under the guidance of sgRNA, and then cut the target site, causing double-strand breaks in DNA. Broken DNA can be repaired by NHEJ (non-homologous end joining) or HDR (homologous-mediated double-stranded DNA), resulting in insertions and deletions (indels) or base mutations. However, while cutting the target site, Cas9 may also bind to other sites and cut them, causing off-target.

[0004] To reduce the probability of off-target effects, some researchers have reduced off-target effects by engineering Cas9 or shortening the length of sgRNA; reducing the half-life of Cas9 in cells can also reduce off-target effects; and inhibiting the activity of Cas9 in a specific editing time window can also reduce off-target effects without affecting the targeting activity. Therefore, it is necessary to develop Cas9 inhibitors to control the activity of Cas9 and thus improve the safety of CRISPR-based gene therapy.

[0005] Anti-CRISPR (Acr) proteins are the most widely studied Cas protein inhibitors. About 50 Acr proteins have been identified that can inhibit different CRISPR / Cas systems. These Acr proteins can inhibit CRISPR / Cas activity by inhibiting Cas protein binding or cutting target DNA. However, Acr proteins act slowly and have difficulty penetrating cell membranes; they are also immunogenic and therefore cannot be administered repeatedly; Acr proteins cannot be supplied on a large scale, and as protein products, Acr proteins have high storage and transportation costs. However, compared with the Acr protein toolbox, there are few small molecule inhibitors of CRISPR proteins. BRD0539 is a small molecule inhibitor of Streptococcus pyogenes Cas9 (SpCas9) that works by inhibiting the binding of SpCas9 protein to DNA. However, the IC of BRD0539 50 Very high (IC 50=22 μM), its inhibitory mechanism and its impact on off-target effects are also unclear. In addition, the affinity of CRISPR proteins for different target DNAs is different. Therefore, it is necessary to develop small molecule inhibitors with different affinities for CRISPR proteins to respond to different cleavage events. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing CRISPR-Cas protein small molecule inhibitors, and to provide a Cas protein activity inhibitor and its application.

[0007] The first object of the present invention is to provide a Cas protein activity inhibitor.

[0008] The second object of the present invention is to provide the use of the inhibitor in inhibiting the activity of Cas protein or preparing an agent for inhibiting the activity of Cas protein.

[0009] The third object of the present invention is to provide the use of the inhibitor in regulating the CRISPR / Cas gene editing system or in preparing a reagent for regulating the CRISPR / Cas gene editing system.

[0010] The fourth object of the present invention is to provide a drug for inhibiting the activity of SpCas9 protein.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a Cas protein activity inhibitor, wherein the inhibitor is a compound having any of the following structural formulas or a pharmaceutically acceptable salt thereof;

[0013]

[0014] Among them, R1 is a single substituent or a multiple substituent at any position of the benzene ring, and R2 is a substituent on a carbon atom; R1 and R2 are one of -H, C1-6 alkyl, halogen hydroxyl, cyanonitromethyl, cycloalkyl, nitro, hydroxyl, nitrogen heterocycle, alkoxy, cyano, halogen, sulfonic acid, and keto; R1 and R2 are independently selected from the above groups.

[0015] Specifically, in the inhibition, two adjacent substituents and the connecting atom may form a five-membered or multi-membered ring structure.

[0016] More specifically, R1 and R2 are -H, methyl, nitro, hydroxyl, -F, -Cl, -Br, methoxy, cyano, -COOR, One of the following; R1 and R2 are independently selected from the above groups; R in the -COOR is an alkyl group.

[0017] Specifically, the Cas protein inhibitor of the present invention is any of the following compounds or pharmaceutically acceptable salts thereof:

[0018]

[0019]

[0020]

[0021] Specifically, the Cas protein that can be inhibited by the above-mentioned inhibitor is SpCas9 protein.

[0022] The present invention finds through in vitro cleavage experiments, EGFP reporter cell lines and high-throughput sequencing that the inhibitor of the present invention can inhibit the cleavage activity of SpCas9 protein. Therefore, the present invention requests protection for the use of the inhibitor in inhibiting the activity of Cas protein or preparing an agent for inhibiting the activity of Cas protein.

[0023] Specifically, the Cas protein is SpCas9 protein.

[0024] The present invention also seeks to protect the use of the inhibitor in regulating the CRISPR / Cas gene editing system or in preparing a reagent for regulating the CRISPR / Cas gene editing system.

[0025] Specifically, the CRISPR / Cas gene editing system is a CRISPR / SpCas9 gene editing system.

[0026] The present invention also provides a drug for inhibiting the activity of SpCas9 protein, wherein the drug contains the inhibitor of the present invention.

[0027] Specifically, the drug comprises at least one active ingredient and one or more pharmaceutically acceptable carriers or excipients, and the active ingredient is selected from one or more of the inhibitors of the present invention.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides a Cas protein activity inhibitor, which is a compound having a structural formula shown in any one of formula (I) to formula (V) or a pharmaceutically acceptable salt thereof. The Cas protein activity inhibitor of the present invention can inhibit the activity of SpCas9 protein both in vitro and in cells, reduce the off-target cleavage activity of SpCas9 protein, increase the safety of SpCas9 protein in clinical use, and can be used to regulate the CRISPR / SpCas9 system, reduce the off-target cleavage activity of SpCas9 protein, and also help to broaden the scope of application of SpCas9 in clinical practice. In addition, the Cas protein activity inhibitor of the present invention has a simple structure and is worth continuing to develop and transform. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the inhibition rate of the compound of the present invention on SpCas9 in reporter cells at concentrations of 10 μM and 40 μM.

[0031] Figure 2 The figure shows the effect of the compound of the present invention on the expression of GFP in the reporter cell line at a concentration of 40 μM.

[0032] Figure 3 Figure 5 shows the effects of compound V-1 and compound IV-25 on SpCas9 cutting different target sites and off-target sites; Figure A shows the effects of compound V-1 and compound IV-25 on SpCas9 cutting EMX1-1 target site and off-target site; Figure B shows the effects of compound V-1 and compound IV-25 on SpCas9 cutting EMX1-2 target site and off-target site; Figure C shows the effects of compound V-1 and compound IV-25 on SpCas9 cutting VEGFA-site3 target site and off-target site; Figure D shows the effects of compound V-1 and compound IV-25 on SpCas9 cutting ZSCAN2 target site and off-target site; in the figure, ns means no significant difference; * means significant difference, P < 0.05; ** means significant difference, P < 0.01; *** means extremely significant difference, P < 0.001. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The preparation of all compounds involved in the present invention can refer to the following documents:

[0036] (1) Al-Saleh, B., El-Apasery, MA, Hilmy, NM & Elnagdi, MH Microwaves inorganic synthesis: Synthesis of pyridazinones, phthalazinones and pyridopyridazinones from 2-oxo-arylhydrazones under microwaveirradiation. Journal of Heterocyclic Chemistry 43, 1575-1581 (2006).

