A compound capable of targeted degradation of myc and uses thereof

Abstract

The application discloses a compound capable of targeted degradation of MYC and application thereof, and belongs to the technical field of medicines. The compound can target degradation of MYC after entering a patient's body, thereby effectively inhibiting the proliferation of tumor cells such as lymphoma and osteosarcoma and cancer cells such as colorectal cancer, and having good anti-tumor effect and anticancer effect; the compound can also inhibit the proliferation of myelomonocytic leukemia cells in the patient's body, thereby providing a new treatment scheme for treating myelomonocytic leukemia; the compound has a synergistic effect when combined with doxorubicin, has better anti-tumor activity, and has a wide application prospect in the field of medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a compound that can target and degrade MYC and its applications. Background Technology

[0002] Many diseases are caused by mutations or accumulation of specific proteins, and specifically degrading these pathogenic proteins can fundamentally intervene in or treat these diseases. Targeted protein degradation (TPD) provides a promising new intervention approach by hijacking endogenous protein degradation mechanisms, inducing the consumption or reduction of pathogenic proteins, thus offering a different approach from traditional drugs. This novel drug development includes protein degradation-targeting chimeras (PROTAC), molecular gels, lysosome-targeting chimeras (LYTAC), perspectives of autophagy-tethering compounds (ATTEC), autophagy-targeting chimeras (AUTAC and AUTOTAC), and antibody-drug conjugates (Degrader-antibody). There are various types of conjugates (DACs), etc. Targeted protein degradation currently mainly involves the degradation of target proteins through ubiquitin-proteasomes and lysosomes, with molecular gels and PROTAC technologies being the fastest developing. PROTAC uses a linker group to chemically link the target protein ligand to the E3 ligase ligand, "recruiting" the E3 ligase to the vicinity of the target protein, and utilizing the intracellular ubiquitin-proteasome system to achieve ubiquitination labeling and protein degradation of the target protein. Molecular gels are small molecules that induce access, allowing for precise time control of various biological processes, such as signal transduction, transcription, chromatin regulation, and protein folding, localization, and degradation. Molecular gels have a small molecular weight, making their physicochemical properties easy to optimize. Molecular gels mainly induce or stabilize protein-protein interactions between ubiquitin ligases and substrate proteins, leading to protein degradation. They can degrade inaccessible target proteins without requiring a binding pocket on the target protein. In summary, the unique mechanism of TPD drugs provides a very promising prospect for the drug development of these undrugable proteins.

[0003] MYC, a nuclear transcription factor, typically refers to c-Myc, which regulates growth and development in normal cells, promoting cell metabolism and proliferation. However, when MYC expression and function are abnormal, excessive cell proliferation leads to homeostasis imbalance. MYC can not only cause normal cells to become cancerous but also promote cancer cells' escape from immune responses, closely related to tumor recurrence, metastasis, and drug resistance. Furthermore, MYC is a key stem cell regulator, possessing the ability to "reprogram" adult cells into pluripotent stem cells. Studies show that 70-80% of tumors exhibit dysregulation or overexpression of MYC protein; this persistent high expression leads to abnormal activation of tumor cells, becoming a driving factor in tumor development and progression. Therefore, the development of drugs targeting MYC can provide more treatment options for clinical cancer patients, possessing significant biological and clinical importance. However, MYC as a drug target has inherent limitations: 1) Antibody drugs cannot target it; 2) MYC's smooth binding surface lacks traditional drug-binding pockets, making it difficult for small molecule inhibitors to bind, and existing traditional drugs struggle to directly target MYC; 3) Biotechnology drugs such as siRNA have also failed in Phase I / II clinical trials. Multiple studies have shown that post-translational modifications of MYC, such as phosphorylation, ubiquitination, and acetylation, regulate its transcriptional activity by affecting the formation of the MYC transcriptional complex or its degradation by the proteasome. Dysregulation of post-translational modifications leading to excessive MYC accumulation is a key cause of downstream proliferation, metastasis, and activation of stem signaling pathways, suggesting that targeting MYC degradation is a novel strategy for tumor suppression.

[0004] Therefore, it is necessary to develop a compound that can target and degrade MYC to solve the above problems. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a compound that can target and degrade MYC, providing a new treatment option for cancer, tumors and leukemia.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A compound capable of targeted degradation of MYC, the structure of which is as follows:

[0008]

[0009] Among them, R 1 Selected from 2-(4-methylpiperazin-1-yl)acetyl, cyclobutyryl, 3,3-dimethylbutyryl, 5-chloro-2-methoxy-4-(methylamino)benzoyl, oxazol-4-carboxyl, 2-cyclopropyl-4-fluorobenzene-1-carboxyl, 4-cyclopropyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid;

[0010] R 2 Selected from 2-cyclopropyl-4-fluorophenyl.

[0011] The present invention also provides a pharmaceutical composition comprising the compounds described above.

[0012] Furthermore, the pharmaceutical composition further includes a pharmaceutically acceptable pharmaceutical excipient, which is a pharmaceutically acceptable salt, excipient, or carrier.

[0013] Furthermore, the carrier is any one or more of a filler, wetting agent, adhesive, disintegrant, or lubricant.

[0014] Furthermore, the pharmaceutical composition is formulated in one or more of the following forms: aqueous extract, powder, lotion, tincture, oil, emulsion, ointment, plaster, or aerosol.

[0015] Another object of the present invention is to provide the use of the MYC-targeting compounds described above in the preparation of antitumor drugs, anticancer drugs, drugs for treating leukemia, and drugs for antitumor treatment in combination with doxorubicin.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention provides a compound that can target and degrade MYC. After entering the patient's body, this compound can target and degrade MYC, thereby effectively inhibiting the proliferation of tumor cells such as lymphoma and osteosarcoma, as well as cancer cells such as colorectal cancer, and has good anti-tumor and anti-cancer effects. This compound can also inhibit the proliferation of myeloid monocytic leukemia cells in the patient's body, providing a new treatment option for myeloid monocytic leukemia. This compound has a synergistic effect when used in combination with doxorubicin, exhibiting superior anti-tumor activity and has broad application prospects in the medical field.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1This is a time- and dose-dependent statistical graph of the degradation of MYC protein expression in Daudi cells by a series of compounds at different times and concentrations, using Western blotting technology.

[0021] Figure 2 This is a diagram showing the results of in vivo studies on the inhibitory effect of different concentrations of compound 8 on the growth of Daudi cell tumors, conducted using a Daudi subcutaneous tumor model.

[0022] Figure 3 To construct a Daudi mid-to-late stage xenograft model (>500mm) 3 (Figure showing the results of detecting the inhibitory effect of compound 30 on the growth of Daudi cell tumors)

[0023] Figure 4 This is a graph showing the results of constructing a Daudi cell subcutaneous tumor model to detect the antitumor effect of compound 30 in combination with doxorubicin, a first-line drug for lymphoma. Detailed Implementation

[0024] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0026] The following examples pertain to the intermediate compounds and final products identified in the specification and synthetic regimens. The preparation of the compounds of the present invention is described in detail using the following examples, but the described chemical reactions are disclosed in accordance with their general applicability to the preparation of the compounds of the present invention. Sometimes, the reactions may not be applicable to every compound within the scope of the present invention as described. Compounds in which this may occur are readily identifiable to those skilled in the art. In these cases, the reactions can be successfully carried out with conventional modifications known to those skilled in the art. In all preparation methods, all starting materials are known or can be readily prepared using known starting materials. All temperatures are given in degrees Celsius, and unless otherwise explicitly stated, all parts and percentages are in moles when referring to yields, and all parts are in volumes when referring to solvents and eluents.

[0027] The abbreviations used in the following examples are as follows:

[0028] Et3N is triethylamine; Toluene is toluene; Pd(PPh3)4 is tetratetraphenylphosphine palladium; Na2CO3 is sodium carbonate; ACN is acetonitrile; HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DMF is dimethylformamide; PBS is phosphate buffer solution; K2CO3 is potassium carbonate; Boc is tert-butyloxycarbonyl; OH is hydroxyl; TFA is trifluoroacetic acid; DCM is dichloromethane; Pyridine is pyridine; AcOH is acetic acid; PBST is phosphate buffer; DMSO is dimethyl sulfoxide; PVDF is polyvinylidene fluoride; GAPDH is olealdehyde-3-phosphate dehydrogenase; PEG is polyethylene glycol.

