A compound containing a fused ring, its use and a composition containing the same

Abstract

The application discloses a kind of compounds containing fused ring, its application and the composition containing it.The application provides a kind of compound containing fused ring as shown in formula I or its pharmaceutically acceptable salt.The structure of such compound is novel, has preferable STAT5 inhibitory activity.

Description

Technical Field

[0001] This invention provides a compound containing a fused ring, its application, and compositions containing it. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by inflammation of the synovial membrane of the joints. Its incidence is approximately 1% in the adult population. It primarily manifests as symmetrical, polyarticular, recurrent arthritis, with the small joints of the hands and feet most commonly affected. Early or acute onset often presents with redness, swelling, heat, pain, and limited movement of the joints; late stages can lead to joint destruction, stiffness, deformity, and loss of function, along with osteoporosis and skeletal muscle atrophy. Throughout the course of the disease, it may be accompanied by fever, anemia, weight loss, vasculitis, and subcutaneous nodules, and can also affect multiple organs throughout the body. It is a highly disabling disease and significantly increases patient mortality. Modern medicine does not fully understand the pathogenesis of rheumatoid arthritis, and there is currently no complete cure, imposing a heavy health and financial burden on patients worldwide.

[0003] Over the past two decades, our understanding of the pathogenesis of arthritis has deepened. It is believed that the overactivation of immune cells and the increase of inflammatory cytokines lead to symptoms in joints and other parts of the body. Significant progress has been made in its treatment, and a series of drugs to combat rheumatoid factors have been developed and produced, including methotrexate, anti-tumor necrosis factor (TNFα) antibodies, interleukin (IL)1, interleukin-6 antibodies, and JAK kinase inhibitors.

[0004] These antirheumatic drugs can control disease progression and improve patients' quality of life to some extent. However, their sustained effects are not ideal, and they are only effective for a small number of patients, accompanied by side effects of varying severity. For example, methotrexate, as an anticancer drug, has significant non-specific cytotoxicity and causes great damage to the gastrointestinal tract, liver, and kidneys. Due to insufficient efficacy or strong adverse reactions, about half of the patients will discontinue the drug within one year of starting treatment. Anti-TNFα antibodies are only effective for 30-40% of patients, and are also accompanied by random infections and cancer risks. There are reports that they increase the possibility of developing multiple sclerosis, and long-term use produces neutralizing antibodies in the body, leading to loss of efficacy [Ann Rheum Dis. 2014 Mar; 73(3):529-35]. We urgently need to find new disease pathogenesis mechanisms and develop more targeted and better products.

[0005] The pathogenesis of rheumatoid arthritis involves a severely dysregulated innate and adaptive immune response, leading to chronic inflammation. Helper T cells play a crucial role in the initiation and progression of arthritis. When their function is impaired, persistent activation and abnormal proliferation of T lymphocytes, along with the corresponding proliferation of fibroblasts in joint tissues, contribute to the superficial symptoms of arthritis. The JAK-STAT pathway plays an important role in regulating T cells and other immune cells. Among them, STAT5 plays a key role in the development and maturation of various T cell subtypes.

[0006] Recently, our team discovered a novel helper T cell, named Th-GM. This cell plays a driving role in many inflammatory diseases, representing a new mechanism in the pathogenesis of inflammation. Th-GM cells are characterized by the STAT5 signaling pathway activated by interleukin-7, leading to high expression of downstream colony-stimulating factor (GM-CSF) and interleukin-3. Furthermore, RNA sequencing experiments revealed that Th-GM cells exhibit a unique gene expression profile, distinct from the established Th-1 and Th-17 helper T cell types. Genetic experiments also demonstrated that knocking out the STAT5 gene significantly reduced neurological inflammation and arthritis severity in mice with multiple sclerosis (references CN107002037A, PCT / US2018 / 031217). Given the crucial regulatory role of STAT5 in Th-GM cells, and considering its potential role in other inflammatory cells, we believe that STAT5 inhibitors are promising candidates for treating arthritis.

[0007] In this patent, we elucidate the design and synthesis of a series of novel STAT5 inhibitors. Summary of the Invention

[0008] The technical problem to be solved by the present invention is the deficiency of the relatively simple structure of existing STAT5 inhibitors. To this end, the present invention provides a compound containing a fused ring, its application and a composition containing the compound. This type of compound has a novel structure and better STAT5 inhibitory activity.

[0009] The present invention provides a compound containing a fused ring as shown in Formula I, or a pharmaceutically acceptable salt thereof;

[0010]

[0011] Among them, R 1 For hydrogen or

[0012] R 2 It is hydrogen or chlorine;

[0013] R 3It is a C1-C3 alkyl group in which hydrogen is replaced by one or more halogens;

[0014] R 4 for

[0015] n is 0, 1, 2, 3 or 4;

[0016] R 4-1 It is independently a cyano, a halogen, a C1-C3 alkyl group, or a C1-C3 alkyl group substituted with one or more halogens;

[0017] R 4-2 and R 4-3 It is independently a C1 to C3 alkyl group;

[0018] m can be 0, 1, 2, 3 or 4 (m refers to the number of substituents on the benzene ring);

[0019] R 4-4 It is independently a cyano, a halogen, a C1-C3 alkyl group, or a C1-C3 alkyl group substituted with one or more halogens;

[0020] Ring A is a 5- or 6-membered heteroaryl group with 1, 2, or 3 heteroatoms selected from one or more of N, O, and S;

[0021] t is 0, 1, 2, 3 or 4 (t refers to the number of substituents on ring A);

[0022] R 4-5 It is independently a cyano, hydroxyl, halogen, C1-C3 alkyl, C1-C3 alkoxy or a C1-C3 alkyl substituted with one or more halogens;

[0023] However, the compound containing a fused ring as shown in Formula I is not one of the following compounds:

[0024]

[0025] In one embodiment, certain groups in the compound with a fused ring as shown in Formula I or a pharmaceutically acceptable salt thereof may be defined as follows, and groups not covered in this embodiment may be defined as described in any of the embodiments above (hereinafter referred to as "in one embodiment"): R 2 It is hydrogen.

[0026] In one particular scheme, R 1 for

[0027] In one particular scheme, R 3 It is a C1 to C3 alkyl group that has been substituted with one or more halogens.

[0028] In one particular scheme, n is 0.

[0029] In one particular scheme, R 4 for

[0030] In a certain scheme, m is either 0 or 1.

[0031] In one particular scheme, R 4-4 It is independently a halogen or a C1-C3 alkyl group.

[0032] In a certain scheme, t is 0 or 1.

[0033] In one particular scheme, R 4-5 It is independently a cyano, halogen, C1-C3 alkyl, C1-C3 alkoxy, or a C1-C3 alkyl substituted with one or more halogens.

[0034] In one particular scheme, R 4-5 It is a halogen on its own.

[0035] In one scheme, when the R 1 When the hydrogen is present, m+t is greater than or equal to 1.

[0036] In one scheme, when the R 1 When the hydrogen is present, m+t=1.

[0037] In one particular scheme, R 1 For hydrogen or

[0038] R 2 It is hydrogen;

[0039] R 3 It is a C1-C3 alkyl group substituted with one or more halogens;

[0040] R 4 for

[0041] n is 0;

[0042] R 4-2 and R 4-3 It is independently a C1 to C3 alkyl group;

[0043] m is 0 or 1;

[0044] R 4-4 It is a halogen or a C1-C3 alkyl group;

[0045] Ring A is a 5- or 6-membered heteroaryl group with 1, 2, or 3 heteroatoms selected from one or more of N, O, and S;

[0046] t is 0 or 1;

[0047] R 4-5 It is a cyano, halogen, C1-C3 alkyl, C1-C3 alkoxy, or a C1-C3 alkyl substituted with one or more halogens;

[0048] However, the compound containing a fused ring as shown in Formula I is not one of the following compounds:

[0049]

[0050] In one particular scheme, R 1 For hydrogen or

[0051] R 2 It is hydrogen;

[0052] R 3 It is a C1-C3 alkyl group substituted with one or more halogens;

[0053] R 4 for

[0054] m is 0 or 1;

[0055] R 4-4 It is a halogen or a C1-C3 alkyl group;

[0056] t is 0 or 1;

[0057] R 4-5 It is a halogen;

[0058] However, the compound containing fused rings as shown in Formula I is not...

[0059] In one particular scheme, R 1 for

[0060] R 2 It is hydrogen;

[0061] R 3 It is a C1-C3 alkyl group substituted with one or more halogens;

[0062] R 4 for

[0063] n is 0;

[0064] R 4-2 and R 4-3 It is independently a C1 to C3 alkyl group;

[0065] m is 0 or 1;

[0066] R 4-4It is a halogen or a C1-C3 alkyl group;

[0067] Ring A is a 5- or 6-membered heteroaryl group with 1, 2, or 3 heteroatoms selected from one or more of N, O, and S;

[0068] t is 0 or 1;

[0069] R 4-5 It is a cyano, halogen, C1-C3 alkyl, C1-C3 alkoxy, or a C1-C3 alkyl substituted with one or more halogens;

[0070] However, the compound containing a fused ring as shown in Formula I is not one of the following compounds:

[0071]

[0072] In one particular scheme, R 1 for

[0073] R 2 It is hydrogen;

[0074] R 3 It is a C1-C3 alkyl group substituted with one or more halogens;

[0075] R 4 for

[0076] m is 0 or 1;

[0077] R 4-4 It is a halogen or a C1-C3 alkyl group;

[0078] t is 0 or 1;

[0079] R 4-5 It is a halogen;

[0080] However, the compound containing fused rings as shown in Formula I is not...