[0037] (2)Al-Awadi,N.A.,Ibrahim,M.R.,Al-Etaibi,A.M.&Elnagdi,M.H.Improvedsynthesis of 2-arylhydrazono-3-hydroxy-1-propanones and their utility inefficient synthesis of pyridazine derivatives.Arkivoc 2011,310-321(2011).

[0038] (3)Zhang,C.Y.,Liu,X.H.,Wang,B.L.,Wang,S.H.&Li,Z.M.Synthesis andantifungal activities of new pyrazole derivatives via 1,3-dipolarcycloaddition reaction.Chem Biol Drug Des 75,489-493(2010).

[0039] (4)Barker,D.et al.Synthesis of 3-Amino-2-carboxamideTetrahydropyrrolo[2,3-b]quinolines.Synlett 27,2811-2814(2016).

[0040] (5)Mavrova,A.,Wesselinova,D.,Tsenov,Y.A.&Denkova,P.Synthesis,cytotoxicity and effects of some 1,2,4-triazole and 1,3,4-thiadiazolederivatives on immunocompetent cells.Eur J Med Chem 44,63-69(2009).

[0041] (6)Xu,H.et al.Discovery of thiadiazole amides as potent,S1P(3)-sparing agonists of sphingosine-1-phosphate 1(S1P(1))receptor.Bioorganic&medicinal chemistry letters 22,2456-2459(2012).

[0042] Example 1 Preparation of Compounds I-1 to I-19 and Compounds II-1 and II-2

[0043] The synthetic routes of compounds Ⅰ-1 to Ⅰ-19, and compounds Ⅱ-1 and Ⅱ-2 are as follows:

[0044]

[0045] Specifically, the compound can be obtained by the following three-step reaction:

[0046] Step S1: Synthesis of N-(4-chlorophenyl)-2-chloroacetamide

[0047] Weigh 2.55 g (20 mmol) of p-chloroaniline (Material A) into a 100 mL eggplant-shaped bottle, add a magnetic bar, measure 50 mL of 1,2-dichloroethane solvent and add it to the reaction bottle, place the reaction bottle in an ice-water bath, and when the solution temperature is 0-5°C, measure 3.97 mL (50 mmol) of chloroacetyl chloride and add it dropwise. After the addition is completed, return to room temperature, heat reflux, and monitor the reaction progress with thin layer chromatography. The reaction is completed in about 3.5 hours; the obtained compound is filtered and recrystallized with ethanol.

[0048] Step S2: Synthesis of 2-azido-N-(4-chlorophenyl)acetamide

[0049] Weigh 1.01 g (5 mmol) of N-(4-chlorophenyl)-2-chloroacetamide into a 50 mL eggplant-shaped bottle, add a magnetic bar, measure 10 mL of N,N-dimethylformamide solvent and add it to the reaction bottle, add 2.93 g (4.5 mmol) of sodium azide, and stir at room temperature; monitor the reaction progress by thin layer chromatography, and the reaction is completed in about 12 hours; filter the obtained compound and recrystallize it with ethanol.

[0050] Step S3: Synthesis of target product

[0051] Weigh 0.42 g (2 mmol) of 2-azido-N-(4-chlorophenyl)acetamide into a 50 mL eggplant-shaped bottle, add a magnetic bar, measure 10 mL of tetrahydrofuran solvent and add it to the reaction bottle, add 0.27 g (2 mmol) of p-methylphenylacetylene (Material B), then add 0.2 mmol of cuprous iodide and 2.4 mmol of triethylamine in sequence, and stir at room temperature; monitor the reaction progress by thin layer chromatography, and the reaction is completed in about 12 hours; the obtained compound is filtered, recrystallized with ethanol, and finally dried to obtain the pure target product.

[0052] The present invention synthesizes compound Ⅰ-1 through the above steps, with a yield of 78%.

[0053] Compound Ⅰ-1: N-(4-chlorophenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide

[0054] ; White solid; Yield: 78%; mp: 265.8-267.9℃;

[0055] 1 H NMR (600 MHz, DMSO-d 6 )δ10.69(s,1H),8.56(s,1H),7.78(d,J=7.7Hz,2H),7.65(d,J=8.5Hz,2H ),7.42(d,J=8.7Hz,2H),7.28(d,J=7.5Hz,2H),5.40(s,2H),2.34(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ164.39,146.28,137.35,137.14,129.46,128.84,127.92,127.38,125.07,122.62,120.80,52.33,20.82.MS(ESI)m / z:327.1002[M+H] + .

[0056] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-2 was prepared using p-chloroaniline (Material A) and ethyl propiolate (Material B) as raw materials; the yield was 65%.

[0057] Compound I-2: ethyl 1-(2-((4-chlorophenyl)amino)-2-oxoethyl)-1H-1,2,3-triazole-4-carboxylate; White solid; Yield: 65%; mp: 231.2-232.4℃;

[0058] 1 H NMR(600MHz,DMSO-d6)δ10.67(s,1H),8.79(s,1H),7.61(d,J=8.6Hz,2H),7.4 1(d,J=8.6Hz,2H),5.43(s,2H),4.42–3.76(m,2H),1.32(t,J=7.1Hz,3H).13C NMR(151MHz,DMSO-d6)δ163.95,160.23,138.67,137.25,130.77,128.85,127.42,120.80,60.55,52.30,14.16.MS(ESI)m / z:309.0741[M+H]+.

[0059] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-3 was prepared using p-chloroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 68%.

[0060] Compound I-3: N-(4-chlorophenyl)-2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)acetamide; Gray solid; Yield: 68%; mp: 261.8-263.6℃;

[0061] 1 H NMR (600 MHz, DMSO-d 6 )δ10.67(s,1H),8.67(s,1H),7.91(s,1H),7.64(s,2H),7.53(s,2H),7.41(s,2H),5.42(s,2H).MS(ESI)m / z:369.0274[M+Na] + .

[0062] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-4 was prepared using 1,4-diaminobenzene (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 79%.