[0029] A compound capable of targeted degradation of MYC, the structure of which is as follows:

[0030]

[0031] Among them, R 1 Selected from 2-(4-methylpiperazin-1-yl)acetyl, cyclobutyryl, 3,3-dimethylbutyryl, 5-chloro-2-methoxy-4-(methylamino)benzoyl, oxazol-4-carboxyl, 2-cyclopropyl-4-fluorobenzene-1-carboxyl, 4-cyclopropyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid;

[0032] R 2 Selected from 2-cyclopropyl-4-fluorophenyl.

[0033] Example 1: Synthesis of Compound 1

[0034]

[0035] N-(2-Cyclopropyl-4-fluorophenyl)-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide

[0036] Synthesis scheme:

[0037]

[0038] Step 1: Synthesis of 2-cyclopropyl-4-fluoroaniline

[0039]

[0040] Cyclopropylboronic acid (4.97 g, 57.89 mmol), 2-bromo-4-fluoroaniline (10.00 g, 52.63 mmol), potassium phosphate (27.93 g, 131.57 mmol), toluene (100 mL), and purified water (20 mL) were added to the reaction flask. The mixture was stirred at room temperature for 10 min. Palladium acetate (0.60 g, 2.63 mmol) and tricyclohexylphosphine (1.48 g, 5.26 mmol) were then added to the reaction flask, and nitrogen was used to replace the nitrogen atmosphere. The mixture was stirred at 90 °C for 8 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, and the mixture was extracted with dichloromethane, concentrated, and purified by column chromatography to obtain 2-cyclopropyl-4-fluoroaniline as a brown liquid (7.00 g, yield 88%, purity 95.3%). Characterization: LCMS m / z = 152.08 [M+1] + .

[0041] Step 2: Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0042]

[0043] Methyl 6-amino-4'-methyl-[1,1'-biphenyl]-3-carboxylic acid (7.00 g, 46.30 mmol), 4-fluoro-7-nitrobenzo[c][1,2,5]oxadiazole (9.33 g, 50.93 mmol), acetonitrile (28 mL), and purified water (7 mL) were added to the reaction flask. The mixture was stirred at 75 °C for 8 h. After the reaction was complete, the mixture was cooled to room temperature, and a large amount of red solid precipitated. The solid was filtered and washed with acetonitrile to give N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine, which was a bright red solid (14.26 g, yield: 98%, purity: 99.1%). It was characterized as follows: LCMS m / z = 315.08 [M+1]+.

[0044] Step 3: Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)acetamide

[0045]

[0046] Acetic acid (76 mg, 1.27 mmol), TCTU (566 mg, 1.59 mmol), DMF (2 ml), and triethylamine (193 mg, 1.91 mmol) were added to the reaction flask, and the mixture was stirred at room temperature for 5 min. N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide (200 mg, 0.64 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 4 h. The mixture was diluted with purified water, extracted with dichloromethane, and the organic phase was concentrated to obtain a brown liquid. The liquid was purified by column chromatography (n-heptane:ethyl acetate = 1:2) to obtain an oily substance. Preparative HPLC was used to purify N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide, which was a pale yellow solid (122 mg, yield: 54%, purity 100%). The expression is: LCMS m / z = 357.09[M+1] + .

[0047] Example 2: Synthesis of Compound 2

[0048]

[0049] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2-fluoro-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0050] N-(2-cyclopropyl-4-fluorophenyl)-2-fluoro-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 437.10 [M+1] +

[0051] Example 3: Synthesis of Compound 3

[0052]

[0053] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3-fluoro-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0054] N-(2-cyclopropyl-4-fluorophenyl)-3-fluoro-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 437.10 [M+1] +

[0055] Example 4: Synthesis of Compound 4

[0056]

[0057] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2,6-difluoro-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0058] N-(2-cyclopropyl-4-fluorophenyl)-2,6-difluoro-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 455.09 [M+1] +

[0059] Example 5: Synthesis of Compound 5

[0060]

[0061] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)cyclohexaneformamide

[0062] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclohexaneformamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows:

[0063] 1 H NMR (400MHz, CDCl3) δ8.41(d,J=8.0Hz,1H),7.11(d,J=8.0Hz,1H),6.98(ddd,J=8.6,7.6,2.9Hz,1H),6.66(dd,J=9.7,2.9Hz,1H),2.4 8-2.28(m,1H),1.97-1.53(m,12H),1.37-1.19(m,4H),1.17-0.95(m,3H),0.81-0.69(m,2H),0.67-0.52(m,1H).LCMSm / z=425.15[M+1] +

[0064] Example 6: Synthesis of Compound 6

[0065] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopentanamide.

[0066] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopentanamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 411.14 [M+1] +

[0067]

[0068] Example 7 Synthesis of Compound 7

[0069]

[0070] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-2-(piperidin-1-yl)acetamide

[0071] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-2-(piperidin-1-yl)acetamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 440.17 [M+1] +

[0072] Example 8: Synthesis of Compound 8

[0073]

[0074] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2-(4-methylpiperazin-1-yl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide

[0075] N-(2-cyclopropyl-4-fluorophenyl)-2-(4-methylpiperazin-1-yl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 455.18 [M+1] +

[0076] Example 9: Synthesis of Compound 9

[0077]

[0078] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropaneformamide

[0079] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropaneformamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 383.11 [M+1] +

[0080] Example 10: Synthesis of Compound 10

[0081]

[0082] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclobutyramide

[0083] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclobutyramide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 397.12 [M+1] +

[0084] Example 11 Synthesis of Compound 11

[0085] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide.

[0086]

[0087] N-(2-cyclopropyl-4-fluorophenyl)-2,2,3,3-tetramethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows:

[0088] 1 H NMR (400MHz, CDCl3) δ8.42(d,J=8.1Hz,1H),7.10(d,J=8.1Hz,1H),7.05-6.97(td,1H),6.67(dd,J=9.7,2.9Hz,1H),1.88-

[0089] 1.76(m,1H),1.39(s,3H),1.32(s,4H),1.08(s,3H),1.01(s,5H),0.79(ddd,J=1 1.3,9.9,5.1Hz,1H),0.71-0.61(m,1H),0.43(ddt,J=9.6,6.5,4.9Hz,1H).LCMS m / z=438.17[M+1] +

[0090] Example 12 Synthesis of Compound 12

[0091]

[0092] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2-morpholino-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide

[0093] N-(2-cyclopropyl-4-fluorophenyl)-2-morpholino-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows:

[0094] 1 H NMR (400MHz, CDCl3) δ8.48(d,J=8.0Hz,1H),7.49(dd,J=8.4,5.5Hz,2H),7.02(ddd,J=8.7,7.5,2.8Hz,1H),6.66(dd,J=9.5,2.8Hz,1H),4.22(d ,J=16.4Hz,1H),4.05(t,J=4.7Hz,4H),3.78(d,J=16.4Hz,1H),1.97-1. 87(m,1H),1.18-1.07(m,1H),0.86-0.72(m,2H),0.62-0.49(m,1H).LCMS m / z=442.14[M+1] +

[0095] Example 13 Synthesis of Compound 13

[0096]

[0097] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0098] N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows:

[0099] 1 H NMR (400MHz, CDCl3) δ8.44(d,J=8.0Hz,1H),7.28-7.21(m,1H),7.14(d,J=8.0Hz,1H),7.00(ddd,J=8.6,7.6,2.9Hz,1H),6.66(dd,J=9 .7,2.9Hz,1H),2.29(q,J=14.8Hz,2H),1.95-1.85(m,1H),1.15(s,8H),1.12-1.05(m,1H),0.84-0.74(m,2H),0.65-0.56(m,1H).LCMS m / z=399.14[M+1] +

[0100] Example 14 Synthesis of Compound 14

[0101]

[0102] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-7-nitro-N-(pyrimidin-2-yl)benzo[c][1,2,5]oxadiazole-4-amine

[0103] N-(2-cyclopropyl-4-fluorophenyl)-7-nitro-N-(pyrimidin-2-yl)benzo[c][1,2,5]oxadiazole-4-amine was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 393.10 [M+1] +

[0104] Example 15 Synthesis of Compound 15

[0105] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0106]

[0107] N-(2-cyclopropyl-4-fluorophenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 413.15 [M+1] +

[0108] Example 16 Synthesis of Compound 16

[0109]

[0110] Synthesis of N-(2-cyclohexyl-4-fluorophenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0111] N-(2-cyclohexyl-4-fluorophenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using similar steps to steps 1-3 of Example 1.

[0112] Characterized as: 1H NMR (400MHz, CDCl3) δ8.42(d,J=8.0Hz,1H),7.35(dd,J=8.6,5.3Hz,1H),7.15-7.05(m,2H),7.04(d,J=8.0Hz,1H),2.74(td,J=11.9,2.0Hz,1 LCMS m / z=455.20[M+1] +

[0113] Example 17 Synthesis of Compound 17

[0114]

[0115] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3,5-difluoro-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0116] N-(2-cyclopropyl-4-fluorophenyl)-3,5-difluoro-N-(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using similar steps to steps 1-4 of Example 1.