[0081] In one scheme, when the R 3 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the plurality of alkyl groups may be two or three.

[0082] In one scheme, when the R 3 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the halogen can be fluorine or chlorine, or it can be fluorine.

[0083] In one scheme, when the R 3When the C1-C3 alkyl group is substituted with one or more halogens, the C1-C3 alkyl group may be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0084] In one scheme, when the R 3 When the alkyl group is a C1-C3 alkyl group substituted with multiple halogens, the "C1-C3 alkyl group substituted with multiple halogens" may be trifluoromethyl.

[0085] In one scheme, when the R 4-2 When the alkyl group is C1 to C3, the C1 to C3 alkyl group can be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0086] In one scheme, when the R 4-3 When the alkyl group is C1 to C3, the C1 to C3 alkyl group can be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0087] In one scheme, when the R 4 for At that time, the aforementioned Can be

[0088] In one scheme, when the R 4-1 When the halogen is halogen, the halogen can be fluorine or chlorine, or it can be chlorine.

[0089] In one scheme, when the R 4-1 When the alkyl group is C1 to C3, the C1 to C3 alkyl group can be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0090] In one scheme, when the R 4-1 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the plurality of alkyl groups may be two or three.

[0091] In one scheme, when the R 4-1 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the halogen can be fluorine or chlorine, or it can be fluorine.

[0092] In one scheme, when the R 4-1 When the C1-C3 alkyl group is substituted with one or more halogens, the C1-C3 alkyl group may be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0093] In one scheme, when the R 4-1 When the alkyl group is a C1-C3 alkyl group substituted with multiple halogens, the "C1-C3 alkyl group substituted with multiple halogens" may be trifluoromethyl.

[0094] In one scheme, when the R 4-4 When the halogen is halogen, the halogen can be fluorine or chlorine, or it can be chlorine.

[0095] In one scheme, when the R 4-4 When the alkyl group is C1 to C3, the C1 to C3 alkyl group can be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0096] In one scheme, when the R 4-4 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the plurality of alkyl groups may be two or three.

[0097] In one scheme, when the R 4-4 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the halogen can be fluorine or chlorine, or it can be fluorine.

[0098] In one scheme, when the R 4-4 When the C1-C3 alkyl group is substituted with one or more halogens, the C1-C3 alkyl group may be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0099] In one scheme, when the R 4-4 When the alkyl group is a C1-C3 alkyl group substituted with multiple halogens, the "C1-C3 alkyl group substituted with multiple halogens" may be trifluoromethyl.

[0100] In one embodiment, when the ring A is a 5- or 6-membered heteroaryl group having one, two, or three heteroatoms selected from one or more of N, O, and S, the 5- or 6-membered heteroaryl group having one, two, or three heteroatoms selected from one or more of N, O, and S can be a thiophene ring, a pyrrole ring, a pyridine ring, or a pyrazine ring.

[0101] In one scheme, when the R 4 for At that time, the aforementioned Can be

[0102] In one scheme, when the R 4-5 When the halogen is halogen, the halogen can be fluorine or chlorine, or it can be chlorine.

[0103] In one scheme, when the R 4-5 When the alkyl group is C1 to C3, the C1 to C3 alkyl group can be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0104] In one scheme, when the R4-5 When the alkoxy group is C1 to C3, the C1 to C3 alkoxy group can be methoxy, ethoxy, n-propoxy, or isopropoxy, or it can be methoxy.

[0105] In one scheme, when the R 4-5 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the plurality of alkyl groups may be two or three.

[0106] In one scheme, when the R 4-5 When the alkyl group is a C1-C3 alkyl group substituted with one or more halogens, the halogen can be fluorine or chlorine, or it can be fluorine.

[0107] In one scheme, when the R 4-5 When the C1-C3 alkyl group is substituted with one or more halogens, the C1-C3 alkyl group may be methyl, ethyl, n-propyl or isopropyl, or methyl.

[0108] In one scheme, when the R 4-5 When the alkyl group is a C1-C3 alkyl group substituted with multiple halogens, the "C1-C3 alkyl group substituted with multiple halogens" may be trifluoromethyl.

[0109] In one scheme, when the R 1 for At that time, the pharmaceutically acceptable salt may be a sodium salt, that is, the salt that is pharmaceutically acceptable may be a sodium salt. One or two hydrogen atoms are replaced with sodium ions.

[0110] In one embodiment, the compound containing a fused ring as shown in Formula I is any of the following compounds:

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] In one embodiment, the pharmaceutically acceptable salt of the compound with a fused ring as shown in Formula I is the following compound:

[0118]

[0119]

[0120] The present invention also provides a pharmaceutical composition comprising substance X and pharmaceutical excipients;

[0121] The substance X is the compound with a fused ring as shown in Formula I above, or a pharmaceutically acceptable salt thereof.

[0122] The present invention also provides the application of substance X in the preparation of STAT5 inhibitor or TNF-α generation inhibitor, wherein the STAT5 inhibitor is used in vitro and the TNF-α generation inhibitor is used in vitro;

[0123] The substance X is the compound with a fused ring as shown in Formula I above, or a pharmaceutically acceptable salt thereof.

[0124] This invention also provides the application of substance X in the preparation of pharmaceuticals;

[0125] The substance X is the compound with a fused ring as shown in Formula I above, or a pharmaceutically acceptable salt thereof;

[0126] The drug mentioned is:

[0127] (1) Medications used to treat and / or prevent diseases related to STAT5;

[0128] or,

[0129] (2) Medications used to treat and / or prevent any of the following conditions: arthritis, atherosclerosis or psoriasis.

[0130] In the described applications, the diseases associated with STAT5 may be arthritis, atherosclerosis, or psoriasis.

[0131] In the described application, the arthritis may be rheumatoid arthritis, adjuvant arthritis, or collagen-induced arthritis.

[0132] In the aforementioned application, the psoriasis may be imiquimod-induced psoriasis.

[0133] Unless otherwise specified, the terms used in this invention have the following meanings:

[0134] The term "multiple" refers to 2, 3, 4, or 5.

[0135] The term "pharmaceutical acceptable" means that salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for patient use.

[0136] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, etc. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentian acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0137] When any variable (e.g., R) 4-1 When a variable appears multiple times in the definition of a compound, the definition at each position is independent of the definitions at the other positions; their meanings are independent and do not affect each other. Therefore, if a group is surrounded by one, two, or three R... 4-1 Group substitution, meaning that the group can be replaced by up to 3 R groups. 4-1 Replace, the position R 4-1 Definition and other positions R 4-1The definitions are independent of each other. Furthermore, combinations of substituents and / or variables are only permitted if the combination produces a stable compound.

[0138] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0139] The term "alkyl" refers to a straight-chain or branched alkyl group having a specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, etc.

[0140] The term "alkoxy group" refers to the group -OR X , where R X It is an alkyl group as defined above.

[0141] The term "heteroaryl" refers to an aromatic group containing heteroatoms, preferably containing one, two or three aromatic 5-6 membered monocyclic rings independently selected from nitrogen, oxygen and sulfur, such as furanyl, pyridinyl, pyridinyl, pyrazinyl, thiophene, etc.

[0142] The term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. It includes all substances contained in pharmaceutical preparations, excluding the active ingredient. See also the Pharmacopoeia of the People's Republic of China (2015 Edition), Volume IV, or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0143] The term “treatment” refers to a therapeutic approach. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0144] The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0145] The term "STAT5 inhibitor" refers to substances that can ultimately specifically inhibit the activity of STAT5 in the body environment.

[0146] The term "patient" refers to any animal, preferably a mammal, that is about to receive or has already received administration of the compound according to embodiments of the invention, with humans being the most preferred. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0147] The term "therapeutic effective amount" refers to an amount of compound sufficient to effectively treat the disease or condition described herein when administered to a patient. The "therapeutic effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but may be adjusted as needed by those skilled in the art.

[0148] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0149] The reagents and raw materials used in this invention are all commercially available.

[0150] The positive and progressive effects of this invention are that the compounds have a novel structure and exhibit better STAT5 inhibitory activity. Attached Figure Description

[0151] Figure 1 These are representative images of arterial plaques in mice.

[0152] Figure 2 This is a typical image of the left ventricular outflow tract in a mouse. Detailed Implementation

[0153] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0154] Example 1: Synthesis of a General Intermediate for Raw Material Preparation

[0155]

[0156] Step 1. Synthesis of 4-((2-nitro-6-(trifluoromethyl)phenyl)amino)piperidine-1-carboxylic acid tert-butyl ester (GEN1-013-1)

[0157]

[0158] 2-Fluoro-1-nitro-3-(trifluoromethyl)benzene (1.00 g, 4.80 mmol), tert-butyl 4-aminopiperidine-1-carboxylic acid (961 mg, 4.80 mmol), and K₂CO₃ (995 mg, 7.20 mmol) were suspended in DMF (10 mL) and stirred at 85 °C for 2 hours. After cooling to room temperature, the reaction mixture was injected with water (50 mL) and extracted with EA (25 mL × 3). The combined organic layers were washed with water (25 mL), brine (25 mL), dried over MgSO₄, and concentrated to give the desired compound (1.86 g, 100% yield) as a yellow solid. 1HNMR (400MHz, CDCl3): δ8.08 (dd, J=8.0, 1.2Hz, 1H), 7.78 (dd, J=7.6, 1.2Hz, 1H), 6.96 (t, J=8.0Hz, 1H), 5.93 (d, J=9. 6Hz, 1H), 4.10-3.93 (m, 2H), 3.46-3.36 (m, 1H), 2.84-2.78 (m, 2H), 1.94-1.91 (m, 2H), 1.45 (s, 9H), 1.39-1.29 (m, 2H).