[0063] Compound I-4: N,N'-(1,3-phenylene)bis(2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)acetamide); White solid; Yield: 79%; mp: >300℃;

[0064] 1 H NMR (600MHz, DMSO-d6) δ10.59(s,1H),8.64(s,1H),8.00(s,1H),7.89(s,2H),7.51(s,2H),7.31(d,J=16.9Hz,3H),5.39(s,50H). 13 C NMR (151 MHz, DMSO-d 6 )δ164.58,145.53,139.24,132.68,130.00,129.77,129.39,127.22,123.77,115.08,110.67,52.85.MS(ESI)m / z:547.1145[M+H] + .

[0065] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-5 was prepared using 3,4-dichloroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 64%.

[0066] Compound I-5: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(3,4-dichlorophenyl)acetamide; White solid; Yield: 64%; mp: 271.2-272.3℃;

[0067] 1 H NMR (600 MHz, DMSO-d 6 )δ10.83(s,1H),8.64(s,1H),7.96(s,1H),7.88(s,2H),7.58(s,1H),7.48(s,3H),5.42(s,2H). 13 C NMR (151 MHz, DMSO-d 6)δ169.84,150.26,143.49,137.38,136.23,135.97,134.62,134.07,131 .89,130.41,128.49,125.56,124.38,57.47.MS(ESI)m / z:381.0068[M+H] + .

[0068] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-6 was prepared using 3,4-dichloroaniline (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 66%.

[0069] Compound Ⅰ-6: N-(3,4-dichlorophenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 66%; mp: 264.8-266℃;

[0070] 1 H NMR (600 MHz, DMSO-d 6 )δ10.85(s,1H),8.55(s,1H),7.99(s,1H),7.78(s,2H),7.63(s,1H),7.51(s,1H),7.28(s,2H),5.42(s,2H),2.34(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ169.94,151.39,143.52,142.26,136.22,135.98,134.56,132.95,130.38 ,130.14,127.71,125.55,124.38,57.40,25.92.MS(ESI)m / z:361.0622[M+H] + .

[0071] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-7 was prepared using 2,4,6-trichloroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 53%.

[0072] Compound I-7: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2,4,6-trichlorophenyl)acetamide; White solid; Yield: 53%; mp: 273.2-275℃;

[0073] 1 H NMR (600MHz, DMSO-d6) δ10.53(s,1H),8.66(s,1H),7.75(s,4H),7.52(s,2H),5.49(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.95,147.57,136.59,135.22,134.65,133.81,131.90,131.34,130.78,129.23,125.80,53.96.MS(ESI)m / z:414.9673[M+H] + .

[0074] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-8 was prepared using 2,4,6-trichloroaniline (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 76%.

[0075] Compound I-8: 2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-N-(2,4,6-trichlorophenyl)acetamide; White solid; Yield: 76%; mp: 270.9-271.7℃;

[0076] 1 H NMR (600 MHz, DMSO-d 6 )δ10.52(s,1H),8.54(s,1H),7.79(s,4H),7.27(s,2H),5.47(s,2H),2.33(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ167.02,148.70,139.56,136.60,135.20,133.83,131.83,130.78,130.22,127.48,125.05,53.88,23.21.MS(ESI)m / z:395.0221[M+H] + .

[0077] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-9 was prepared using p-chloroaniline (Material A) and (E)-1-(2-(4-ethynylphenyl)hydrazine)propan-2-one (Material B) as raw materials; the yield was 78%.

[0078] Compound Ⅰ-9: (E)-N-(4-chlorophenyl)-2-(4-(4-(2-(2-oxopropylidene)hydrazinyl)phenyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 78%; mp: >300℃;

[0079] 1 H NMR (600 MHz, DMSO-d 6 )δ11.16(s,1H),8.68(s,1H),8.28(s,2H),7.93(s,2H),7.85(s,2H),7.54(s,2H),5.52(s,2H).MS(ESI)m / z:397.1166[M+H] + .

[0080] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-10 was prepared using p-nitroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 87%.

[0081] Compound I-10: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(4-nitrophenyl)acetamide; White solid; Yield: 87%; mp: 263-264.3℃;

[0082] 1 H NMR (600 MHz, DMSO-d 6 )δ11.15(s,1H),8.57(s,1H),8.28(s,2H),7.87(s,2H),7.77(s,2H),7.28(s,2H),5.49(s,2H),2.34(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ165.58,145.52,144.76,142.93,132.64,129.85,129.31,127.15,125.44,123.74,119.38,52.83.MS(ESI)m / z:358.0706[M+H] + .

[0083] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-11 was prepared using p-nitroaniline (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 71%.

[0084] Compound I-11: N-(4-nitrophenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 71%; mp: 265.1-267.3℃;

[0085] 1 H NMR (600 MHz, DMSO-d 6 )δ11.13(s,1H),8.25(d,J=8.0Hz,4H),7.85(s,3H),7.76(d,J=6.7Hz,4H),7.26(d,J=7.1Hz,4H),5.47(s,3H).MS(ESI)m / z:338.1243[M+H] + .

[0086] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-12 was prepared using 2-amino-5-nitrobenzonitrile (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 59%.

[0087] Compound I-12: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2-cyano-4-nitrophenyl)acetamide; White solid; Yield: 59%; mp: 256.7-257.3℃;

[0088] 1 H NMR (600 MHz, DMSO-d 6 )δ11.20(s,1H),8.74(s,1H),8.65(s,1H),8.50(s,1H),8.08(s,1H),7.89(s,2H),7.50(s,2H),5.61(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.42,147.71,145.83,134.83,132.00,131.47,129.32,126.82,125.94,117.43,107.85,105.91,54.88.

[0089] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-13 was prepared using 2-amino-5-nitrobenzonitrile (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 57%.

[0090] Compound I-13: N-(2-cyano-4-nitrophenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 57%; mp: 258.4-259.3℃;

[0091] 1 H NMR (600 MHz, DMSO-d 6 )δ11.16(s,1H),8.73(s,1H),8.53(s,1H),8.50(s,1H),8.09(s,1H),7.73(s,2H),7.24(s,2H),5.57(s,2H),2.31(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.38,146.75,145.61,143.72,137.60,132.67,129.92,129.88,129.46,128.25 ,125.47,124.72,123.07,115.37,105.73,52.71,21.23.MS(ESI)m / z:363.1191[M+H] + .

[0092] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-14 was prepared using 2-fluoro-4-nitroaniline (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 81%.