[0117] The expression is: LCMS m / z = 455.09[M+1] +

[0118] Example 18 Synthesis of Compound 18

[0119]

[0120] Synthesis of 5-fluoro-2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)phenol

[0121] 5-Fluoro-2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)phenol was prepared using steps similar to steps 1-4 of Example 1.

[0122] The expression is: LCMS m / z = 291.05[M+1] +

[0123] Example 19 Synthesis of Compound 19

[0124]

[0125] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-4-fluoro-N-(7-nitrobenzyl[c][1,2,5]oxadiazol-4-yl)benzamide

[0126] N-(2-cyclopropyl-4-fluorophenyl)-4-fluoro-N-(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps similar to steps 1 and 3-4 in Example 1.

[0127] The expression is: LCMS m / z = 437.10[M+1] +

[0128] Example 20 Synthesis of Compound 20

[0129]

[0130] Synthesis of 4-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)carbamoyl)benzoic acid

[0131] 4-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamoyl)benzoic acid was prepared using steps 1 and 3-4 of Example 1.

[0132] The expression is: LCMS m / z = 463.10[M+1] +

[0133] Example 21 Synthesis of Compound 21

[0134]

[0135] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3-hydroxy-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0136] N-(2-cyclopropyl-4-fluorophenyl)-3-hydroxy-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using similar steps to steps 1-3 of Example 1.

[0137] The expression is: LCMS m / z = 415.13[M+1] +

[0138] Example 22 Synthesis of Compound 22

[0139]

[0140] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide

[0141] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide was prepared using similar steps to steps 1-3 of Example 1.

[0142] Characterized as: 1 H NMR(600MHz,DMSO)δ9.81(s,1H),9.08(s,1H),8.49(d,J=8.9Hz,1H),7.33(dd,J=8.8,5.7Hz,1H),6.96(td,J=8.5,2.9Hz,1H), 6.76-6.68(m,2H),2.90(s,2H),1.93(td,J=8.4,4.4Hz,1H),1.65(s,6H),0.86(tt,J=12.2,6.1Hz,2H),0.67-0.62(m,2H).LCMS m / z=426.06[M+1] +

[0143] Example 23 Synthesis of Compound 23

[0144]

[0145] Synthesis of methyl (1-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzol-[c][1,2,5]oxadiazol-4-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate

[0146] Methyl(1-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate was prepared using similar steps to steps 1-3 of Example 1.

[0147] The expression is: LCMS m / z = 472.16[M+1] +

[0148] Example 24 Synthesis of Compound 24

[0149]

[0150] Synthesis of 2-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide

[0151] 2-Amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide was prepared using similar steps to those in steps 1-3 of Example 1. It was characterized as follows: LCMS m / z = 436.11 [M+1] +

[0152] Example 25 Synthesis of Compound 25

[0153]

[0154] Synthesis of 3-amino-N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0155] 3-Amino-N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using similar steps to steps 1-3 of Example 1. It was characterized as follows:

[0156] 1 H NMR (400MHz, CDCl3) δ8.46(d,J=8.0Hz,1H),7.57(q,J=3.1Hz,2H),7.10(d,J=8.0Hz,1H),6.94(ddd,J=8.6,7.7,2.9Hz,1H),6.74(dd, J=9.7,2.9Hz,1H),1.96-1.88(m,1H),0.89-0.82(m,2H),0.77-0.69(m,1H),0.64-0.56(m,1H),0.47(ddt,J=9.7,6.4,4.9Hz,1H).LCMS m / z=414.16[M+1] +

[0157] Example 26 Synthesis of Compound 26

[0158]

[0159] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3,4-dimethoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide

[0160] N-(2-cyclopropyl-4-fluorophenyl)-3,4-dimethoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide was prepared using steps 1 and 3-4 of Example 1. It was characterized as follows:

[0161] 1H NMR (400MHz, CDCl3) δ8.41(d,J=8.0Hz,1H),7.23(m,1H),7.12(dd,J=8.7,5.2Hz,1H),6.90-6.84(m,2H),6.73(d,J=8.4Hz,1H),6.62(dd,J=9.7,2. 8Hz,1H),3.88(s,3H),3.79(s,3H),1.87(ddd,J=14.2,8.9,5.6Hz,2H),0 .98-0.91(m,1H),0.73(m,1H),0.63-0.55(m,1H),0.47-0.39(m,1H).LCMS m / z=479.13[M+1] +

[0162] Example 27 Synthesis of Compound 27

[0163]

[0164] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3,4,5-trimethoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide

[0165] N-(2-cyclopropyl-4-fluorophenyl)-3,4,5-trimethoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using similar steps to steps 1-3 of Example 1.

[0166] 1 H NMR (400MHz, CDCl3) δ8.43(d,J=8.0Hz,1H),7.12(dd,J=8.7,5.2Hz,1H),6.94-6.85(m,4H),6.60(dd,J=9.6,2.8Hz,1H),3.86(s,3H),3.7 2(s,6H),1.94-1.85(m,1H),0.99-0.91(m,1H),0.73(tt,J=10.0,5.2Hz,1H),0.64-0.56(m,1H),0.43(ddd,J=11.3,9.6,4.9Hz,1H).LCMS m / z=509.14[M+1] +

[0167] Example 28 Synthesis of Compound 28

[0168]

[0169] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-5-(ethylsulfonyl)-2-methoxy-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0170] 4-Amino-N-(2-cyclopropyl-4-fluorophenyl)-5-(ethylsulfonyl)-2-methoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamide was prepared following steps 1-3 of Example 1. It was characterized as follows:

[0171] 1 H NMR (600MHz, DMSO) δ8.59(d,J=8.1Hz,1H),7.73(s,1H),7.27(dd,J=8.7,5.5Hz,1 H),7.20(d,J=8.1Hz,1H),6.98(td,J=8.4,2.8Hz,1H),6.81(dd,J=10.3,2.8Hz,1 H),6.52(s,2H),6.20(s,1H),3.42(s,3H),3.14(q,J=7.3Hz,2H),1.92-1.88(m,1 H),1.01(t,J=7.3Hz,3H),0.88-0.80(m,4H),0.55-0.48(m,1H),0.33(s,1H).LCMS m / z = 556.12 [M+1] +

[0172] Example 29 Synthesis of Compound 29

[0173]

[0174] Synthesis of 4-bromo-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide

[0175] 4-Bromo-N-(2-Cyclopropyl-4-fluorophenyl)-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide was prepared using steps 1-3 of Example 1.

[0176] Characterized as: 1H NMR (600MHz, CDCl3) δ8.47(d,J=8.0Hz,1H),7.45(s,1H),7.15(d,J=8.0Hz,1H),6.97-6.93(m,1H),6.73(dd,J=9. 7,2.8Hz,1H),1.92-1.86(m,1H),0.92-0.86(m,1H),0.74-0.70(m,1H),0.66-0.61(m,1H),0.54-0.49(m,1H).LCMS m / z=505.98[M+1] +

[0177] Example 30 Synthesis of Compound 30

[0178]

[0179] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-4,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide

[0180] N-(2-cyclopropyl-4-fluorophenyl)-4,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide was prepared using steps 1-3 of Example 1.

[0181] The expression is: LCMS m / z = 454.09[M+1] +

[0182] Example 31 Synthesis of Compound 31

[0183]

[0184] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-5-methyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide

[0185] N-(2-cyclopropyl-4-fluorophenyl)-5-methyl-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)thiazole-2-carboxamide was prepared using steps 1-3 of Example 1.

[0186] The expression is: LCMS m / z = 440.09[M+1] +

[0187] Example 32 Synthesis of Compound 32

[0188]

[0189] Synthesis of 2-amino-N-(2-cyclopropyl-4-fluorophenyl)-4-methyl-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide

[0190] 2-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-4-methyl-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide was prepared using steps 1-3 of Example 1.