[0159] Step 2. Synthesis of 4-((2-amino-6-(trifluoromethyl)phenyl)amino)piperidine-1-carboxylic acid tert-butyl ester (GEN1-013-2)

[0160]

[0161] Pd / C (10%, 186 mg) was added to a methanol (20 mL) solution of 4-((2-nitro-6-(trifluoromethyl)phenyl)amino)piperidine-1-carboxylic acid tert-butyl ester (1.86 g, 4.78 mmol) under a nitrogen atmosphere. The resulting mixture was degassed with H2 and stirred overnight at 35 °C under a hydrogen atmosphere. The reaction mixture was filtered, and the filtrate was concentrated to give the desired compound (1.66 g, 97% yield) as a brown oil. 1 HNMR (400MHz, CDCl3): δ6.97 (dd, J=7.6, 1.6Hz, 1H), 6.92 (t, J=8.0Hz, 1H), 6.86 (dd, J=8.0, 1.6Hz, 1H), 4.11 (br s,2H),3.88(s,2H),3.38-3.24(m,2H),2.73-2.66(m,2H),1.84-1.81(m,2H),1.46(s,9H),1.40-1.31(m,2H).

[0162] Step 3. Synthesis of tert-butyl 4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylic acid (GEN1-013-3)

[0163]

[0164] Triphosgene (206 mg, 0.694 mmol) was added to a suspension of 4-((2-amino-6-(trifluoromethyl)phenyl)amino)piperidine-1-carboxylic acid tert-butyl ester (500 mg, 1.39 mmol) and NaHCO3 (584 mg, 6.95 mmol) in DCM (25 mL) at 7 °C. The mixture was stirred for 2 hours. The reaction mixture was poured into water (50 mL), extracted with DCM (25 mL × 3), and the resulting organic layer was washed with brine (25 mL), dried on MgSO4, and concentrated to give the desired compound (510 mg, 95% yield) as a white solid. 1 HNMR (400MHz, CDCl3): δ10.16(s,1H),7.40(d,J=8.0Hz,1H),7.30(d,J=7.6Hz,1H),7.14 (t,J=8.0Hz,1H),4.42-4.25(m,3H),2.82-2.70(m,4H),1.76-1.73(m,2H),1.50(s,9H).

[0165] Step 4. Synthesis of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1H-benzo[d]imidazol-2(3H)-one (GEN1-013-4)

[0166]

[0167] 4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylic acid tert-butyl ester (510 mg, 1.32 mmol) was added to DCM (6 ml), followed by TFA (2 ml) at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated, and the residue was dissolved in DCM (10 ml). Na2CO3 (sat., 10 mL) was added to the solution, and the mixture was stirred for 20 minutes. The mixture was filtered, and the filter cake was dried to give the desired compound (340 mg, 67% yield) as a white solid. 1 HNMR(400MHz,DMSO-d6):δ8.41(br s,1H),7.37(d,J=8.0Hz,1H),7.31(d,J=7.2Hz,1H),7.18(t,J=8.0Hz,1H),4.29-4.18(m ,1H),3.43-3.40(m,2H),2.97-2.80(m,4H),1.88-1.85(m,2H).MS(ESI)Rt=1.50min,m / z 286.3[M+H] + Purity: 98%@254nm, 100%@214nm.

[0168] Step 5.1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1H-benzo[d]imidazol-2(3H)-one (GEN1-117)

[0169]

[0170] Compounds 1-(piperidin-4-yl)-7-(trifluoromethyl)-1H-benzo[d]imidazol-2(3H)-one 2,2,2-trifluoroacetate (100 mg, 250 μmol, see the final report of GEN1-013-4 for synthesis details), EDCI (72 mg, 0.36 mmol), HOBT (51 mg, 0.38 mmol), and DIEA (162 mg, 1.25 mmol) were dissolved in DMF (2 mL) and 2-(6-quinolinyl)acetic acid (56 mg, 0.30 mmol) was added. After the addition was complete, the resulting mixture was stirred at room temperature for 16 hours. The mixture was then purified by preparative methods (Preparation of 5Xbridge C). 18 The desired compound (88 mg, yield 77%) was obtained as a white solid at 5 μm 19*150 mm, 30-70% B; A: H2O (0.1% NH4HCO3), B: CH3CN; 214 nm, flow rate: 15 mL / min. 1 HNMR (400MHz, DMSO-d6): δ11.45(s,1H),8.87(dd,J=4.4,1.6Hz,1H),8.33(d,J=7.2Hz,1H),7.97(d ,J=8.4Hz,1H),7.82(s,1H),7.66(dd,J=8.8,2.0Hz,1H),7.52(dd,J=8.4,4.4Hz,1H),7.36(d,J=8. 4Hz,1H),7.28(d,J=7.2Hz,1H),7.16(t,J=8.0Hz,1H),4.64(d,J=10.0Hz,1H),4.29-4.20(m,2H),4 .01-3.96(m,2H),3.09-3.02(m,1H),2.61-2.54(m,3H),1.72(s,2H).LCMS(ESI):Rt=3.694min,m / z 455.1[M+H] + Purity: 98.00% @ 254nm, 99.57% @ 214nm.

[0171] Example 1

[0172] (2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl dihydrophosphate (GEN1-284)

[0173]

[0174] Step 1. Di-tert-butyl((2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)phosphate (GEN1-284-1)

[0175]

[0176] 120 mg (0.26 mmol) of 1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one and cesium carbonate (258 mg, 0.79 mmol) were dissolved in DMF (2.0 mL). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 2.5 h. Then, di-tert-butyl (chloromethyl)phosphate (88 mg, 0.34 mmol) was added, and stirring was continued at room temperature under nitrogen atmosphere for 48 h. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The mixture was washed with water (25 mL) and saturated brine (25 mL), and the combined organic layers were dried over anhydrous sodium sulfate and concentrated to give a pale yellow oil. The crude product was purified by silica gel column chromatography, eluting with (MeOH / (MeOH+DCM) = 5%) to give di-tert-butyl((2-oxo-3-(1-(2-(quinoline-6(-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)phosphate (188 mg), a pale yellow oil. LCMS (ESI): Rt = 1.63 min, m / z 677.3 [M+H] + Purity: 63%@254nm, 85%@214nm;

[0177] Step 2. (2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl dihydrophosphate (GEN1-284)

[0178]

[0179] Di-tert-butyl((2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)phosphate (188 mg) was dissolved in a mixed solution of acetic acid (2.0 mL) and water (0.5 mL). The resulting mixture was stirred at 65 °C for 1.0 h. The resulting mixture was then purified directly by reversed-phase rapid chromatography (C1). 18 Reverse silica column chromatography; mobile phase CH3CN, water, 0% to 70% gradient, 40 min; detection, UV 214 nm) yielded the desired compound (60 mg, yield 40%) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ:8.87(d,J=4.1Hz,1H),8.35(d,J=8.2Hz,1H),7.98(d,J=8.6H z,1H),7.83(s,1H),7.69–7.59(m,2H),7.57–7.43(m,2H),7.29(t,J=8.0Hz,1H),5.62( d,J=8.0Hz,2H),4.63(d,J=12.6Hz,1H),4.37–4.16(m,2H),4.09–3.91(m,2H),3.07(t, J=13.2Hz,1H),2.69–2.50(m,3H),1.75(d,J=12.1Hz,2H).LCMS(ESI):Rt=1.16min,m / z 565.2[M+H] + Purity: One main peak.

[0180] Example 2

[0181] (2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl phosphate disodium salt (GEN1-284 salt)

[0182]

[0183] Step 1. Disodium salt of (2-oxo-3-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl phosphate (GEN1-284 salt)

[0184]

[0185] (2-oxo-3-(1-(2-(quinoline-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl dihydrophosphate (26.2 mg, 0.046 mmol) was dissolved in methanol (0.5 mL), and 0.01 N standard sodium hydroxide aqueous solution (9.29 mL, 0.093 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for 0.25 h. The resulting mixture was lyophilized to give a white solid disodium salt of (2-oxo-3-(1-(2-(quinoline-6-yl)acetyl)piperidin-4-yl)-4-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl phosphate (28 mg, 100% yield). 1 H NMR(400MHz,D2O)δ:8.72–8.65(m,1H),8.23(d,J=8.3Hz,1H),7.90(d,J=8.7Hz,1H),7.66(s,1H),7.57(d,J=8. 3Hz,2H),7.48–7.35(m,2H),7.21(d,J=8.0Hz,1H),5.56–5.40(m,2H),4.55(d,J=13.6Hz,1H),4.39–4.27(m,1H) 4.12 (d, J = 14.0 Hz, 1H), 4.04–3.91 (m, 2H), 3.13 (t, J = 13.2 Hz, 1H), 2.73 (t, J = 13.1 Hz, 1H), 2.53–2.20 (m, 2H), 1.81 (d, J = 12.6 Hz, 1H), 1.67 (d, J = 12.7 Hz, 1H). LCMS (ESI): Rt = 3.355 min (Note: The liquid chromatography-mass spectrometry analysis conditions for this compound differ from those described in GEN1-284), m / z 565.1 [M+H] + Purity: 95.45%@254nm, 81.76%@214nm.