[0093] Compound I-14: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2-fluoro-4-nitrophenyl)acetamide; White solid; Yield: 81%; mp: 264.2-265.8℃;

[0094] 1 H NMR (600 MHz, DMSO-d 6)δ10.89(s,1H),8.64(s,1H),8.37(s,1H),8.22(d,J=9.3Hz,1H),8.15–8.00(m,1H),7.88(d,J=6.7Hz,2H),7.50(d,J=6.8Hz,2H),5.58(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.24,152.58,150.92,145.60,143.11,132.72,129.92,129.38,127.22, 123.84,122.18,121.10,112.04,103.83,52.84.MS(ESI)m / z:376.0611[M+H] + .

[0095] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-15 was prepared using 2-amino-5-methylphenol (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 78%.

[0096] Compound I-15: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2-hydroxy-4-methylphenyl)acetamide; White solid; Yield: 78%; mp: 263.4-265.9℃;

[0097] 1 H NMR (600 MHz, DMSO-d 6 )δ9.81(s,1H),9.66(s,1H),8.64(s,1H),7.91(d,J=7.7Hz,2H),7.69(d,J=8.3Hz,1H),7.53(d, J=7.9Hz,2H),6.72(s,1H),6.59(s,1H),5.47(s,2H),2.20(s,3H).MS(ESI)m / z:343.0949[M+H] + .

[0098] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-16 was prepared using 2-amino-5-methylphenol (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 64%.

[0099] Compound I-16: N-(2-hydroxy-4-methylphenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 64%; mp: 261.3-262.8℃;

[0100] 1 H NMR (600 MHz, DMSO-d 6 )δ9.79(s,1H),9.60(s,1H),8.54(s,1H),7.77(d,J=7.2Hz,2H),7.69(d,J=7.4Hz,1H),7.26(d,J=14.0Hz,2 H),6.71(s,1H),6.58(d,J=7.6Hz,1H),5.44(s,2H),2.33(s,3H),2.20(s,3H).MS(ESI)m / z:323.1502[M+H] + .

[0101] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-17 was prepared using 2-methoxy-5-nitroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 67%.

[0102] Compound I-17: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2-methoxy-5-nitrophenyl)acetamide; White solid; Yield: 67%; mp: 265.3-266.4℃;

[0103] 1 H NMR (600 MHz, DMSO-d 6 )δ10.31(s,1H),8.67(s,1H),8.31(s,1H),7.92(s,4H),7.48(s,2H),5.64(s,2H),4.04(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.07,151.27,147.68,145.59,135.91,134.79,132.04,131.46,129.31,125.94,122.25,119.28,108.58,59.04,55.05.

[0104] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-18 was prepared using 3-aminophenol (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 78%.

[0105] Compound I-18: 2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)-N-(3-hydroxyphenyl)acetamide; White solid; Yield: 78%; mp: 269-271.2℃;

[0106] 1 H NMR (600 MHz, DMSO-d 6 )δ10.29(s,1H),9.30(s,1H),8.66(s,1H),7.93(d,J=8.3Hz,2H),7.54(d ,J=8.4Hz,2H),7.40(d,J=8.7Hz,2H),6.75(d,J=8.7Hz,2H),5.35(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.44,158.88,154.03,150.24,146.72,137.39,135.12,134.77,134.11,131.96,128.50,126.15,120.37,57.48.

[0107] Referring to the synthesis method of compound Ⅰ-1, compound Ⅰ-19 was prepared using 2-fluoro-4-nitroaniline (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 66%.

[0108] Compound I-19: N-(2-fluoro-4-nitrophenyl)-2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetamide; White solid; Yield: 66%; mp: 254.2-256.1℃;

[0109] 1 H NMR (600 MHz, DMSO-d 6)δ10.88(s,1H),8.53(s,1H),8.38(t,J=7.9Hz,2H),8.22(d,J=10.3Hz,1H),8.11(d, J=8.5Hz,1H),7.74(d,J=7.8Hz,2H),7.24(d,J=7.7Hz,2H),5.56(s,2H),2.31(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.28,152.57,150.91,146.74,143.11,137.59,132.91,129.87,128.26,125 .47,123.07,122.14,121.11,111.96,52.76,21.23.MS(ESI)m / z:356.1153[M+H] + .

[0110] Referring to the synthesis method of compound Ⅰ-1, compound Ⅱ-1 was prepared using 4-chlorobenzamide (Material A) and p-chlorophenylacetylene (Material B) as raw materials; the yield was 54%.

[0111] Compound II-1: 4-chloro-N-(2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)acetyl)benzamide; White solid; Yield: 54%; mp: 267.7-268.3℃;

[0112] 1 H NMR (600 MHz, DMSO-d 6 )δ11.76(s,1H),8.61(s,1H),8.04(s,2H),7.92(s,2H),7.66(s,2H),7.54(s,2H),5.85(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ174.20,170.96,150.19,143.19,137.37,136.28,135.61,134.65,134.09,133.80,131.91,59.43.MS(ESI)m / z:375.0411[M+H] + .

[0113] Referring to the synthesis method of compound Ⅰ-1, compound Ⅱ-2 was prepared using 4-chlorobenzamide (Material A) and p-methylphenylacetylene (Material B) as raw materials; the yield was 63%.

[0114] Compound II-2: 4-chloro-N-(2-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)acetyl)benzamide; White solid; Yield: 63%; mp: 269-271.2℃;

[0115] 1 H NMR (600 MHz, DMSO-d 6 )δ11.74(s,1H),8.48(s,1H),8.02(d,J=8.1Hz,2H),7.76(d,J=7.6Hz,2H ),7.65(d,J=8.9Hz,2H),7.27(d,J=7.7Hz,2H),5.81(s,2H),2.34(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ174.25,171.02,151.33,143.16,142.24,136.33,135.62,134.57,133.80,132.98,130.15,127.73,59.35,25.93.

[0116] Example 2 Preparation of Compounds III-1, III-2 and III-3

[0117] The synthetic routes of compounds III-1, III-2 and III-3 are as follows:

[0118]

[0119] For the synthesis of the intermediate 5-aminothiadiazole chromone, the specific steps can be divided into the following two steps:

[0120] Step 1: Synthesis of 3-(aminothioformylhydrazomethyl)chromone

[0121] 1 g (5.5 mmol) of chromone-3-carboxaldehyde and 470 g (5.7 mmol) of thiosemicarbazide were dissolved in 50 mL of anhydrous ethanol; refluxed; the reaction progress was monitored by thin layer chromatography (TLC), and heating was stopped after the reaction of chromone 3-carboxaldehyde was complete; a light yellow solid was precipitated after cooling, and the filter cake was recrystallized with a mixed solvent of V (EtOH): V (DMF) = 1:1; filtered and dried to obtain 1.2 g of light yellow solid powder (yield: 90%).