[0191] The expression is: LCMS m / z = 450.12[M+1] +

[0192] Example 33 Synthesis of Compound 33

[0193]

[0194] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-4-hydroxy-2-methyl-N-(7-nitrobenzyl[c][1,2,5]oxadiazol-4-yl)benzamide

[0195] N-(2-cyclopropyl-4-fluorophenyl)-4-hydroxy-2-methyl-N-(7-nitrobenzon[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0196] The expression is: LCMS m / z = 449.12[M+1] +

[0197] Example 34 Synthesis of Compound 34

[0198]

[0199] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-2-methoxy-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0200] 4-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-2-methoxy-N-(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0201] The expression is: LCMS m / z = 464.13[M+1] +

[0202] Example 35 Synthesis of Compound 35

[0203]

[0204] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)-1H-pyrazole-4-carboxamide

[0205] N-(2-cyclopropyl-4-fluorophenyl)-3-methyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)-1H-pyrazole-4-carboxamide was prepared using steps 1-3 of Example 1.

[0206] The expression is: LCMS m / z = 423.11[M+1] +

[0207] Example 36 Synthesis of Compound 36

[0208]

[0209] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-3,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)-1H-pyrazole-4-carboxamide

[0210] N-(2-cyclopropyl-4-fluorophenyl)-3,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)-1H-pyrazole-4-carboxamide was prepared using steps 1-3 of Example 1.

[0211] The expression is: LCMS m / z = 437.13[M+1] +

[0212] Example 37 Synthesis of Compound 37

[0213]

[0214] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-4-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)pentanamide

[0215] 4-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-4-methyl-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)pentanamide was prepared using steps 1-3 of Example 1.

[0216] Characterized as: 1H NMR (400MHz, DMSO) δ8.67(d,J=8.0Hz,1H),7.80(s,3H),7.62(dd,J=8.6,5.5Hz,1H),7.46(d,J=8.0Hz,1H),7.18(td,J=8.4,2.7Hz,1H),6.95(dd,J= 10.2,2.6Hz,1H),3.94(s,5H),2.42-2.30(m,2H),1.90(dd,J=16.3,7.5Hz ,4H),1.15(m,7H),0.95(m,1H),0.86-0.78(m,1H),0.66-0.53(m,2H).LCMS m / z=428.17[M+1] +

[0217] Example 38 Synthesis of Compound 38

[0218]

[0219] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-3,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0220] 4-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-3,5-dimethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0221] The expression is: LCMS m / z = 462.15[M+1] +

[0222] Example 39 Synthesis of Compound 39

[0223]

[0224] Synthesis of 2-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide

[0225] 2-Amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide was prepared using steps 1-3 of Example 1.

[0226] The expression is: LCMS m / z = 372.10[M+1] +

[0227] Example 40 Synthesis of Compound 40

[0228]

[0229] Synthesis of 4-amino-5-chloro-N-(2-cyclopropyl-4-fluorophenyl)-2-methoxy-N-(7-nitrobenzol[c][1,2,5]oxadiazol-4-yl)benzamide

[0230] 4-Amino-5-chloro-N-(2-cyclopropyl-4-fluorophenyl)-2-methoxy-N-(7-nitrobenzon[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0231] Characterized as: 1 H NMR (600MHz, DMSO) δ8.58(d,J=8.1Hz,1H),7.43(s,1H),7.22(dd,J=8.6,5.5Hz,1H),7.14(d,J=8.1Hz,1H),6.98(td,J=8.4,2.8Hz,1H), 6.81(dd,J=10.3,2.8Hz,1H),6.14(s,1H),3.24(s,3H),1.94-1.89(m,1H),0.87-0.78(m,2H),0.55-0.48(m,1H),0.42-0.34(m,1H).LCMS m / z=498.09[M+1] +

[0232] Example 41 Synthesis of Compound 41

[0233]

[0234] Synthesis of 5-chloro-N-(2-cyclopropyl-4-fluorophenyl)-2-methoxy-4-(methylamino)-N-(7-nitrobenzol[c][1,2,5]oxadiazol-4-yl)benzamide

[0235] 5-Chloro-N-(2-cyclopropyl-4-fluorophenyl)-2-methoxy-4-(methylamino)-N-(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0236] The expression is: LCMS m / z = 512.11[M+1] +

[0237] Example 42 Synthesis of Compound 42

[0238]

[0239] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-2,3,5,6-tetrafluoro-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0240] 4-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-2,3,5,6-Tetrafluoro-N-(7-Nitrobenzo[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 1-3 of Example 1.

[0241] Characterized as: 1 H NMR(600MHz,DMSO)δ8.63(d,J=8.0Hz,1H),7.40-7.33(m,2H),7.07(td,J=8.4,2.9Hz,1H ),6.85(dd,J=10.2,2.8Hz,1H),6.65(s,2H),1.90-1.84(m,1H),0.89-0.79(m,2H),0.67-

[0242] 0.60(m,1H),0.46-0.39(m,1H).LCMS m / z=506.09[M+1] +

[0243] Example 43 Synthesis of Compound 43

[0244]

[0245] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)oxazol-4-carboxamide

[0246] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazole-4-yl)oxazole-4-carboxamide was prepared using steps 1-3 of Example 1.

[0247] Characterized as: 1 H NMR (400MHz, DMSO) δ8.68-8.61(m,1H),8.29(s,1H),7.42(dd,J=8.6,5.5Hz,1H),7.28(d,J=8.0Hz,1H),7.09(td,J=8. 4,2.7Hz,1H),6.95(dd,J=10.1,2.5Hz,1H),1.89-1.78(m,1H),1.23(s,1H),0.82-0.68(m,1H),0.58-0.44(m,1H).LCMS m / z=410.08[M+1] +

[0248] Example 44 Synthesis of Compound 44

[0249]

[0250] Synthesis of 3,5-diamino-6-chloro-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)pyrazine-2-carboxamide

[0251] 3,5-Diamino-6-chloro-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)pyrazine-2-carboxamide was prepared using steps 1-3 of Example 1.

[0252] The expression is: LCMS m / z = 485.08[M+1] +

[0253] Example 45 Synthesis of Compound 45

[0254]

[0255] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)-1H-imidazol-2-carboxamide

[0256] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)-1H-imidazol-2-carboxamide was prepared using steps 1-3 of Example 1.

[0257] The expression is: LCMS m / z = 409.10[M+1] +

[0258] Example 46 Synthesis of Compound 46

[0259]

[0260] Synthesis scheme:

[0261]

[0262] Step 1: Synthesis of 2-cyclopropyl-4-fluoroaniline: 2-cyclopropyl-4-fluoroaniline was prepared following the procedure in Step 1 of Example 1. Characterization showed: LCMS m / z = 152.08 [M+1] +

[0263] Step 2: Synthesis of N-(2-cyclopropyl-4-fluorophenyl)pyrimidine-2-amine

[0264]

[0265] 2-Bromopyrimidine (1.16 g, 7.28 mmol), 2-bromo-4-fluoroaniline (1.00 g, 6.61 mmol), p-toluenesulfonic acid (1.71 g, 9.92 mmol), and isopropanol (5 mL) were added to the reaction flask. The mixture was heated to 90 °C and stirred for 8 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with pure water, extracted with dichloromethane, concentrated, and purified by column chromatography to give N-(2-cyclopropyl-4-fluorophenyl)pyrimidine-2-amine, a light brown liquid (1.38 g, 91% yield, 94.6% purity). Characterization was: LCMS m / z = 230.10 [M+1] +

[0266] Step 3: Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0267] N-(2-cyclopropyl-4-fluorophenyl)-7-nitro-N-(pyrimidin-2-yl)benzo[c][1,2,5]oxadiazole-4-amine was prepared following step 3 of Example 1. It was characterized as follows: LCMS m / z = 393.10 [M+1] +

[0268] Example 47 Synthesis of Compound 47

[0269]

[0270] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-7-nitro-N-(thiazol-2-yl)benzo[c][1,2,5]oxadiazole-4-amine

[0271] N-(2-cyclopropyl-4-fluorophenyl)-7-nitro-N-(thiazo-2-yl)benzo[c][1,2,5]oxadiazole-4-amine was prepared following steps 1-3 of Example 46. Characterization showed: LCMS m / z = 397.06 [M+1] +

[0272] Example 48 Synthesis of Compound 48

[0273]

[0274] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(1H-imidazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0275] N-(2-cyclopropyl-4-fluorophenyl)-N-(1H-imidazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine was prepared following steps 1-3 of Example 46. Characterization showed: LCMS m / z = 381.10 [M+1] +

[0276] Example 49 Synthesis of Compound 49

[0277]

[0278] Synthesis of 6-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pyrimidine-2,4(1H,3H)-dione

[0279] 6-((2-cyclopropyl-4-fluorophenyl)(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pyrimidine-2,4(1H,3H)-dione was prepared using steps 1-3 of Example 46.