[0186] Example 3

[0187] 4-Chloro-1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-295)

[0188]

[0189]

[0190] Step 1. Synthesis of tert-butyl 4-((3-chloro-2-nitrophenyl)amino)piperidine-1-carboxylate (GEN1-295-1)

[0191]

[0192] At room temperature, K₂CO₃ (1.1 g, 8 mmol) and KI (66.4 mg, 0.4 mmol) were added to a DMF (20 mL) solution of 1-chloro-3-fluoro-2-nitrobenzene (702.16 mg, 3 mmol), tert-butyl 4-aminopiperidine-1-carboxylate (880.23 mg, 4.4 mmol). The resulting mixture was then stirred at 80 °C for 3 hours. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (30 mL × 3). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated and purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (1.2 g, yield 84.3%) as a yellow oil. MS (ESI) RT = 1.79 min, m / z 354 [MH] - ,purity:90.58%@254nm,73.24%@214nm.

[0193] Step 2. Synthesis of tert-butyl 4-((2-amino-3-chlorophenyl)amino)piperidine-1-carboxylate (GEN1-295-2)

[0194]

[0195] Fe (1.888 g, 33.72 mmol) was added to a suspension of tert-butyl 4-((3-chloro-2-nitrophenyl)amino)piperidine-1-carboxylate (1.2 g, 3.372 mmol), NH4Cl (3.607 g, 67.44 mmol), i-PrOH (25 mL), and H2O (5 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was concentrated to give a crude product. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 1:1) to give the desired compound (1.0 g, yield 91%) as a brown solid. MS (ESI) RT = 1.58 min, purity: 100% @ 254 nm, 100% @ 214 nm.

[0196] Step 3. Synthesis of tert-butyl 4-(4-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylate (GEN1-295-3)

[0197]

[0198] Under nitrogen protection and controlled temperature of 0°C, triphosgene (72.86 mg, 0.246 mmol) was added to a DCM solution of 4-((2-amino-3-chlorophenyl)amino)piperidin-1-carboxylic acid tert-butyl ester (200 mg, 0.614 mmol), NaHCO3 (515.76 mg, 6.14 mmol), and stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was concentrated to give the crude product. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 1:1) to give the desired compound (210 mg, yield 97.2%) as a brown solid. MS (ESI) RT = 1.50 min, purity: 64.2% @ 254 nm, 89.68% @ 214 nm.

[0199] Step 4. Synthesis of 4-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-295-4)

[0200]

[0201] TFA (4 mL) was added to a DCM (10 mL) solution of tert-butyl 4-(4-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylate (100 mg, 0.284 mmol) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h. The reaction solution was concentrated. The resulting residue was freshly dissolved in ACN (40 mL) and concentrated again. A crude product (100 mg) was given, which could be used directly for the next step without purification. MS (ESI) RT = 1.36 min, m / z 252.2 [M+H] + ,purity:100%@254nm,100%@214nm.

[0202] Step 5. Synthesis of 4-chloro-1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-295)

[0203]

[0204] DIPEA (146.8 mg, 1.136 mmol) was added to a DMF (2 mL) solution of 4-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2- (100 mg, crude, 0.284 mmol), 2-(quinoline-6-yl)acetic acid (63.84 mg, 0.341 mmol), HOBT (57.56 mg, 0.426 mmol), and EDCI (81.66 mg, 0.341 mmol). The resulting mixture was stirred at room temperature for 1 h. The crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (30 mg, two-step yield 25.1%) was obtained as a white solid. 1 HNMR (400MHz, DMSO-d6) δ11.41(s,1H),8.96–8.78(m,1H),8.37(d,J=8.3Hz,1H),8.00(d,J=8. 6Hz,1H),7.88(s,1H),7.71(dd,J=8.8,1.9Hz,1H),7.56-7.49(m,1H),7.05–6.82(m,3H),4.61( d,J=13.1Hz,1H),4.48–4.32(m,1H),4.16(d,J=13.9Hz,1H),4.10-3.92(m,2H),3.19(t,J=13.0 Hz,1H),2.71(t,J=12.8Hz,1H),2.17-1.87(m,2H),1.76-1.57(m,2H).MS(ESI)RT=1.32min,m / z 421.2[M+H] + ,purity:100%@254nm,100%@214nm.

[0205] Example 4

[0206] 1-(1-(1-(2-(1H-indol-5-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-296)

[0207]

[0208] Step 1. Synthesis of methyl 2-(4-amino-3-bromophenyl)acetate (GEN1-296-1)

[0209]

[0210] NBS (3.23 g, 18.16 mmol) was added to a solution of methyl 2-(4-aminophenyl)acetate (3 g, 18.16 mmol) in 20 mL of ACN under nitrogen protection. The resulting mixture was stirred at room temperature for 16 h. The reaction solution was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography, eluting with (PE:EA = 5:1) to give the desired compound (4.2 g, yield 94.75%) as a pale yellow oil. MS (ESI) RT = 1.36 min, m / z 244.0 [M+H] + ,purity:83.91%@254nm,71.46%@214nm.

[0211] Step 2. Synthesis of methyl 2-(4-amino-3-((trimethylsilyl)ethynyl)phenyl)acetate (GEN1-296-2)

[0212]

[0213] Pd(PPh3)2Cl2 (575 mg, 0.819 mmol) was added to a TEA (10 mL) solution of methyl 2-(4-amino-3-bromophenyl)acetate (2.0 g, 8.19 mmol), trimethylsilylacetylene (8.125 g, 81.9 mmol), and DMAP (100 mg, 0.819 mmol) at room temperature and under nitrogen protection. The resulting mixture was stirred at 70 °C for 16 h. The mixture was concentrated, diluted with water (50 mL), and extracted with DCM (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 20:1) to give the desired compound (1.16 g, yield 54%) as a yellow oil.

[0214] Step 3. Synthesis of methyl 2-(1H-indole-5-yl)acetate (GEN1-296-3)

[0215]

[0216] CuI (80 mg, 0.42 mmol) was added to a DMF (20 mL) solution of methyl 2-(4-amino-3-(((trimethylsilyl)ethynyl)phenyl)acetate (1.1 g, 4.2 mmol) at room temperature and under nitrogen protection. The resulting mixture was stirred at 90 °C for 16 h. The reaction was then quenched with water (50 mL). The mixture was extracted with DCM (50 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated to give the crude product. The crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H₂O, gradient: 5% to 60% for 50 min, monitoring wavelength: 214 nm, yielding the desired compound (400 mg, yield 50.24%) as a yellow oil. MS (ESI) RT = 2.343 min, m / z 190.1 [M + H] + ,purity:55.31%@254nm,40.54%@214nm.

[0217] Step 4. Synthesis of 2-(1H-indol-5-yl)acetic acid (GEN1-295-4)

[0218]

[0219] NaOH (169.12 mg, 4.228 mmol) was added to a solution of methyl 2-(1H-indol-5-yl)acetate (400 mg, 2.114 mmol) in THF (10 mL) and H₂O (5 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 h. The pH of the mixture was then adjusted to 4, and purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H₂O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (300 mg, yield 81%) was obtained as a white solid. MS (ESI) RT = 1.68 min, m / z 176.1 [M+H] + ,purity:97.25%@254nm,80.46%@214nm.

[0220] Step 5. Synthesis of 1-(1-(1-(2-(1H-indol-5-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-296)

[0221]

[0222] DIPEA (258.5 mg, 0.2 mmol) was added to a DMF (1 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (81.4 mg, 0.2854 mmol), 2-(1H-indol-5-yl)acetic acid (50 mg, 0.2854 mmol), HOBT (57.84 mg, 0.4281 mmol), and EDCI (82.07 mg, 0.4281 mmol). The resulting mixture was stirred at room temperature for 1 h. The crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H2O, gradient: 5% to 50%; 40 min; monitoring wavelength: 214 nm. The desired compound (50 mg, yield 39.6%) was obtained as a white solid. 1 H NMR(400MHz,Methanol-d4)δ7.48(s,1H),7.44–7.36(m,2H),7.33–7.28(m,1H) ,7.26–7.16(m,2H),7.06(dd,J=8.4,1.7Hz,1H),6.44(dd,J=3.1,0.9Hz,1H),4. 86-4.78(m,1H),4.52-4.38(m,1H),4.34–4.25(m,1H),3.93(d,J=4.4Hz,2H),3. 13-3.01(m,1H),2.79–2.52(m,3H),1.88–1.68(m,2H).MS(ESI)RT=2.63min,m / z 443.2[M+H] + ,purity:100%@254nm,93.97%@214nm.

[0223] Example 5

[0224] 1-(1-(2-(3-(dimethylamino)phenyl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-297)

[0225]

[0226] Step 1. Synthesis of 2-(3-(dimethylamino)phenyl)acetic acid (GEN1-297-1)

[0227]

[0228] At 0 °C, 37% formaldehyde (202.91 mg, 2.5 mmol) was added to a solution of 2-(3-aminophenyl)acetic acid (151.17 mg, 1 mmol) in MeOH (3 mL). The resulting mixture was stirred at room temperature for 6 h. The mixture was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H₂O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound was obtained (90 mg, yield 50.21%). MS (ESI) RT = 0.27 min, m / z 180.1 [M+H] + ,purity:100%@254nm,100%@214nm.

[0229] Step 2.1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-297-2)

[0230]

[0231] TFA (2 ml) was added to a DCM (10 ml) solution of 200 mg (0.52 mmol) of tert-butyl 4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-1-carboxylate (200 mg, 0.52 mmol). The resulting mixture was stirred at room temperature for 0.5 h. The reaction mixture was then concentrated and redissolved in DCM (20 ml). The solution was concentrated again to give a crude product as a yellow solid. The crude product can be used directly in the next step without purification.