[0122] Step 2: Synthesis of 5-aminothiadiazolylchromone

[0123] Take 1g of 3-(aminothioformylhydrazonemethyl)chromone and add it to a two-necked round-bottom flask filled with 35mL of water. There is a large amount of undissolved light yellow solid at the bottom. Add 3 times the equivalent of powdered anhydrous FeCl 3 , set up the reflux device, reflux at 110°C for 4h; the previously undissolved light yellow solid becomes a brown solid, and the reaction progress is monitored by TLC until the reaction is completed; after cooling, filter and wash 3 times with 30mL of water, and the resulting filter cake is the pure product; for the purple filtrate, 2.2 equivalents of citric acid and 2 equivalents of sodium citrate can be added, and stirred at room temperature for 30min; the solution changes from purple to yellow, and then ammonia water is added dropwise to pH=7, a precipitate is precipitated, filtered, and washed with water; the filter cakes obtained twice are both the target product, combined and dried to obtain 1.5g of light brown solid powder (yield is 85%).

[0124] The synthesis of the target product N-(chromone thiadiazole) benzamide compounds, the specific experimental steps are as follows:

[0125] Take 5mmol 5-aminothiadiazolylchromone and dissolve it in 25mL dichloromethane. Add 5mmol substituted benzoyl chloride (Material C) dropwise, then add catalytic amount of triethylamine. The color of the solution changes from brown to milky white. Stir at room temperature for 12h. White insoluble matter is generated. Monitor by TLC until the reaction is basically complete. Filter by suction. For the obtained filter cake, use V(CH 2 Cl 2 ):V(MeOH)=1:1 mixed solvent for recrystallization, and drying the filter cake to obtain a pure powder solid.

[0126] Compound III-1 was obtained by referring to the synthesis method of Example 2; the yield was 60%.

[0127] Compound III-1: N-(5-(4-oxo-4H-chromen-3-yl)-1,3,4-thiadiazol-2-yl)benzamide; White solid; Yield: 60%;

[0128] 1H NMR (600MHz, DMSO-d6) δ13.02(s,1H,-NHCO),9.43(s,1H,-C=CH),8.22(d,J=8.0Hz,1H,ArH),8.13(d,J=7.5Hz,2H,ArH),7.90(t, J=7.6Hz,1H,ArH),7.80(d,J=8.3Hz,1H,ArH),7.64(d,J=7.3Hz,1H,ArH),7.60(t,J=7.5Hz,1H,ArH),7.55(t,J=7.5Hz,2H,ArH).

[0129] Referring to the synthesis method of Example 2, compound III-2 was prepared using 4-fluorobenzoyl chloride (Material C) as a raw material; the yield was 40%.

[0130] Compound III-2: 2-fluoro-N-(5-(4-oxo-4H-chromen-3-yl)-1,3,4-thiadiazol-2-yl)benzamide; White solid; Yield: 40%;

[0131] 1 H NMR (400MHz, DMSO-d6) δ13.06(s,1H,-NHCO),9.46(s,1H,-CH=C),8.26(d,J=8.7Hz,1H,ArH),7.97–7 .91(m,1H,ArH),7.84(d,J=9.0Hz,2H,ArH),7.70–7.61(m,2H,ArH),7.39(q,J=9.3,8.0Hz,2H,ArH).

[0132] Referring to the synthesis method of Example 2, compound III-3 was prepared using 2-fluorobenzoyl chloride (Material C) as a raw material; the yield was 43%.

[0133] Compound III-3: 4-fluoro-N-(5-(4-oxo-4H-chromen-3-yl)-1,3,4-thiadiazol-2-yl)benzamide; White solid; Yield: 43%;

[0134] 1H NMR (400MHz, DMSO-d6) δ13.15(s,1H,-NHCO),9.44(s,1H,-C=CH),8.26(d,J=8.9Hz,1H,ArH),8.18(d,J=8.6Hz,2H, ArH),7.94(d,J=8.5Hz,2H,ArH),7.83(d,J=8.5Hz,1H,ArH),7.64(d,J=8.4Hz,2H.ArH),7.56(d,J=8.5Hz,2H,ArH).

[0135] Example 3 Preparation of Compounds IV-1, IV-4 to IV-6, IV-11 to IV-14, IV-28 and IV-29

[0136] The synthetic routes of compounds IV-1, IV-4 to IV-6, IV-11 to IV-14, IV-28 and IV-29 are as follows:

[0137]

[0138] Specifically, 10 mmol of ethyl (4-bromobenzoyl)acetate (Material E) was weighed in a 250 mL conical flask, a magnet was added, and KOH aqueous solution (3.5 g, 100 mL of water) was added to the eggplant bottle and stirred at room temperature for 24 h; cooled to 0 ° C, 4.5 mL of concentrated HCl and 15 mL of ice water were slowly added; 10 mmol of p-nitroaniline (Material D) was weighed and a 100 mL double-necked flask was added, a magnet was added, 10 mL of water and 3 mL of concentrated hydrochloric acid were added, cooled to 0 ° C, and stirred for 30 min; 10 mmol of NaNO was slowly added 2 The aqueous solution was stirred at a temperature not exceeding 5°C for 30 min; the newly prepared diazonium salt was added dropwise to the 250 mL eggplant flask and the solution was kept at 0-5°C; the solution became an orange-yellow turbid solution and CH 3 COONa aqueous solution (8.2 g, 100 mL ice water) was added dropwise and stirred at 0-5°C for 2 h; the reaction was nearly completed, and the solution was allowed to stand to precipitate a solid to obtain a yellow solid.

[0139] Referring to the synthesis method of Example 3, compound IV-1 was prepared using 4-chlorobenzamide (Material D) and p-chlorophenylacetylene (Material E) as raw materials; the yield was 79%.

[0140] Compound IV-1: 1-(2-(2-methoxy-5-nitrophenyl)hydrazono)propan-2-one; Yellow solid; Yield: 79%;

[0141] 1 H NMR (600 MHz, DMSO-d 6 )δ8.08–8.06(m,1H),8.05(s,2H),7.82(s,1H),7.59(d,J=8.8Hz,1H),2.38(s,3H),2.36(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ202.15,152.67,145.46,144.96,131.45,128.36,117.90,108.56,29.46,22.71.MS(ESI)m / z:224.0687[M+Na] + .

[0142] Referring to the synthesis method of Example 3, compound IV- was prepared using 4-chlorobenzamide (Material D) and p-chlorophenylacetylene (Material E) as raw materials; the yield was 67%.