[0280] Characterized as: 1 H NMR (400MHz, DMSO) δ10.95(s,1H),10.58(s,1H),8.68(d,J=7.7Hz,1H),8.43(s,1H),7.71(d,J=7.7Hz,1H),7.05(dd,J=8.7,5.6 Hz,1H),6.85(td,J=8.5,2.9Hz,1H),6.61(dd,J=10.4,2.9Hz,1H),2.01-1.99(m,1H),0.99-0.92(m,2H),0.69-0.67(m,2H).LCMS m / z=425.09[M+1] +

[0281] Example 50 Synthesis of Compound 50

[0282]

[0283] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(1-methyl-1H-1,2,3-triazol-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine

[0284] N-(2-cyclopropyl-4-fluorophenyl)-N-(1-methyl-1H-1,2,3-triazol-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine was prepared using steps 1-3 of Example 46.

[0285] The expression is: LCMS m / z = 396.11[M+1]+

[0286] Example 51 Synthesis of Compound 51

[0287]

[0288] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(5-methylthiazolyl-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0289] N-(2-cyclopropyl-4-fluorophenyl)-N-(5-methylthiazolyl-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0290] The expression is: LCMS m / z = 412.08[M+1] +

[0291] Example 52 Synthesis of Compound 52

[0292]

[0293] Synthesis of N-(5-chlorothiazol-2-yl)-N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0294] N-(5-chlorothiazol-2-yl)-N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0295] The expression is: LCMS m / z = 432.03[M+1] +

[0296] Example 53 Synthesis of Compound 53

[0297]

[0298] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(4,5-dimethylthiazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0299] N-(2-cyclopropyl-4-fluorophenyl)-N-(4,5-dimethylthiazolyl-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0300] The expression is: LCMS m / z = 426.10[M+1] +

[0301] Example 54 Synthesis of Compound 54

[0302]

[0303] Synthesis of N-(4-chlorothiazol-2-yl)-N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0304] N-(4-chlorothiazol-2-yl)-N-(2-cyclopropyl-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0305] The expression is: LCMS m / z = 432.03[M+1] +

[0306] Example 55 Synthesis of Compound 55

[0307]

[0308] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(2-methylthiazolyl-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0309] N-(2-cyclopropyl-4-fluorophenyl)-N-(2-methylthiazolyl-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0310] The expression is: LCMS m / z = 412.08[M+1] +

[0311] Example 56 Synthesis of Compound 56

[0312]

[0313] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(1,3-dimethyl-1H-pyrazol-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0314] N-(2-cyclopropyl-4-fluorophenyl)-N-(1,3-dimethyl-1H-pyrazol-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0315] The expression is: LCMS m / z = 409.13[M+1] +

[0316] Example 57 Synthesis of Compound 57

[0317]

[0318] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(4,5-dimethyloxazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0319] N-(2-cyclopropyl-4-fluorophenyl)-N-(4,5-dimethyloxazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0320] Characterized as: 1 H NMR (600MHz, DMSO) δ8.65(d,J=8.5Hz,1H),7.45(dd,J=8.7,5.4Hz,1H),7.17(td,J=8.4,2.9Hz,1H),7.11(d,J= 8.5Hz,1H),7.01(dd,J=10.1,2.8Hz,1H),2.20(s,3H),2.04(s,3H),1.78-1.71(m,1H),0.77-0.60(m,4H).LCMS m / z=410.12[M+1] +

[0321] Example 58 Synthesis of Compound 58

[0322]

[0323] Synthesis of N-(4-fluoro-2-methylphenyl)-7-nitro-N-(oxazol-2-yl)benzo[c][1,2,5]oxadiazole-4-amine

[0324] N-(4-fluoro-2-methylphenyl)-7-nitro-N-(oxazol-2-yl)benzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0325] The expression is: LCMS m / z = 356.07[M+1] +

[0326] Example 59 Synthesis of Compound 59

[0327]

[0328] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(4-methylthiazolyl-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine

[0329] N-(2-cyclopropyl-4-fluorophenyl)-N-(4-methylthiazolyl-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 1-3 of Example 46.

[0330] The expression is: LCMS m / z = 412.08[M+1] +

[0331] Example 60: Synthesis of Compound 60

[0332]

[0333] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(5-methyl-1,3,4-oxadiazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine

[0334] N-(2-cyclopropyl-4-fluorophenyl)-N-(5-methyl-1,3,4-oxadiazol-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine was prepared using steps 1-3 of Example 46.

[0335] The expression is: LCMS m / z = 397.10[M+1] +

[0336] Example 61 Synthesis of Compound 61

[0337]

[0338] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)-2-(trifluoromethyl)pyrimidine-5-carboxamide

[0339] N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-2-(trifluoromethyl)pyrimidine-5-carboxamide was prepared using steps 1-3 of Example 1.

[0340] Characterized as: 1 H NMR (400MHz, DMSO) δ9.22(s,1H),8.71(d,J=8.0Hz,1H),7.63(dd,J=11.2,5.7Hz,1H),7.10(td,J=8.4,2.6Hz,1H), 6.85(dd,J=10.1,2.4Hz,1H),2.04-1.93(m,1H),0.91-0.95(m,1H),0.73-0.64(m,1H),0.57(d,J=4.0Hz,1H).LCMS m / z=489.09[M+1]+

[0341] Example 62 Synthesis of Compound 62

[0342]

[0343] Synthesis of 4-amino-N-(2-cyclopropyl-4-fluorophenyl)-2-methyl-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide

[0344] 4-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-2-methyl-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)pyrimidine-5-carboxamide was prepared using steps 1-3 of Example 1.

[0345] Characterized as: 1 H NMR (600MHz, DMSO) δ12.89(s,1H),10.66(s,1H),9.34(s,1H),8.80(dd,J=22.3,8.5Hz,2H),7.36(dd,J=8.5,5.7Hz,1H),7.09(td, J=8.4,2.8Hz,1H),6.85(dd,J=10.3,2.6Hz,1H),2.74(s,3H),2.09-2.01(m,1H),0.93(t,J=6.4Hz,2H),0.71(d,J=4.4Hz,2H).LCMS m / z=450.12[M+1] +

[0346] Example 63 Synthesis of Compound 63

[0347]

[0348] Synthesis of 5-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)pyridazine-4-carboxamide

[0349] 5-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)pyridazine-4-carboxamide was prepared using steps 1-3 of Example 1.

[0350] Characterized as: 1H NMR (400MHz, DMSO) δ10.97(s,1H),8.51(d,J=8.8Hz,1H),7.38(dd,J=8.7,5.6Hz,1H),7.15(td,J=8.4,2.9Hz,1H), 6.92(dd,J=10.4,2.9Hz,1H),6.01(d,J=8.8Hz,1H),1.98-1.89(m,1H),0.91-0.85(m,2H),0.77-0.71(m,2H).LCMS m / z=436.11[M+1] +

[0351] Example 64 Synthesis of Compound 64

[0352]

[0353] Synthesis of 2-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)-4-(trifluoromethyl)pyrimidine-5-carboxamide

[0354] 2-Amino-N-(2-Cyclopropyl-4-fluorophenyl)-N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)-4-(trifluoromethyl)pyrimidine-5-carboxamide was prepared using steps 1-3 of Example 1.

[0355] The expression is: LCMS m / z = 504.10[M+1] +

[0356] Example 65 Synthesis of Compound 65

[0357]

[0358] Synthesis of 6-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)pyrazine-2-carboxamide

[0359] 6-amino-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)pyrazine-2-carboxamide was prepared using steps 1-3 of Example 1.

[0360] The expression is: LCMS m / z = 436.11[M+1] +

[0361] Example 66 Synthesis of Compound 66

[0362]

[0363] Synthesis scheme:

[0364]

[0365] Synthesis of (R)-N-(4-fluoro-2-methylphenyl)-7-nitro-N-(oxetane-2-ylmethyl)benzo[c][1,2,5]oxadiazole-4-amine

[0366] (R)-N-(4-fluoro-2-methylphenyl)-7-nitro-N-(oxetane-2-ylmethyl)benzo[c][1,2,5]oxadiazole-4-amine was prepared using steps 2-3 of Example 46.

[0367] The expression is: LCMS m / z = 359.11[M+1] +

[0368] Example 67 Synthesis of Compound 67

[0369]

[0370] Synthesis of 3-methoxy-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carbamate)benzoic acid

[0371] 3-Methoxy-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carbamate)benzoic acid was prepared using steps 2-3 of Example 1.

[0372] The expression is: LCMS m / z = 455.15[M+1] +

[0373] Example 68 Synthesis of Compound 68

[0374]

[0375] Synthesis of 3-cyclopropyl-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide)benzamide

[0376] 3-Cyclopropyl-4-(2,2,3,3-Tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide)benzamide was prepared using steps 1-3 of Example 1.