[0232] Step 3. Synthesis of 1-(1-(2-(3-(dimethylamino)phenyl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-297)

[0233]

[0234] DIPEA (193.86 mg, 1.5 mmol) was added to a DMF (1 mL) solution of 2-(3-(dimethylamino)phenyl)acetic acid (200 mg, crude, 0.5 mmol), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (90 mg, 0.5 mmol), HOBT (101.4 mg, 0.75 mmol), and EDCI (143.78 mg, 0.75 mmol). The resulting mixture was stirred at room temperature for 1 h. The mixture was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (50 mg, two-step yield 22.4%) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.47(s,1H),7.38(d,J=8.1Hz,1H),7.30(d,J=7.7Hz,1H),7.23-7.09(m,2H),6.66–6.54(m,3H),4.65(d,J=10.0Hz,1H) ,4.34–4.09(m,2H),3.79–3.54(m,2H),2.99(t,J=13.1Hz,1H),2.61-2.5 6(m,1H),2.52-2.42(m,2H),1.78-1.64(m,2H).MS(ESI)RT=2.60min,m / z 447.2[M+H] + ,purity:100%@254nm,93.4%@214nm.

[0235] Example 6

[0236] 1-(1-(2-(benzo[b]thiophene-5-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-298)

[0237]

[0238] Step 1.5-(bromomethyl)benzo[b]thiophene (GEN1-298-1)

[0239]

[0240] NBS (370 mg, 2.08 mmol) and AIBN (34 mg, 0.11 mmol) were added to a CCl4 (12 mL) solution of 5-methylbenzo[b]thiophene (280 mg, 1.89 mmol). The mixture was stirred and heated under reflux for 3 hours, then cooled to room temperature overnight. The mixture was filtered, and the filtrate was evaporated to dryness under vacuum at 40 °C. The mixture was filtered again, and the residue was treated with n-hexane and heated until dissolved, then cooled. The solid was then separated by filtration to give 5-(bromomethyl)benzo[b]thiophene (490 mg, 86% yield) as a white solid. TLC: PE = 100%, Rf = 0.6

[0241] Step 2.2 - (benzo[b]thiophene-5-yl)acetonitrile (GEN1-298-2)

[0242]

[0243] At 0 °C, K₂CO₃ (3.05 mg, 2.2 mmol) and TMSCN (164 mg, 1.7 mmol) were added to a solution of 5-(bromomethyl)benzo[b]thiophene (250 mg, 1.1 mmol) in ACN (10 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (20 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), respectively, dried over Na₂SO₄, and concentrated to give a yellow oil. The crude product was purified by reversed-phase rapid chromatography (C₂CO₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(benzo[b]thiophene-5-yl)acetonitrile (84 mg, yield 44%) as a white solid. MS (ESI) RT = 2.67 min, m / z 174.1 [M+H] + ,purity:46.06%@254nm,83.97%@214nm.

[0244] Step 3.2 - (benzo[b]thiophene-5-yl)acetic acid (GEN1-298-3)

[0245]

[0246] Under a nitrogen atmosphere, NaOH (190 mg, 4.75 mmol) was added to a mixed solution of 2-(benzo[b]thiophene-5-yl)acetonitrile (60 mg, 0.35 mmol) in THF (5 mL) and H₂O (5 mL). The mixture was stirred at 80 °C for 18 hours. It was then diluted with H₂O (20 mL) and washed with EA (20 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (20 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(benzo[b]thiophene-5-yl)acetic acid (57 mg, 86% yield) as a white solid. MS (ESI) RT = 2.35 min, m / z 193.0 [M+H] + ,purity:100%@254nm,96.39%@214nm.

[0247] Step 4.1-(1-(2-(benzo[b]thiophene-5-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-298)

[0248]

[0249] To a mixture of 2-(benzo[b]thiophene-5-yl)acetic acid (57 mg, 0.30 mmol) in DMF (5 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (85 mg, 0.30 mmol), EDCI (68 mg, 0.36 mmol), HOBT (48 mg, 0.36 mmol), and DIEA (77 mg, 0.60 mmol) were added. The mixture was stirred at room temperature for 1 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(benzo[b]thiophene-5-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (25 mg, yield 18%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ11.46 (s, 1H), 7.93 (d, J = 8.4Hz, 1H), 7.77-7.73 (m, 2H), 7.43(d,J=5.6Hz,1H),7.35(d,J=8.0Hz,1H),7.27-7.23(m,2H),7.15(t,J=8.0H z,1H),4.62(d,J=10.4Hz,1H),4.31-4.15(m,2H),3.89(q,J=15.2Hz,2H),3.00( t,J=12.8Hz,1H),2.63-2.51(m,3H),1.83-1.63(m,2H).MS(ESI)RT=2.98min,m / z 460.2 [M+H] + ,purity:96.67%@254nm,95.17%@214nm.

[0250] Example 7

[0251] 1-(1-(2-(isoquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-299)

[0252]

[0253]

[0254] Step 1. Isoquinoline-6-ylboronic acid (GEN1-299-1)

[0255]

[0256] To a solution of 6-bromoisoquinoline (1 g, 4.8 mmol) in 1,4-dioxane (10 mL), KOAc (1.41 g, 14.4 mmol), 4,4,4',4',5,5,5,5'-octamethyl-2,2'-bi(1,3,2-dioxaborane) (1.83 g, 7.2 mmol), and Pd(dppf)Cl2·CH2Cl2 (392 mg, 0.48 mmol) were added. The mixture was stirred at 100 °C for 2 hours. The reaction mixture was poured into water (100 mL) and extracted with EA (100 mL x 3). The combined organic layers were washed with water (100 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow oil. The crude product was purified by reversed-phase rapid chromatography (C10). 18Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded isoquinoline-6-ylboronic acid (590 mg, yield 71%). MS (ESI) RT = 0.43 min, m / z 174.1 [M+H] + ,purity:7.59%@254nm,63.95%@214nm.

[0257] Step 2.2 - (isoquinoline-6-yl)ethyl acetate (GEN1-299-2)

[0258]

[0259] To a solution of Pd(OAc)₂ (27 mg, 0.12 mmol) in THF (10 mL), K₃PO₄ (1.27 g, 5.99 mmol), isoquinoline-6-ylboronic acid (300 mg, 1.73 mmol), ethyl 2-bromoacetate (200 mg, 1.2 mmol), and tris(1-naphthyl)phosphine (150 mg, 0.36 mmol) were added. The mixture was stirred overnight at 73 °C. The reaction mixture was poured into water (20 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na₂SO₄, and concentrated to give the crude product. The crude product was purified by reversed-phase rapid chromatography (C₂SO₄). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded ethyl 2-(isoquinoline-6-yl)acetate (49 mg, yield 19%) as a yellow liquid. MS (ESI) RT = 1.50 min, m / z 216.1 [M+H] + ,purity:50.64%@254nm,84.37%@214nm.

[0260] Step 3.2 - (isoquinoline-6-yl)acetic acid (GEN1-299-3)

[0261]

[0262] LiOH (24 mg, 1 mmol) was added to a solution of ethyl 2-(isoquinoline-6-yl)acetate (49 mg, 0.23 mmol) in MeOH (8 mL) and H₂O (2 mL). The mixture was stirred at room temperature for 1 h. It was then concentrated and diluted with H₂O (20 mL), and washed with EA (20 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (20 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(isoquinoline-6-yl)acetic acid (42 mg, 99% yield) as a white solid. MS (ESI) RT = 0.59 min, m / z 188.1 [M + H₂] + ,purity:100%@254nm,43.80%@214nm.

[0263] Step 4.1-(1-(2-(isoquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-299)

[0264]

[0265] To a DMF (3 mL) solution of 2-(isoquinoline-6-yl)acetic acid (42 mg, 0.22 mmol), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (70 mg, 0.25 mmol), EDCI (52 mg, 0.27 mmol), HOBT (36 mg, 0.27 mmol), and DIEA (58 mg, 0.45 mmol) were added. The mixture was stirred at room temperature for 5 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(isoquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (40 mg, yield 39%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ11.46(s,1H),9.27(s,1H),8.48(d,J=5.6Hz,1H),8.07(d,J=8.4Hz,1H) ,7.89–7.73(m,2H),7.58(dd,J=8.4,1.6Hz,1H),7.36(d,J=8.0Hz,1H),7.27(d,J=7.6Hz,1H),7 .16(t,J=8.0Hz,1H),4.63(d,J=11.2Hz,1H),4.24(dt,J=17.6,10.8Hz,2H),4.02(d,J=2.4Hz,2 H),3.06(t,J=13.2Hz,1H),2.66–2.50(m,3H),1.72(d,J=12.0Hz,2H).MS(ESI)RT=2.54min,m / z 455.2[M+H] + ,purity:100%@254nm,99.57%@214nm.

[0266] Example 8

[0267] 1-(1-(2-(2-(2-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-300)

[0268]

[0269] Step 1. Synthesis of 6-(bromomethyl)-2-methylquinoline (GEN1-300-1)

[0270]

[0271] At room temperature, NBS (1.245 g, 7 mmol) was added to a CCl4 (35 mL) solution of 2,6-dimethylquinoline (1.0 g, 6.36 mmol), AIBN (104.44 mg, 0.636 mmol), and 2,6-dimethylquinoline (1.0 g, 6.36 mmol), and AIBN (104.44 mg, 0.636 mmol). The resulting mixture was stirred at 77 °C for 3 hours. The reaction mixture was quenched with water (50 mL). The resulting mixture was extracted with DCM (40 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated to give a brown solid. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (1.0 g, yield 66.59%) as a pale yellow solid. MS (ESI) RT = 1.602 min, m / z 236 [M+H] + Purity: 94.84% @ 254nm.