[0143] Compound IV-4: 2-(2-(3-nitrophenyl)hydrazono)-1-phenylbutane-1,3-dione; Yellow solid; Yield: 67%;

[0144] 1 H NMR (600 MHz, DMSO-d 6 )δ11.76(s,1H),8.12(s,1H),7.93–7.76(m,4H),7.71(s,1H),7.64–7.52(m,3H),2.54(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ196.11,148.90,145.64,140.90,135.87,134.83,131.08,129.48,121.21,116.94,109.44.MS(ESI)m / z:334.0792[M+Na] + .

[0145] Referring to the synthesis method of Example 3, compound IV-5 was prepared using 2-amino-4-nitrophenol (Material D) and ethyl 2-benzoyl-3-oxobutyrate (Material E) as raw materials; the yield was 70%.

[0146] Compound IV-5: 2-(2-(2-hydroxy-5-nitrophenyl)hydrazono)-1-phenylbutane-1,3-dione; Yellow solid; Yield: 70%

[0147] 1 H NMR (600 MHz, DMSO-d 6 )12.18(s,1H),7.90(s,3H),7.71(d,J=6.2Hz,1H),7.68(s,1H),7.53(d,J=17.8Hz,2H),7.08(d,J=7.7Hz,1H),2.54(d,J=9.9Hz,3H). 13 C NMR (151 MHz, DMSO-d 6 )198.06,191.82,152.48,140.57,138.29,134.29,132.62,130.60,130. 37,128.23,121.68,115.90,110.22,30.71.MS(ESI)m / z:350.0744[M+Na] + .

[0148] Referring to the synthesis method of Example 3, compound IV-6 was prepared using 3-chlorobenzamide (Material D) and ethyl 2-benzoyl-5,5,5-trifluoro-3-oxypentanoate (Material E) as raw materials; the yield was 75%.

[0149] Compound IV-6: 2-(2-(3-chlorophenyl)hydrazono)-5,5,5-trifluoro-1-phenylpentane-1,3-dione; Gray solid; Yield: 75%;

[0150] 1 H NMR (600 MHz, DMSO-d 6 )δ12.34(s,1H),7.77(d,J=7.8Hz,2H),7.51–7.29(m,5H),7.23(d,J=7.9Hz,1H),2.42(s,2H). 13 C NMR (151 MHz, DMSO-d 6)δ188.74,153.31,149.01,144.06,143.48,134.50,131.51,130.42,129.52,127.96,119.29,118.70,21.66.MS(ESI)m / z:391.0430[M+Na] + .

[0151] Referring to the synthesis method of Example 3, compound IV-11 was prepared using 4-methylbenzamide (Material D) and ethyl 3-(3,4-dichlorophenyl)-3-oxopropionate (Material E) as raw materials; the yield was 69%.

[0152] Compound IV-11: 1-(2-(4-nitrophenyl)hydrazono)-3,4-dichlor-acetophenone; Yellow solid; Yield: 69%; mp: 216.3-218°C;

[0153] 1 H NMR (400 MHz, DMSO-d 6 )δ12.08(s,1H),8.20(d,J=9.2Hz,2H),8.13(s,1H),7.95(d,J=10.2Hz,1H),7.82 (d,J=8.3Hz,1H),7.76(s,1H),7.20(d,J=9.1Hz,2H).MS(ESI)m / z:335.9957[MH] + .

[0154] Referring to the synthesis method of Example 3, compound IV-12 was prepared using 4-chlorobenzamide (Material D) and ethyl (4-bromobenzoyl)acetate (Material E) as raw materials; the yield was 65%.

[0155] 1 H NMR (400 MHz, DMSO-d 6 )δ11.57(s,1H),7.88(d,J=8.4Hz,2H),7.71(d,J=8.5Hz,2H),7.65(s,1H),7.33(d,J=8.8Hz,2H),7.08(d,J=8.8Hz,2H). 13 C NMR (151 MHz, DMSO-d 6)δ188.66,142.44,136.57,134.53,131.87,131.63,129.75,126.41,125.74,115.65.MS(ESI)m / z:358.9592[M+Na] + .

[0156] Compound IV-13 was obtained by referring to the synthesis method of Example 3; the yield was 77%.

[0157] Compound IV-13: 1-(2-(4-nitrophenyl)hydrazono)-4-bromo-acetophenone; Yellow solid; Yield: 77%; mp: 223.3-224°C;

[0158] 1 H NMR (400 MHz, DMSO-d 6 )δ12.01(s,1H),8.19(d,J=7.4Hz,2H),7.91(d,J=8.5Hz,2H),7.77(d,J=4.7Hz,2H),7.74(s,1H),7.20(d,J=9.1Hz,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ188.72,149.23,141.27,137.95,136.02,131.98,131.81,126.96,126.42,113.80.MS(ESI)m / z:369.9792[M+Na] + .

[0159] Referring to the synthesis method of Example 3, compound IV-14 was prepared using 2-amino-4-nitrophenol (Material D) and (4-bromobenzoyl)ethyl acetate (Material E) as raw materials; the yield was 76%.

[0160] 1 H NMR (600 MHz, DMSO-d 6 )δ11.66(s,1H),11.33(s,1H),8.11(s,1H),7.97(s,1H),7.92(d,J=8.1Hz,1H),7.76(t,J=9.9Hz,2H),7.01(d,J=8.8Hz,3H). 13 C NMR (151 MHz, DMSO-d 6)δ188.96,151.23,140.77,136.64,136.31,132.39,131.87,131.53,126.52,118.57,115.17,108.89.MS(ESI)m / z:385.9750[M+Na] + .

[0161] Referring to the synthesis method of Example 3, compound IV-28 was prepared using 3-chloroaniline (Material D) and ethyl 3-(2,4-difluorophenyl)-3-oxo-2-(1H-1,2,4-triazol-1-yl)propanoate (Material E) as raw materials; the yield was 60%.

[0162] Compound IV-28: 2-(2-(3-chlorophenyl)hydrazono)-1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone; Brown solid; Yield: 60%;

[0163] 1 H NMR (600 MHz, DMSO-d 6 )δ11.31(s,1H),8.99(s,1H),8.46(s,1H),7.84(s,1H),7.52(s,1H),7.43–7.27(m,2H),7.19(s,1H),7.12(d,J=23.3Hz,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ182.85,153.12,147.06,144.02,134.25,133.17,131.58,128.15,123. 59,122.99,115.19,113.97,112.01,104.90.MS(ESI)m / z:236.0613[M+H] + .