[0377] The expression is: LCMS m / z = 464.19[M+1] +

[0378] Example 69: Synthesis of Compound 69

[0379]

[0380] Synthesis of N-(4-fluoro-2-methylphenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide

[0381] N-(4-fluoro-2-methylphenyl)-3,3-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butyramide was prepared using steps 2-3 of Example 1.

[0382] The expression is: LCMS m / z = 387.14[M+1] +

[0383] Example 70 Synthesis of Compound 70

[0384]

[0385] Synthesis of 4-methyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid

[0386] 4-Methyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid was prepared using steps 2-3 of Example 1.

[0387] The expression is: LCMS m / z = 389.15[M+1] +

[0388] Example 71 Synthesis of Compound 71

[0389]

[0390] Synthesis of 3-methyl-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid

[0391] 3-Methyl-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid was prepared using steps 2-3 of Example 1.

[0392] The expression is: LCMS m / z = 389.15[M+1] +

[0393] Example 72 Synthesis of Compound 72

[0394]

[0395] Synthesis of 3-cyclopropyl-4-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid

[0396] 3-Cyclopropyl-4-(2,2,3,3-Tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid was prepared using steps 1-3 of Example 1.

[0397] The expression is: LCMS m / z = 465.17[M+1] +

[0398] Example 73 Synthesis of Compound 73

[0399]

[0400] Synthesis of N-(2-cyclopropyl-5-fluorophenyl)-2,2,3,3-tetramethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide

[0401] N-(2-cyclopropyl-5-fluorophenyl)-2,2,3,3-tetramethyl-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamide was prepared using steps 1-3 of Example 1.

[0402] The expression is: LCMS m / z = 439.17[M+1] +

[0403] Example 74 Synthesis of Compound 74

[0404]

[0405] Synthesis of 2-cyclopropyl-4-fluoro-N-(4-fluoro-2-methylphenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)benzamide

[0406] 2-Cyclopropyl-4-fluoro-N-(4-fluoro-2-methylphenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazole-4-yl)benzamide was prepared using steps 2-3 of Example 1.

[0407] The expression is: LCMS m / z = 451.11[M+1] +

[0408] Example 75 Synthesis of Compound 75

[0409]

[0410] Synthesis of 4-cyclopropyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid

[0411] 4-Cyclopropyl-3-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid was prepared using steps 1-3 of Example 1.

[0412] The expression is: LCMS m / z = 465.17[M+1] +

[0413] Example 76 Synthesis of Compound 76

[0414]

[0415] Synthesis of 2-fluoro-4-methyl-5-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid

[0416] 2-Fluoro-4-methyl-5-(2,2,3,3-tetramethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)cyclopropane-1-carboxamido)benzoic acid was prepared using steps 2-3 of Example 1.

[0417] The expression is: LCMS m / z = 457.14 [M+1] +

[0418] Example 77 Synthesis of Compound 77

[0419]

[0420] Synthesis of 3-(2,4-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamido)-4-methylbenzoic acid

[0421] 3-(2,4-dimethyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzamido)-4-methylbenzoic acid was prepared using steps 2-3 of Example 1.

[0422] The expression is: LCMS m / z = 447.12[M+1] +

[0423] Example 78 Synthesis of Compound 78

[0424]

[0425] Synthesis of methyl (2-cyclopropyl-4-fluorophenyl)(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)carbamate

[0426] Methyl (2-cyclopropyl-4-fluorophenyl)(7-nitrobenzo[c][1,2,5]oxadiazole-4-yl) carbamate was prepared using steps 1-3 of Example 1.

[0427] The expression is: LCMS m / z = 373.09[M+1] +

[0428] Example 79: Synthesis of Compound 79

[0429]

[0430] Synthesis of (2-cyclopropyl-4-fluorophenyl)(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)carbamate

[0431] (2-Cyclopropyl-4-fluorophenyl)(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamate was prepared using steps 1-3 of Example 1.

[0432] The expression is: LCMS m / z = 387.10[M+1] +

[0433] Example 80: Synthesis of Compound 80

[0434]

[0435] Synthesis of 2,5-dichloro-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenz[c][1,2,5]oxadiazol-4-yl)thiazole-4-carboxamide

[0436] 2,5-Dichloro-N-(2-cyclopropyl-4-fluorophenyl)-N-(7-nitrobenzon[c][1,2,5]oxadiazol-4-yl)thiazolyl-4-carboxamide was prepared following steps 1-3 of Example 1. It was characterized as follows:

[0437] 1H NMR (400MHz, DMSO) δ8.66(d,J=8.0Hz,1H),7.40(dd,J=8.7,5.5Hz,1H),7.32(d,J=8.0Hz,1H),7.10(td,J=8.4,2.9Hz,1H),6.92(dd,J=10.2,2 .9Hz,1H),1.89-1.80(m,1H),0.85(tt,J=6.3,4.5Hz,1H),0.78(dt,J=10.6,4.3Hz,1H),0.63-0.55(m,1H),0.51(dq,J=10.8,4.9Hz,1H).LCMS m / z=493.98[M+1] +

[0438] Example 81 Synthesis of Compound 81

[0439]

[0440] Synthesis of N-(2-cyclopropyl-4-fluorophenyl)-2-hydroxy-6-methyl-N-(7-nitrobenzyl[c][1,2,5]oxadiazol-4-yl)isonicotinamide

[0441] N-(2-cyclopropyl-4-fluorophenyl)-2-hydroxy-6-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)isonicotinamide was prepared using steps 1-3 of Example 1.

[0442] Characterized as: 1 H NMR (400MHz, DMSO) δ8.66(d,J=8.0Hz,1H),7.51(dd,J=8.5,5.5Hz,1H),7.36(d,J=8.0Hz,1H),7.08(td,J=8.3,2.5Hz,1H),6.87(dd,J=1 0.1,2.3Hz,1H),6.27(s,1H),6.14(s,1H),2.12(s,3H),1.86-1.82(m,1H),0.96-0.93(m,1H),0.81-0.73(m,1H),0.56-0.54(m,2H).LCMS m / z=450.11[M+1] +

[0443] Example 82

[0444] This invention discloses the inhibitory activity of the compound on human Burkitt's lymphoma cells (Daudi cells) and their proliferation.

[0445] Daudi cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640 basic (C11875500BT) complete medium containing 10% fetal bovine serum. On the first day of the experiment, Daudi cells were seeded at a density of 10,000 cells / well in RPMI 1640 medium containing 10% fetal bovine serum in 96-well plates, with 100 μL of cell suspension per well. 300 μL of PBS was added to each well, and the plates were incubated overnight at 37°C with 5% CO2. On the second day, 100 μL of different concentrations of the test compound prepared in medium was added to each well. The final concentrations of the compound were set at seven points: 25 μM, 10 μM, 7.5 μM, 5 μM, 3.75 μM, 2.5 μM, and 1 μM (adjusted as needed according to experimental objectives). A blank control was also included. The plates were incubated at 37°C with 5% CO2 for 24 hours. On the third day, the 96-well plate was removed, and all culture medium from the experimental and blank control wells was discarded. CCK-8 was prepared at a ratio of 10:1 (RPMI 1640: Cell Counting Kit-8, CK04), and 100 μL was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, the luminescence signal values ​​were read using a multi-label microplate reader (BioTek, Synergy H1). The IC50 of the compound's inhibitory activity was calculated using Graphpad Prism software based on the compound concentration and luminescence signal values. 50 value.

[0446] This invention discloses the inhibitory effect of compounds on the proliferation of Daudi cells (IC50). 50 The values ​​are shown in Table 1 below.

[0447] Table 1 shows the inhibitory IC50 values ​​of the compounds disclosed in this invention on Daudi cell proliferation. 50 Value (μM)

[0448]

[0449]

[0450] The above results indicate that the compound provided by this invention has a good inhibitory effect on the proliferation of Daudi cells.

[0451] Example 83

[0452] The present invention discloses the inhibitory activity of the compound on the proliferation of human colorectal cancer cells (A2780 cells), as shown in Table 2 below.

[0453] Following the experimental steps of Example 82, the inhibitory effect of the compound disclosed in this invention on the proliferation of A2780 cells was determined. The A2780 cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640 basic (C11875500BT) complete medium containing 10% fetal bovine serum.

[0454] This invention discloses the inhibitory effect of the compound on the proliferation of A2780 cells (IC50). 50 The values ​​are shown in Table 2 below.