[0272] Step 2.2 - (2-methylquinoline-6-yl)acetonitrile (GEN1-300-2)

[0273]

[0274] Under nitrogen protection at room temperature, TMS-CN (143.85 mg, 1.45 mmol) was added to a suspension of 6-(bromomethyl)-2-methylquinoline (236.11 mg, 1 mmol), K₂CO₃ (158.94 mg, 1.15 mmol) in ACN (5 mL). The resulting mixture was stirred at 60 °C for 3 h. The mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (125 mg, yield 68.59%) as a white solid. MS (ESI) RT = 0.91 min, m / z 183.1 [M+H] + Purity: 78.01% @ 214nm.

[0275] Step 3. Synthesis of 2-(2-methylquinoline-6-yl)acetic acid (GEN1-300-3)

[0276]

[0277] At room temperature, NaOH (33.25 mg, 0.831 mmol) was added to a solution of 2-(2-methylquinoline-6-yl)acetonitrile (100 mg, 0.554 mmol) in H₂O (2.5 mL) and THF (2.5 mL). The resulting mixture was stirred at 90 °C for 3 h. The reaction mixture was quenched with water (25 mL). The mixture was extracted with EtOAc (15 mL × 3). The aqueous phase was adjusted to pH 4 with 1 N HCl. It was then extracted with EtOAc (15 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated to give the desired compound (110 mg, crude product). The crude product was used directly in the next step. MS (ESI) RT = 0.571 min, m / z 202.1 [M + H] + Purity: 83.06% @ 214nm.

[0278] Step 4. Synthesis of 1-(1-(2-(2-(2-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-300-3)

[0279]

[0280] DIPEA (142 mg, 1.1 mmol) was added to a DMF (5 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (156.9 mg, 0.55 mmol), 2-(2-methylquinoline-6-yl)acetic acid (110 mg, 0.55 mmol), EDCI (158.2 mg, 0.825 mmol), and HOBT (114.5 mg, 0.825 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with water (50 mL). The mixture was extracted with EtOAc (30 mL x 3). The combined organic phases were washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated to give a brown solid. The crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN:H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (20 mg, two-step yield 7.76%) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),8.20(d,J=8.4Hz,1H),7.86(d,J=8.6Hz,1H),7.75(d ,J=1.9Hz,1H),7.59(dd,J=8.6,2.0Hz,1H),7.43–7.31(m,2H),7.27(d,J=7.7Hz,1H),7.15( t,J=7.9Hz,1H),4.63(d,J=11.2Hz,1H),4.32-4.15(m,2H),3.96(d,J=4.3Hz,2H),3.04(t,J =13.2Hz,1H),2.64(s,3H),2.62–2.51(m,3H),1.78-1.63(m,2H).MS(ESI)RT=1.930min,m / z 469.2[M+H] + Purity: 81.51% @ 214nm.

[0281] Example 9

[0282] 1-(1-(2-(4-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-301)

[0283]

[0284] Step 1. 6-(bromomethyl)-4-chloroquinoline (GEN1-301-1)

[0285]

[0286] NBS (442 mg, 2.49 mmol) and AIBN (37 mg, 0.23 mmol) were added to a CCl4 (20 mL) solution of 4-chloro-6-methylquinoline (400 mg, 2.26 mmol). The mixture was stirred and refluxed overnight. The mixture was filtered, and the filtrate was evaporated to dryness under vacuum at 40 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow oil. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 2:1) to give 6-(bromomethyl)-4-chloroquinoline (316 mg, 55% yield) as a white solid. MS(ESI)RT=2.92min, m / z 255.9[M+H]+, purity:95.13%@254nm, 98.35%@214nm.

[0287] Step 2.2 - (4-chloroquinoline-6-yl)acetonitrile (GEN1-301-2)

[0288]

[0289] At 0 °C, K₂CO₃ (274 mg, 1.98 mmol) and TMSCN (184 mg, 1.86 mmol) were added to a solution of 6-(bromomethyl)-4-chloroquinoline (316 mg, 1.24 mmol) in ACN (6 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄ and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C₂CO₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(4-chloroquinoline-6-yl)acetonitrile (140 mg, yield 56%) as a yellow liquid. MS (ESI) RT = 2.27 min, m / z 203.0 [M+H] + ,purity:77.17%@254nm,77.68%@214nm.

[0290] Step 3.2 - (4-chloroquinoline-6-yl)acetic acid (GEN1-301-3)

[0291]

[0292] To a mixture of 2-(4-chloroquinoline-6-yl)acetonitrile (140 mg, 0.7 mmol) in THF (2.2 mL) and H₂O (2.2 mL), NaOH (111 mg, 2.8 mmol) was added. The mixture was stirred at 80 °C for 18 hours. It was then diluted with H₂O (10 mL) and washed with EA (10 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(4-chloroquinoline-6-yl)acetic acid (100 mg, 65% yield) as a red solid. MS (ESI) RT = 1.92 min, m / z 222.0 [M+H] + ,purity:73.66%@254nm,95.15%@214nm.

[0293] Step 4.1 -(1-(2-(4-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-301)

[0294]

[0295] To a DMF (5 mL) solution of 2-(4-chloroquinoline-6-yl)acetic acid (100 mg, 0.45 mmol), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (129 mg, 0.45 mmol), EDCI (104 mg, 0.54 mmol), HOBT (73 mg, 0.54 mmol), and DIEA (117 mg, 0.9 mmol) were added. The mixture was stirred at room temperature for 1 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(4-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (30 mg, yield 14%) as a red solid. 1H NMR (400MHz, DMSO-d6) δ11.45 (s, 1H), 8.81 (d, J = 4.8Hz, 1H), 8.12-8.00 (m, 2H), 7.83-7.71(m,2H),7.36(dd,J=8.0,1.2Hz,1H),7.32-7.24(m,1H),7.16(t,J=8.0 Hz,1H),4.62(d,J=12.0Hz,1H),4.34-4.18(m,2H),4.09(s,2H),3.07(t,J=12.8 Hz,1H),2.56(d,J=12.4Hz,3H),1.73(d,J=11.2Hz,2H).MS(ESI)RT=2.73min,m / z 489.2 [M+H] + ,purity:92.20%@254nm,99.14%@214nm.

[0296] Example 10

[0297] 1-(1-(2-(2-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-302)

[0298]

[0299] Step 1. 6-(bromomethyl)-2-chloroquinoline (GEN1-302-1)

[0300]

[0301] NBS (221 mg, 1.24 mmol) and AIBN (19 mg, 0.11 mmol) were added to a CCl4 (6 mL) solution of 2-chloro-6-methylquinoline (200 mg, 1.13 mmol). The mixture was stirred and refluxed for 3 hours, then cooled to room temperature overnight. The mixture was filtered, and the filtrate was evaporated to dryness under vacuum at 40 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow liquid. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 2:1) to give 6-(bromomethyl)-2-chloroquinoline (187 mg, 65% yield) as a white solid. MS (ESI) RT = 3.02 min, m / z 255.9 [M+H] + ,purity:100%@254nm,100%@214nm.

[0302] Step 2.2 - (2-chloroquinoline-6-yl)acetonitrile (GEN1-302-2)

[0303]

[0304] At 0 °C, K₂CO₃ (162 mg, 1.17 mmol) and TMCSN (109 mg, 1.10 mmol) were added to a solution of 6-(bromomethyl)-2-chloroquinoline (187 mmg, 0.73 mmol) in ACN (6 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄ and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C₂CO₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(2-chloroquinoline-6-yl)acetonitrile (120 mg, yield 81%) as a yellow liquid. MS (ESI) RT = 2.46 min, m / z 203.0 [M+H] + ,purity:100%@254nm,100%@214nm.

[0305] Step 3.2-(2-chloroquinoline-6-yl)acetic acid (GEN1-302-3)

[0306]

[0307] In a sealed tube, NaOH (48 mg, 1.2 mmol) was added to 1 mL of THF and 1 mL of H₂O in 60 mg (60 mg, 0.3 mmol) of 2-(2-chloroquinoline-6-yl)acetonitrile. The mixture was stirred at 80 °C for 18 hours under N₂. It was then diluted with 10 mL of H₂O and washed with 10 mL of EA. The aqueous phase was adjusted to pH 4 with 2 M hydrochloric acid and extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(2-chloroquinoline-6-yl)acetic acid (48 mg, 73% yield) as a white solid.

[0308] TLC: MeOH / (DCM+MeOH)=10%, Rf=0.5

[0309] Step 4.1 -(1-(2-(2-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-302)

[0310]

[0311] To a solution of 2-(2-chloroquinoline-6-yl)acetic acid (48 mg, 0.22 mmol) in DMF (5 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (62 mg, 0.22 mmol), EDCI (50 mg, 0.26 mmol), HOBT (35 mg, 0.26 mmol), and DIEA (56 mg, 0.44 mmol) were added. The mixture was stirred at room temperature for 1 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(2-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[dimidazol-2-one (20 mg, yield 19%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ11.46(s,1H),8.42(d,J=8.8Hz,1H),7.99-7.83(m,2H),7.72(dd,J =8.8,2.0Hz,1H),7.57(d,J=8.8Hz,1H),7.43-7.31(m,1H),7.27(d,J=7.6Hz,1H),7.16(t, J=8.0Hz,1H),4.62(d,J=12.0Hz,1H),4.24(q,J=14.0,12.4Hz,2H),4.01(d,J=2.4Hz,2H), 3.10-2.98(m,1H),2.56(d,J=11.2Hz,3H),1.71(d,J=12.0Hz,2H).MS(ESI)RT=2.87min,m / z 489.2[M+H] + ,purity:100%@254nm,99.44%@214nm.