[0164] Referring to the synthesis method of Example 3, compound IV-29 was prepared using 4-nitroaniline (Material D) and ethyl 3-(2,4-difluorophenyl)-3-oxo-2-(1H-1,2,4-triazol-1-yl)propanoate (Material E) as raw materials; the yield was 73%.

[0165] Compound IV-29: 1-(2,4-difluorophenyl)-2-(2-(4-nitrophenyl)hydrazono)-2-(1H-1,2,4-triazol-1-yl)ethanone; Yellow solid; Yield: 73%;

[0166] 1 H NMR (600 MHz, DMSO-d 6 )δ9.36(s,1H),8.53(d,J=7.7Hz,2H),8.44(s,1H),8.29(s,1H),8.04(d,J=7.6Hz,2H),7.54(s,1H),7.36–7.29(m,1H),7.26(d,J=9.8Hz,1H). 13 CNMR (151MHz, DMSO-d 6 )δ182.56,152.75,147.82,146.80,146.78,142.32,132.97,129.31,128. 43,125.71,115.03,111.90,111.76,104.74.MS(ESI)m / z:373.0844[M+H] + .

[0167] Part IV compounds and compound V-1 were purchased from the commercial database http: / / www.specs.net.

[0168] The numbers and ID numbers of the compounds purchased on the SPECS website are shown in the table below:

[0169]

[0170]

[0171] Example 4 Testing the inhibitory effect of compounds on SpCas9 by in vitro cleavage experiment

[0172] The present invention detects the inhibitory effect of the compounds described in Examples 1 to 3 on the nuclease activity of SpCas9 by an in vitro DNA cleavage test. The cleavage experiment was carried out in the buffer of NEBuffer 3. First, 19.2 μL of SpCas9:gRNA (58.8 nM) was incubated with 2.4 μL of the compound diluted with DMSO at room temperature for 10 min; then 2.4 μL of 3.48 nM linearized plasmid (PX458, Apa I) was added as a cleavage substrate and cultured at 37 ° C for 60 min; 0.5 μL of proteinase K (20 mg / mL) (Invitrogen) was added to terminate the reaction, and the reaction was carried out at 55 ° C for 15 min; the results were visualized with 0.7% TAE agarose gel, and the visualized results were quantified in grayscale using Image J software to calculate the inhibition rate of the compound. The inhibition rate calculation formula is as follows:

[0173] E=(C1-C2) / C1×100%

[0174] E represents the inhibition rate; C1 and C2 represent the lysis rates of the DMSO group and the compound-treated group, respectively.

[0175] The cleavage rate calculation formula is as follows:

[0176] C=(B1+B2) / (B0+B1+B2)×100%

[0177] C represents the cleavage rate; B0 represents the grayscale quantitative value of the uncut product; B1 represents the grayscale quantitative value of the upper cut product; B2 represents the grayscale quantitative value of the lower cut product.

[0178] The results of the inhibition of SpCas9 activity by the compounds described in Examples 1 to 3 are shown in Table 1:

[0179] Table 1 Inhibition rate of compounds on SpCas9 in in vitro cleavage experiments (%)

[0180]

[0181]

[0182] As shown in Table 1, compounds I-1, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-12, I-13, I-15, I-16, I-17, I-18, I-19, II-1, II-2, III-1, III-3, IV-11, IV-12, IV-13, IV-24, IV-25, IV-26, IV-31, IV-32, IV-33, IV-34, IV-36 and IV-37 in the compounds of the present invention have an inhibitory effect on SpCas9, and the inhibitory effect on SpCas9 is good, the inhibition rate exceeds 50%, and SpCas9 can be significantly inhibited from cutting DNA. Subsequent intracellular cutting experiments were performed on compounds that showed an inhibitory effect on SpCas9 in vitro cutting experiments.

[0183] Example 5 Detection of the inhibitory effect of compounds on SpCas9 in cells using EGFP reporter cell lines

[0184] In this example, an intracellular cleavage experiment was performed on the compound shown in Example 4 to have an inhibitory effect on SpCas9 in vitro, and the inhibitory effect of the compound on SpCas9 in cells was detected using an EGFP reporter cell line.

[0185] Step S1: Construction of 293T.EGFP-H2AX.iCas9.gEGFP cell line

[0186] The 293T.EGFP-H2AX.iCas9.gEGFP cell line is prepared by fusing the EGFP expression cassette to the N-terminus of H2AX, and then infecting 293T.EGFP-H2AX with a lentivirus expressing SpCas9 driven by a tet-on promoter; after selecting and amplifying monoclonal cells, the cells are further infected with a lentivirus containing sgEGFP, and then screened with 1 μg / mL puromycin for at least 7 days to obtain the 293T.EGFP-H2AX.iCas9.gEGFP cell line.

[0187] Step S2: Detection of the inhibition of SpCas9 nuclease activity by compounds in 293T.EGFP-H2AX.iCas9.gEGFP cells

[0188] 293T.EGFP-H2AX.iCas9.gEGFP cells were seeded into 96-well plates at a density of 10,000 cells per well and cultured overnight; the specified concentration of the compound was added to the culture medium, with DMSO as the control group; 2 μg / mL doxycycline was added to the drug-containing 96-well plate, with the group without doxycycline as the control group; the culture medium containing cells was replaced with 100 μL culture medium containing DMSO or the above compounds. The cells were cultured for another 72 hours; the cells were digested with 0.25% trypsin EDTA and phenol red (Gibco), and then terminated with cell culture medium; the cells were resuspended, and the eGFP fluorescence was analyzed using a CytoFLEX flow cytometer (Beckman Coulter), and the inhibition efficiency of the compound on SpCas9 was calculated.

[0189] The inhibition rates of the compounds described in Examples 1 to 3 of the present invention on SpCas9 in reporter cells at 10 μM and 40 μM are as follows: Figure 1 As shown by Figure 1 It can be seen that compounds Ⅰ-4, Ⅰ-9, Ⅰ-12, Ⅰ-17, Ⅰ-18, Ⅰ-19, Ⅲ-3, Ⅳ-11, Ⅳ-14, Ⅳ-20, Ⅳ-22, Ⅳ-24, Ⅳ-25, Ⅳ-26, Ⅳ-31, Ⅳ-32, Ⅳ-33 and Ⅴ-1 can inhibit the activity of SpCas9 in cells.