[0455] Table 2 shows the inhibitory IC50 values ​​of the compounds disclosed in this invention on the proliferation of A2780 cells. 50 Value (μM)

[0456]

[0457]

[0458] The above results indicate that the compound provided by this invention has a good inhibitory effect on the proliferation of A2780 cells.

[0459] Example 84

[0460] The present invention discloses the inhibitory activity of the compound on the proliferation of human colorectal cancer cells (COLO320DM cells), as shown in Table 3 below.

[0461] Following the experimental procedure of Example 82, the effect of the compound disclosed in this invention on...

[0462] The inhibitory effect of COLO320DM cell proliferation was investigated, in which COLO320DM cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640basic (C11875500BT) complete medium containing 10% fetal bovine serum.

[0463] This invention discloses the inhibitory effect of compounds on the proliferation of COLO320DM cells (ICP-C). 50 The values ​​are shown in Table 3 below. A = compound IC 50 Less than or equal to 0.1 μM, B = compound IC 50 For compounds with a molecular weight greater than 0.1 μM and less than or equal to 1 μM, C = IC. 50 For compounds with a molecular weight greater than 1 μM and less than or equal to 10 μM, D = IC of the compound. 50 Greater than 10 μM.

[0464] Table 3 shows the inhibitory IC50 values ​​of the compounds disclosed in this invention on the proliferation of COLO320DM cells. 50 Value (μM)

[0465]

[0466] The above results indicate that the compound provided by this invention has a good inhibitory effect on the proliferation of COLO320DM cells.

[0467] Example 85

[0468] The present invention discloses the inhibitory activity of the compound on the proliferation of human osteosarcoma cells (SJSA-1 cells), as shown in Table 4 below.

[0469] Following the experimental steps of Example 82, the inhibitory effect of the compound disclosed in this invention on the proliferation of SJSA-1 cells was determined. The SJSA-1 cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640 basic (C11875500BT) complete medium containing 10% fetal bovine serum.

[0470] This invention discloses the inhibitory effect of compounds on the proliferation of SJSA-1 cells (IC50). 50 The values ​​are shown in Table 4 below.

[0471] Table 4 shows the inhibitory IC50 values ​​of the compounds disclosed in this invention on the proliferation of SJSA-1 cells. 50 value

[0472]

[0473] The above results indicate that the compound provided by this invention has a good inhibitory effect on the proliferation of SJSA-1 cells.

[0474] Example 86

[0475] The present invention discloses the inhibitory activity of the compound on the proliferation of human myeloid monocytic leukemia cells (MV4-11 cells), as shown in Table 5 below.

[0476] Following the experimental steps of Example 82, the inhibitory effect of the compound disclosed in this invention on the proliferation of MV4-11 cells was determined. The MV4-11 cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640 basic (C11875500BT) complete medium containing 10% fetal bovine serum.

[0477] This invention discloses the inhibitory effect of compounds on the proliferation of MV4-11 cells (IC50). 50 The values ​​are shown in Table 5 below.

[0478] Table 5 shows the inhibitory IC50 values ​​of the compounds disclosed in this invention on the proliferation of MV4-11 cells. 50 value

[0479]

[0480] The above results indicate that the compound provided by this invention has a good inhibitory effect on the proliferation of MV4-11 cells.

[0481] Example 87

[0482] This invention discloses the inhibitory activity of the compound on MYC expression in Daudi cells.

[0483] 1. Cell culture:

[0484] Daudi cells frozen in liquid nitrogen were thawed and cultured to the logarithmic growth phase. On the first day of the experiment, Daudi cells were seeded in 6-well plates at a density of 2×10^5 cells / well using RPMI 1640 medium containing 10% fetal bovine serum, with 1 mL of cell suspension per well. The plates were then incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0485] 2. Drug treatment

[0486] Dose-effect assay: Weigh an appropriate amount of the compound and dissolve it in DMSO solvent, vortex to mix, and prepare a 12 mM stock solution. Add the solution to treat Daudi cells. The solvent control group was treated with only DMSO, while the drug treatment groups were treated with the corresponding compound. The final concentrations were set at 12 μM, 6 μM, 3 μM, 1.5 μM and 0.75 μM. Cells were collected after 12 h.

[0487] Time of treatment: Weigh an appropriate amount of the compound and dissolve it in DMSO solvent, vortex mix, and prepare a 7.5 mM stock solution. Add the solution to treat Daudi cells. The solvent control group is treated with only DMSO, while the drug treatment group is treated with the corresponding compound. The final concentration of the treatment is set to 7.5 μM (the activity screening inhibition rate reaches more than 90%). Cells are collected after 0 h, 3 h, 6 h, 9 h, 12 h and 24 h.

[0488] 3. Collect cells

[0489] Cells treated with the compound and cells from the solvent control group were digested with 0.25% trypsin and then centrifuged again in 1 mL of RPMI 1640 complete medium at 3000 rpm for 5 min at 4 °C. PBS was completely discarded after centrifugation.

[0490] 4. Lysed cells collect proteins

[0491] Add an appropriate amount of lysis buffer to the collected cells. Add a mixture of protease inhibitors (P6730, 100×) and a mixture of protein phosphatase inhibitors (P1260, 100×) to the lysis buffer. Pipette the cells 20 times with a 1 mL pipette and lyse them on ice for 30 min (shaking once every 5-10 min). Centrifuge at 12000 rpm for 30 min and collect the supernatant.

[0492] 5. BCA method for determining protein concentration

[0493] Prepare standard proteins of varying concentrations. Add 10 μL of each standard to a 96-well plate, making two replicates for each standard. Dissolve 2 μL of protein lysis buffer in 8 μL of lysis buffer, making two replicates for each sample. Add 100 μL of BCA working solution (A:B = 50:1) to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm. Calculate the protein concentration for each sample and the required volume of protein lysis buffer to produce 10 μg of protein.

[0494] 6. Western blot analysis of protein expression levels

[0495] Take the protein lysis buffer, add a constant volume, and then add an appropriate amount of 5× protein loading buffer. Denature at 98℃ for 10 min. Electrophoresis with a 10% precast gel at 80V for 30 min, then adjust to 120V. After the sample reaches the appropriate position, cut off the stacking gel and transfer the protein on the separating gel to a PVDF membrane using a wet transfer method. Incubate at 200mA for 2 h. Block the PVDF membrane in 5% skim milk at room temperature for 1 h. Cut the PVDF membrane according to the molecular weight of the protein marker and place them in MYC and GAPDH antibodies, respectively, and incubate overnight at 4℃. The next day, place the PVDF membrane in TBST and wash three times with shaking for 10 min each time. Place the PVDF membrane in the secondary antibody solutions corresponding to MYC and GAPDH antibodies (goat anti-rabbit and goat anti-mouse), and incubate with shaking at room temperature for 2 h. After washing three times with shaking in TBST, add ECL luminescence solution and perform luminescence imaging in a Tanon 5200 fully automated chemiluminescence imaging system.

[0496] The experimental results are shown in Figure 1 Tables 6 and 7.

[0497] Table 6. Dose-dependent degradation rates of the compounds disclosed in this invention on MYC expression in Daudi cells.

[0498] Table 7. Time-dependent degradation rate of the compounds disclosed in this invention on MYC expression in Daudi cells.

[0499]

[0500] This indicates that the compound provided by the present invention has good degradation activity against Daudi cell MYC protein.

[0501] Example 88: In vivo antitumor inhibitory activity of compound under sustained treatment

[0502] 1. Tumor Model Construction

[0503] Daudi cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI Medium 1640 basic (C11875500BT) complete medium containing 10% fetal bovine serum. After cells reached 80-90% confluence and were passaged to a sufficient number, they were subcutaneously inoculated into BALB / c nud mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) at a rate of 1×10^6 per mouse.

[0504] 2. Laboratory animals

[0505] BALB / C nud mice, 5-6 weeks old and 18-20g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in an independent small animal housing (IVC) system at a temperature of 20-25℃ and humidity of 40%-70%, with 12 hours of light / dark daily. Four mice were per cage, with free access to food and water. Feed was sterilized by UV irradiation, and water was autoclaved. After three days of rearing, mice were divided into groups for experiments, and Daudi cells were directly inoculated subcutaneously into the right axilla of the nud mice.

[0506] 3. Grouping and Dosing Regimen Design

[0507] When the average tumor volume reaches 200 mm 3 Mice were randomly assigned to three groups of five mice each, based on tumor volume and body weight. The groups were labeled and divided into three subgroups: the vehicle group, the compound 8 group (10 mg / kg), and the compound 8 group (15 mg / kg). The solvent was 10% DMSO + 55% PEG400 + 35% (20% β-cyclodextrin-Saline). Administration was intraperitoneal. Tumor size was recorded using calipers.