[0312] Example 11

[0313] 1-(1-(2-(8-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-303)

[0314]

[0315]

[0316] Step 1. 6-Bromo-8-methylquinoline (GEN1-303-1)

[0317]

[0318] Concentrated sulfuric acid (1.15 mL) was slowly added dropwise to a mixture of 4-bromo-2-methylaniline (1.0 g, 5.40 mmol), Fe₂SO₄·7H₂O (531 mg, 1.59 mmol), PhNO₂ (406 mg, 3.30 mmol), and glycerol (2.04 g, 22.17 mmol) in DMF (3.0 mL). The mixture was heated under reflux and stirred for 3 hours. The reaction mixture was cooled and poured into 20 mL of ice water. Saturated NaHCO₃ was added to the solution to pH 8. The solution was filtered and extracted with dichloromethane. The dichloromethane layers were combined, dried over Na₂SO₄, and concentrated. The crude product was purified by silica gel column chromatography, eluting with (EA / (EA+PE) = 15%), to give 6-bromo-8-methylquinoline (808 mg, yield 68%) as a pale yellow solid. MS (ESI) RT = 3.025 min, m / z 222.0 [M+H₂] + ,purity:100%@254nm,100%@214nm.

[0319] Step 2.2-(8-methylquinoline-6-yl)acetic acid (GEN1-303-2)

[0320]

[0321] Under argon protection, 6-bromo-8-methylquinoline (50 mg, 0.23 mmol), Pd2(dba)3 (41 mg, 0.04 mmol), and X-Phos (43 mg, 0.09 mmol) were dissolved in a THF solution of (2-(tert-butoxy)-2-oxoethyl)zinc(II) bromide (1.8 mL, 0.90 mmol, 0.5 N). The resulting mixture was reacted in a microwave reactor at 120 °C for 4 hours. The reaction mixture was filtered, and the filter cake was washed with THF (10 mL × 3). The filtrate was concentrated under reduced pressure to give a crude product (404 mg), which could be used directly for the next step without further purification. MS (ESI) RT = 1.202 min, m / z 202.1 [M+H] + ,purity:66%@254nm,60%@214nm.

[0322] Step 3.1-(1-(2-(8-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-303)

[0323]

[0324] DIEA (353 mg, 2.74 mmol) was added dropwise to a solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (390 mg, 1.37 mmol), 2-(8-methylquinoline-6-acyl)acetic acid (250 mg, 1.24 mmol), EDCI (394 mg, 2.05 mmol), and HOBT (277 mg, 2.05 mmol) in DMF (4.0 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by reversed-phase rapid chromatography (C1). 18 Reverse silica column chromatography; mobile phase CH3CN, water, 5% to 70% gradient, 50 min; detection, UV 214 nm) yielded the desired compound (33 mg, yield 47%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ:11.47(s,1H),8.91–8.85(m,1H),8.30(d,J=8.4,1.7Hz,1H),7.6 4(s,1H),7.55–7.47(m,2H),7.34(d,J=8.0Hz,1H),7.26(d,J=7.7Hz,1H),7.14(t,J=7.9Hz ,1H),4.64(d,J=10.5Hz,1H),4.30–4.14(m,2H),3.94(s,2H),3.04(t,J=13.2Hz,1H),2.7 1(s,3H),2.63–2.51(m,3H),1.70(t,J=11.4Hz,2H).MS(ESI)RT=2.305min,m / z469.2[M+H] + ,purity:100%@254nm,98.8%@214nm.

[0325] Example 12

[0326] 6-(2-oxo-2-(4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)quinoline-4-onitrile (GEN1-304)

[0327]

[0328] Step 1. 6-(bromomethyl)-4-chloroquinoline (GEN1-304-1)

[0329]

[0330] NBS (442 mg, 2.49 mmol) and AIBN (37 mg, 0.23 mmol) were added to a CCl4 (20 mL) solution of 4-chloro-6-methylquinoline (400 mg, 2.26 mmol). The mixture was stirred and heated under reflux overnight. The mixture was filtered, and the filtrate was evaporated to dryness under vacuum at 40 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow liquid. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 2:1) to give 6-(bromomethyl)-4-chloroquinoline (353 mg, yield 61%) as a white solid. MS(ESI)RT=2.93min, m / z 255.9[M+H]+, purity:72.29%@254nm, 49.39%@214nm.

[0331] Step 2.2 - (4-chloroquinoline-6-yl)acetonitrile (GEN1-304-2)

[0332]

[0333] At 0 °C, K₂CO₃ (306 mg, 2.22 mmol) and TMCSN (206 mg, 2.08 mmol) were added to a solution of 6-(bromomethyl)-4-chloroquinoline (353 mg, 1.38 mmol) in ACN (6 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄ and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C₂CO₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(4-chloroquinoline-6-yl)acetonitrile (180 mg, yield 64%), as a yellow liquid. MS (ESI) RT = 2.27 min, m / z 203.0 [M+H] + ,purity:79.08%@254nm,68.48%@214nm.

[0334] Step 3.2 - (4-chloroquinoline-6-yl)acetic acid (GEN1-304-3)

[0335]

[0336] In a sealed tube, 2-(4-chloroquinoline-6-yl)acetonitrile (80 mg, 0.40 mmol) was mixed with THF (1.6 mL) and H₂O (1.6 mL) and then with NaOH (63 mg, 1.58 mmol). The mixture was stirred overnight at 80 °C. It was then diluted with H₂O (10 mL) and washed with EA (10 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(4-chloroquinoline-6-yl)acetic acid (75 mg, 86% yield) as a red solid. MS (ESI) RT = 1.91 min, m / z 222.0 [M+H] + ,purity:34.16%@254nm,43.93%@214nm.

[0337] Step 4.2 - (4-cyanoquinoline-6-yl)acetic acid (GEN1-304-4)

[0338]

[0339] Zn(CN)₂ (44 mg, 0.38 mmol) and Pd(PPh₃)₄ (78 mg, 0.07 mmol) were added to a solution of 2-(4-chloroquinoline-6-yl)acetic acid (75 mg, 0.34 mmol) in DMF (6 mL). The mixture was stirred at 90 °C for 3 hours. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na₂SO₄ and concentrated to give a yellow oil. The crude product was purified by reversed-phase rapid chromatography (C₂SO₄). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(4-cyanoquinoline-6-yl)acetic acid (38 mg, yield 53%) as a yellow solid. MS (ESI) RT = 1.88 min, m / z 213.1 [M+H] + ,purity:7.02%@254nm,20.19%@214nm.

[0340] Step 5.6 -(2-oxo-2-(4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)quinoline-4-onitrile (GEN1-304)

[0341]

[0342] To a solution of 2-(4-cyanoquinolin-6-yl)acetic acid (38 mg, 0.18 mmol) in DMF (6 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (56 mg, 0.2 mmol), EDCI (41 mg, 0.21 mmol), HOBT (29 mg, 0.21 mmol), and DIEA (46 mg, 0.36 mmol) were added. The mixture was stirred overnight at room temperature. It was then diluted with H₂O (20 mL) and extracted with EA (20 mL x 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄, and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C₂SO₄). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 6-(2-oxo-2-(4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin]-1-yl)ethyl)quinoline-4-onitrile (60 mg, 70% yield) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ11.45(s,1H),9.08(d,J=4.4Hz,1H),8.25–8.09(m,2H),8.03(s ,1H),7.85(d,J=8.8Hz,1H),7.36(d,J=8.0Hz,1H),7.27(d,J=7.6Hz,1H),7.16(t,J=8. 0Hz,1H),4.62(d,J=12.0Hz,1H),4.25(d,J=12.8Hz,2H),4.15(s,2H),3.07(t,J=13.2H z,1H),2.77–2.53(m,3H),1.72(d,J=12.0Hz,2H).MS(ESI)RT=2.66min,m / z480.2[M+H] + ,purity:100%@254nm,100%@214nm.

[0343] Example 13

[0344] 1-(1-(2-(2-(2-methoxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-305)

[0345]

[0346] Step 1. 6-(bromomethyl)-2-chloroquinoline (GEN1-305-1)

[0347]

[0348] NBS (442 mg, 2.48 mmol) and AIBN (37 mg, 0.23 mmol) were added to a CCl4 (20 mL) solution of 2-chloro-6-methylquinoline (400 mg, 2.26 mmol). The mixture was stirred and heated under reflux overnight. The mixture was filtered, and the filtrate was evaporated to dryness under vacuum at 40 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow liquid. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 2:1) to give 6-(bromomethyl)-2-chloroquinoline (399 mg, yield 69%) as a white solid. MS (ESI) RT = 3.02 min, m / z 255.9 [M+H] + ,purity:95.79%@254nm,23.70%@214nm.

[0349] Step 2.2 - (2-chloroquinoline-6-yl)acetonitrile (GEN1-305-2)

[0350]

[0351] At 0 °C, K₂CO₃ (345 mg, 2.5 mmol) and TMSCN (232 mg, 2.34 mmol) were added to a solution of 6-(bromomethyl)-2-chloroquinoline (399 mg, 1.56 mmol) in ACN (6 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄ and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C₂CO₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 2-(2-chloroquinoline-6-yl)acetonitrile (250 mg, yield 79%) as a yellow solid. MS (ESI) RT = 2.47 min, m / z 203.0 [M+H] + ,purity:80.78%@254nm,75.07%@214nm.

[0352] Step 3.2-(2-chloroquinoline-6-yl)acetic acid (GEN1-305-3)

[0353]

[0354] In a sealed tube, NaOH (79 mg, 1.98 mmol) was added to a mixture of 2-(2-chloroquinoline-6-yl)acetonitrile (100 mg, 0.5 mmol) in THF (1.6 mL) and H₂O (1.6 mL). The mixture was stirred at 80 °C for 18 hours. It was then diluted with H₂O (10 mL) and washed with EA (10 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(2-chloroquinoline-6-yl)acetic acid (100 mg, 92% yield) as a white solid. MS (ESI) RT = 2.18 min, m / z 222.0 [M+H] + ,purity:50.98%@254nm,14.63%@214nm.