[0190] The results of the effects of the compounds described in Examples 1 to 3 of the present invention on the expression of GFP in the reporter cell line at 40 μM are as follows: Figure 2 As shown by Figure 2 It can be seen that except for compound I-9 and compound I-12, which affect the expression of EGFP, the other compounds do not affect the expression of EGFP, indicating that the compounds do not produce false positive signals by affecting the expression of EGFP, but can indeed inhibit the activity of SpCas9.

[0191] Example 6 High-throughput sequencing to detect the effect of compounds on SpCas9 cleavage of endogenous target sites and off-target sites

[0192] The present invention also uses high-throughput sequencing to detect the effects of compounds V-1 and IV-25 on SpCas9 cleavage of endogenous target sites and off-target sites. The process is as follows:

[0193] The tetracycline response element and doxycycline-driven SpCas9 were cloned into a lentiviral vector with a puromycin resistance cassette, and U2OS cells were impregnated with lentiviral particles containing the above vector. After lentiviral infection, 1 μg / mL puromycin was used to culture until the cells in the negative control group died completely, and monoclonal cells (named U2OS iCas9 cells) were selected for subsequent experiments; sgRNAs targeting EMX1-1, EMX1-2, VEGFA-site3, and ZSCAN2 were cloned into a lentiviral vector containing the blasticidin S deaminase gene, and 293T cells were used to produce lentiviral particles expressing sgRNA. U2OSiCas9 cells were infected with lentivirus and screened with 10 μg / mL blasticidin S HCL (Gibco) until the control cells died completely. The above cells were plated in 12-well plates (Thermo Fisher Scientific) at a density of approximately 40%. After 24 hours, 2 μg / mL doxycycline and the specified concentration of compounds were added, with the DMSO group as the control and the group without doxycycline as the blank control. Each experiment was repeated twice. After 24 hours, the cells were collected and the cell genome was extracted. The targeted and non-targeted sites were amplified using primers with sequencing adapters and the KOD PCR kit (Toyobo) to generate deep sequencing libraries. The samples were sequenced single-end using the 150Hiseq 2000 (Illumina) platform. The sequencing files were split using MATLAB, and the proportion of indels was analyzed using CRISPResso2.

[0194] The DNA sequences of sgRNA targeting EMX1-1, EMX1-2, VEGFA-site3, and ZSCAN2 target sites and off-target sites are shown below (PAM sequences are shown in bold):

[0195] EMX1-1 on-target gRNA:5'-GAGTCCGAGCAGAAGAAGAAGGG-3'

[0196] EMX1-1 off-target gRNA:5'-GAGTTAGAGCAGAAGAAGAAAGG-3'

[0197] EMX1-2 on-target gRNA:5'-GTCACCTCCAATGACTAGGGTGG-3'

[0198] EMX1-2 off-target gRNA:5'-ATCACTTCCAATGACTAAGACGG-3'

[0199] VEGFA-site3 on-target gRNA:5'-GGTGAGTGAGTGTGTGCGTGTGG-3'

[0200] VEGFA-site3 off-target gRNA:5'-GCTGAGTGAGTGTATGCGTGTGG-3'

[0201] ZSCAN2 on-target gRNA:5'-GTGCGGCAAGAGCTTCAGCCGGG-3'

[0202] ZSCAN2 off-target gRNA:5'-GTGTGGCAAGGGCTTCAGCCAGG-3'

[0203] The PCR primer sequences used to amplify the genomic targeted and non-targeted sites are as follows:

[0204] EMX1-1 on-target F:5'-CGATGTCCTCCCCATTGGCCTG-3'

[0205] EMX1-1 on-target R:5'-GGAGCAGCTGGTCAGAGGGG-3'

[0206] EMX1-1 off-target F:5'-GCTTTTATACCATCTTGGGGTTACAG-3'

[0207] EMX1-1 off-target R:5'-GTGGGGAGATTTGCATCTGTGGAGG-3'

[0208] EMX1-2 on-target F:5'-CCAAAGCCTGGCCAGGGAGTG-3'

[0209] EMX1-2 on-target R:5'-GCCCAGGTGAAGGTGTGGTTCC-3'

[0210] EMX1-2 off-target F:5'-CCATCTGAGTCAGCCAGCCTTGTC-3'

[0211] EMX1-2 off-target R:5'-AGGCAAAGATCTAGGACCTGGATGG-3'

[0212] VEGFA-site3 on-target F:5'-GGGGAGAGGGACACACAGAT-3'

[0213] VEGFA-site3 on-target R:5'-TCCAAAGCCCATTCCCTCTT-3'

[0214] VEGFA-site3 off-target F:5'-CTCCTTGAGGTTCATCCCC-3'

[0215] VEGFA-site3 off-target R:5'-GGTTAGGAGAGCTGGCTTGG-3'

[0216] ZSCAN2 on-target F:5'-TCCAGCTAAAGCCTTTCCCACAC-3'

[0217] ZSCAN2 on-target R:5'-GAGTGCCTGACATGTGGGGAGAG-3'

[0218] ZSCAN2 off-target F:5'-ACCGTATCAGTGTGATGCATGTGGT-3'

[0219] ZSCAN2 off-target R:5'-GAACTCTCTGATGCACCTGAAGGCTG-3'

[0220] Effects of compound V-1 and compound IV-25 on SpCas9 cleavage of different target sites and off-target sites Figure 3 As shown; Figure 3 A in it is the effect of compound V-1 and compound IV-25 on SpCas9 cleavage of EMX1-1 target site and off-target site; Figure 3 B in it is the effect of compound V-1 and compound IV-25 on SpCas9 cleavage of EMX1-2 target sites and off-target sites; Figure 3 C in it is the effect of compound V-1 and compound IV-25 on SpCas9 cleavage of VEGFA-site3 target site and off-target site; Figure 3 D in FIG. 1 is the effect of compound V-1 and compound IV-25 on SpCas9 cleavage of ZSCAN2 target sites and off-target sites. Figure 3 The results show that compounds V-1 and IV-25 can reduce the off-target cleavage activity of SpCas9 and improve the specificity of SpCas9.

[0221] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a compound in the preparation of a reagent for inhibiting Cas protein activity, characterized in that: The compound is a compound having any of the following structural formulas or a pharmaceutically acceptable salt thereof; 。 2. The application according to claim 1, characterized in that: The Cas protein is SpCas9 protein.

3. Use of the compound described in claim 1 in the preparation of a reagent for regulating the CRISPR / Cas gene editing system.

4. The use according to claim 3, characterized in that: The CRISPR / Cas gene editing system is a CRISPR / SpCas9 gene editing system.

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