[0508] 4. Statistical Analysis

[0509] Tumor volume was calculated using the formula: length × width × width / 2. Analysis was performed using GraphPad Prism 8.0 software, with significance set at P < 0.05. All quantitative data are expressed as mean ± SEM. Comparisons between two unpaired groups were assessed using either the parametric Student's t-test or the nonparametric Mann-Whitney U test. If the data were normally distributed, one-way ANOVA was used to test for differences between groups. If the data were not normally distributed, the Kruskal-Wallis test was used to test for differences between groups. The Shapiro-Wilk test was used to test for normality of the data. The Levene test was used to test for homogeneity of variance. Unless otherwise stated, all experiments used at least three individual mice or samples.

[0510] Results of compound 8's inhibition of tumor growth over 30 days are shown in [the table]. Figure 2This indicates that compound 8 of the present invention has good in vivo antitumor activity.

[0511] Example 89: In vivo antitumor inhibitory activity of compound 28 after short-term treatment followed by discontinuation of treatment.

[0512] Following the experimental procedure of Example 88, the in vivo antitumor inhibitory activity of the compound disclosed in this invention after short-term treatment followed by discontinuation was determined. When the average tumor volume of nude mice subcutaneously inoculated with Daudi cells reached 200 mm², the activity was further assessed. 3 Mice were randomly assigned to two groups (n=4 per group) based on tumor volume and body weight, and the groups were labeled: a vehicle group and a compound 28 group (15 mg / kg). The vehicle group consisted of 10% DMSO + 55% PEG400 + 35% (20% β-cyclodextrin-Saline). Intraperitoneal administration was administered until the control tumor reached 1500 mm. 3 Discontinue medication. Record tumor size using calipers.

[0513] Results of compound 28's inhibition of tumor growth over 50 days are shown in [the table]. Figure 3 This indicates that compound 28 of the present invention not only has good in vivo antitumor activity during treatment, but also has a long-term tumor-suppressive effect after early treatment is discontinued.

[0514] Example 90: In vivo antitumor inhibitory activity of compound 28 in combination with doxorubicin

[0515] Following the experimental procedure of Example 88, the in vivo antitumor inhibitory activity of compound 30 disclosed in this invention in combination with doxorubicin was determined. When the average tumor volume of nude mice subcutaneously inoculated with Daudi cells reached 200 mm², the tumors were effectively controlled. 3 Mice were randomly assigned to four groups (n=4 per group) based on tumor volume and body weight. The groups were labeled and divided into four groups: a vehicle group, a doxorubicin group (4 mg / kg), a compound 28 group (15 mg / kg), and a combination group of doxorubicin (4 mg / kg) and compound 28 (15 mg / kg). The solvent consisted of 10% DMSO + 55% PEG400 + 35% (20% β-cyclodextrin-Saline). Tumor size was recorded using calipers.

[0516] Results of the inhibition of tumor growth by compound 28 and doxorubicin, a first-line drug for lymphoma, over 30 days are shown in [the table]. Figure 4 This indicates that compound 28 of the present invention significantly reduces tumor growth in mice and has superior antitumor activity when combined with doxorubicin.

[0517] In summary, this invention provides a compound that can target and degrade MYC. Once in the patient's body, this compound can target and degrade MYC, thereby effectively inhibiting the proliferation of tumor cells such as lymphoma and osteosarcoma, as well as cancer cells such as colorectal cancer, exhibiting good anti-tumor and anti-cancer effects. This compound can also inhibit the proliferation of myeloid monocytic leukemia cells in the patient's body, providing a new treatment option for myeloid monocytic leukemia. Furthermore, this compound exhibits a synergistic effect when used in combination with doxorubicin, demonstrating superior anti-tumor activity and showing broad application prospects in the medical field.

[0518] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0519] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A compound capable of targeted degradation of MYC, characterized in that, The structure of the compound is as follows: Among them, R 1 Selected from acetyl, o-fluorobenzoyl, m-fluorobenzoyl, p-fluorobenzoyl, 2,6 Difluorobenzoyl, cyclohexaneformyl, cyclopentaneformyl, 2 (piperidine) 1 2-Acetyl group (4) Methylpiperazine 1 Acetyl, cyclopropaneformyl, cyclobutaneformyl, 2,2,3,3 Tetramethylcyclopropane 1 Formyl, 2 Morpholinylacetyl, 3 Methylbutyryl, 3,3 Dimethylbutyryl, 3,5 Difluorobenzoyl, p-carboxybenzoyl, 3 hydroxyl 3 Methylbutyryl, thiazole 2 Formyl, 3 methyl 2 ((methyl formate)amino)butyryl, 2 amino Pyrimidine 5 Formyl, 3 amino 3 Methylbutyryl, 3,4 Dimethoxybenzoyl, 3,4,5 Trimethoxyphenylpropionyl, 4 amino 5 (Ethylsulfonyl) benzoyl, 4 hydroxyl 2,6 dimethylbenzoyl, 4 Bromothiazide 2 Formyl, 4,5 dimethylthiazole 2 Formyl, 5 Methylthiazole 2 Formyl, 2 Aminopyrimidine 5 Formyl, 4 hydroxyl 2 Methylbenzoyl, 4 amino 2 Methylbenzoyl, 4 amino 2 Methoxybenzoyl, 3 methyl 1H pyrazole 4 Formyl, 3,5 dimethyl 1H pyrazole 4 Formyl, 4 amino 4 methyl valeryl, 4 amino 3,5 dimethylbenzoyl, 2 aminoacetyl, 4 amino 5 chlorine 2 Methoxyphenylpropionyl, 5 chlorine 2 methoxy 4 (Methamido)benzoyl, 4 amino 2,3,5,6 Tetrafluorobenzoyl, oxazole 4 Formyl, 3,5 Diamino 6 Chlorpyrazine 2 Formyl group, 1H imidazole 2 formyl, pyrimidine 2 basalt, thiazole 2 base, 1H imidazole 2 2,4(1H,3H) Dicarbonylpyrimidine 6 Base, 1 methyl 1H 1,2,3 Triazole 4 Base, 5 Chlorthiazide 2 Base, 4,5 dimethylthiazole 2 Base, 4 Chlorthiazide 2 Base, 2 Methylthiazole 4 Base, 1,3 dimethyl 1H pyrazole 4 Base, 4,5 dimethyloxazole 2 basalt, oxazole 2 Base, 4 Methylthiazole 2 Base, 5 methyl 1,3,4 Oxadiazole 2 Base, 2 (trifluoromethyl)pyrimidine 5 Formyl, 4 amino 2 Methylpyrimidine 5 Formyl, 5 amino pyridazine 4 Formyl, 2 amino 4 (trifluoromethyl)pyrimidine 5 Formyl, 6 amino Pyrazine 2 Formyl group, 2,2,3,3 Tetramethylcyclopropane 1 Formyl, 3,3 Dimethylbutyryl, 2 Cyclopropyl 4 fluorobenzene 1 Formyl, 2 (methoxycarbonyl)aminobutyryl, 2,4 Dimethylbenzoyl, methyl formate 1 ethyl formate 1 Base, 2,5 dichloro Thiazole 4 Formyl, 6 hydroxyl 2 methyl Nicotinyl, 2 hydroxyl 6 Methylisonicotinyl; R 2 Selected from 2 Cyclopropyl 4 Fluorophenyl, 2 Cyclopentyl 4 Fluorophenyl, 2 Cyclohexyl 4 Fluorophenyl, 2 methyl 4 Fluorophenyl, 2 methoxy 4-Carboxyphenyl, 2 methyl 5 Carboxyphenyl, 2 Cyclohexyl 4 Formic acid phenyl, 2 Cyclohexyl 5-Carboxyphenyl, 2 methyl 4 fluorine 5 Carboxyphenyl; Or the compound is:

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients, which are excipients or carriers.

4. The pharmaceutical composition according to claim 3, characterized in that, The carrier is any one or more of a filler, wetting agent, adhesive, disintegrant, or lubricant.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is formulated in one or more of the following forms: aqueous extract, powder, lotion, tincture, oil, emulsion, ointment, plaster, or aerosol.

6. The use of the compound that can target and degrade MYC as described in claim 1 in the preparation of antitumor drugs.

7. The use of the compound that can target and degrade MYC as described in claim 1 in the preparation of anticancer drugs.

8. The use of the compound that can target and degrade MYC as described in claim 1 in the preparation of a medicament for treating leukemia.

9. The use of the compound that can target and degrade MYC as described in claim 1 in the preparation of a drug for antitumor treatment in combination with doxorubicin.

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