[0355] Step 4.2-(2-Methoxyquinoline-6-yl)acetic acid (GEN1-305-4)

[0356]

[0357] 2-(2-chloroquinoline-6-yl)acetic acid (100 mg, 0.45 mmol) was added to a MeONa solution in MeOH (4 mL) and refluxed for 3 hours. The solution was then diluted with H₂O (10 mL) and washed with EA (10 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid (2 M) and extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give 2-(2-methoxyquinoline-6-yl)acetic acid (87 mg, 84% yield) as a white solid. MS (ESI) RT = 2.21 min, m / z 218.1 [M + H₂] + ,purity:78.77%:@254nm,85.78%@214nm.

[0358] Step 5.1 -(1-(2-(2-(2-methoxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-305)

[0359]

[0360] To a solution of 2-(2-methoxyquinoline-6-yl)acetic acid (87 mg, 0.40 mmol) in DMF (5 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (126 mg, 0.44 mmol), EDCI (92 mg, 0.48 mmol), HOBT (65 mg, 0.48 mmol), and DIEA (103 mg, 0.80 mmol) were added. The mixture was stirred at room temperature for 5 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(2-(2-methoxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (50 mg, yield 26%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.45(s,1H),8.20(d,J=8.8Hz,1H),7.73(d,J=8.0Hz,2H),7.55(d d,J=8.8,2.0Hz,1H),7.36(d,J=8.0Hz,1H),7.27(d,J=7.6Hz,1H),7.15(t,J=8.0Hz,1H),7. 00(d,J=8.8Hz,1H),4.63(d,J=11.2Hz,1H),4.30–4.14(m,2H),3.95(d,J=22.8Hz,5H),3.03 (t,J=13.2Hz,1H),2.55(d,J=10.8Hz,3H),1.72(d,J=10.8Hz,2H).MS(ESI)RT=2.91min,m / z 485.3[M+H] + ,purity:95.75%@254nm,99.64%@214nm.

[0361] Example 14

[0362] 1-(1-(2-(5-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-306)

[0363]

[0364]

[0365] Step 1. Synthesis of 5-chloro-6-methylquinoline (GEN1-306-1)

[0366]

[0367] H₂SO₄ (4.6 mL, 86.16 mmol) was added to a mixture of 3-chloro-4-methylaniline (3.05 g, 21.54 mmol), propane-1,2,3-triol (8.16 g, 88.6 mmol), FeSO₄·7H₂O (2.417 g, 8.695 mmol), and nitrobenzene (1.624 g, 13.19 mmol) at room temperature. The resulting mixture was stirred at 140 °C for 3 h. The reaction was quenched with ice water, and the pH was adjusted to alkaline with sodium bicarbonate solution. The resulting mixture was extracted with DCM (80 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated to give a brown solid. The crude product was purified by silica gel column chromatography (PE:EA = 4:1) to give the desired compound (1.55 g, yield 40.51%) as a yellow solid. MS(ESI)RT=2.73min,m / z 178.1[M+H] + ,purity:100%@254nm,63.19%,@214nm.

[0368] Step 2. 6-(bromomethyl)-5-chloroquinoline (GEN1-306-2)

[0369]

[0370] NBS (560.64 mg, 3.15 mmol) was added to a mixture of 5-chloro-6-methylquinoline (532.89 mg, 3 mmol), AIBN (49.26 mg, 0.3 mmol), and CCl4 (10 mL) under nitrogen protection at room temperature. The mixture was then stirred at 75 °C for 6 hours. The reaction mixture was quenched with water (50 mL). The mixture was extracted with DCM (40 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated to give a yellow solid. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (450 mg, yield 58.47%) as a white solid. MS (ESI) RT = 2.873 min, m / z 255.9 [M+H] + Purity: 97.68% @ 254nm.

[0371] Step 3. Synthesis of 2-(5-chloroquinoline-6-yl)acetonitrile (GEN1-306-3)

[0372]

[0373] Under nitrogen protection, TMS-CN (143.85 mg, 1.45 mmol) was added to a suspension of 6-(bromomethyl)-5-chloroquinoline (256.53 mg, 1 mmol), K₂CO₃ (158.94 mg, 1.15 mmol) in ACN (5 mL). The resulting mixture was stirred at 60 °C for 3 h. The mixture was filtered. The filtrate was concentrated and purified by silica gel column chromatography, eluting with (PE:EA = 3:1 to 1:1) to give the desired compound (122 mg, yield 60.2%) as a white solid. MS (ESI) RT = 2.210 min, m / z 203.0 [M+H] + Purity: 100% @ 254nm.

[0374] Step 4. Synthesis of 2-(5-chloroquinoline-6-yl)acetic acid (GEN1-306-4)

[0375]

[0376] To a solution of 2-(5-chloroquinoline-6-yl)acetonitrile (122 mg, 0.6 mmol) in THF (1 mL) and H₂O (1 mL), NaOH (96 mg, 2.4 mmol) was added. The resulting mixture was stirred at 80 °C for 6 h. The mixture was then stirred at 100 °C for 6 h. The solution was adjusted to pH 4 and concentrated. The reaction solution was purified by C18 column chromatography (ACN:H₂O = 0% to 50%) over 45 min to give the desired compound (30 mg, yield 22.56%) as a white solid. MS (ESI) RT = 1.810 min, m / z 222.0 [M+H] + ,purity:16.6%@254nm,31.8%@214nm.

[0377] Step 5. Synthesis of 1-(1-(2-(5-chloroquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-306)

[0378]

[0379] DIPEA (34.87 mg, 0.27 mmol) was added to a DMF (5 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (38.6 mg, 0.135 mmol), 2-(5-chloroquinoline-6-yl)acetic acid (30 mg, 0.135 mmol), EDCI (38.82 mg, 0.2025 mmol), and HOBT (27.36 mg, 0.2025 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with water (50 mL). The mixture was extracted with EA (30 mL x 3). The combined organic phases were washed with brine (20 mL x 2), dried over Na2SO4, and concentrated to give a brown solid. The mixture was then filtered. The filtrate was concentrated, and the crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN:H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (20 mg, yield 30.3%) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.57(s,1H),9.03(dd,J=4.2,1.6Hz,1H),8.66(dt,J=8.7,1.1Hz,1H) ,8.04(d,J=8.6Hz,1H),7.80(d,J=8.7Hz,1H),7.75(dd,J=8.6,4.2Hz,1H),7.42(dd,J=8.1,1. 2Hz,1H),7.35(dd,J=7.8,1.1Hz,1H),7.22(t,J=7.9Hz,1H),4.64(d,J=12.2Hz,1H),4.42–4.0 8(m,4H),3.27–3.12(m,1H),2.85–2.58(m,3H),1.91-1.68(m,2H).MS(ESI):Rt=2.687min; m / z 489.2[M+H] + purity:100%@254nm,99.82%@214nm.

[0380] Example 15

[0381] 1-(1-(2-(8-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-307)

[0382]

[0383] Step 1. Synthesis of 8-chloro-6-methylquinoline (GEN1-307-1)

[0384]

[0385] H₂SO₄ (4.6 mL, 86.16 mmol) was added to a mixture of 2-chloro-4-methylaniline (3.05 g, 21.54 mmol), propane-1,2,3-triol (8.16 g, 88.6 mmol), FeSO₄·7H₂O (2.417 g, 8.695 mmol), and nitrobenzene (1.624 g, 13.19 mmol) at room temperature. The mixture was then stirred at 140 °C for 3 h. The reaction mixture was quenched with ice water and the pH was adjusted to alkaline with sodium bicarbonate solution. The mixture was extracted with DCM (80 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated to give a brown solid. The crude product was purified by column chromatography on silica gel (PE:EA = 3:1) to give the desired compound (3.8 g, 99.3% yield) as a yellow solid. MS(ESI)RT=2.568min,m / z178.1[M+H] + Purity: 94.19% @ 254nm.

[0386] Step 2. 6-(bromomethyl)-8-chloroquinoline (GEN1-307-2)

[0387]

[0388] Under nitrogen protection, NBS (1.12 g, 6.3 mmol) was added to a mixture of 8-chloro-6-methylquinoline (1.066 g, 6 mmol) and AIBN (98.53 mg, 0.6 mmol) in CCl4 (30 mL). The resulting mixture was stirred at 75 °C for 6 hours. The reaction was concentrated and purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (600 mg, yield 38.98%) as a pale yellow solid. MS (ESI) RT = 2.697 min, m / z 255.9 [M+H] + Purity: 93.17% @ 254nm.

[0389] Step 3. Synthesis of 2-(8-chloroquinoline-6-yl)acetonitrile (GEN1-306-3)

[0390]

[0391] Under nitrogen protection at room temperature, TMS-CN (464.3 mg, 4.68 mmol) was added to a suspension of 6-(bromomethyl)-8-chloroquinoline (600 mg, 2.34 mmol), K₂CO₃ (371.9 mg, 2.69 mmol), and ACN (10 mL). The resulting mixture was stirred at 60 °C for 16 h. Then, K₂CO₃ (371.9 mg, 2.69 mmol) and TMS-CN (464.3 mg, 4.68 mmol) were added to the mixture. The resulting mixture was stirred at 60 °C for 6 h. The mixture was filtered. The filtrate was concentrated and purified by silica gel column chromatography, eluting with (PE:EA = 3:1 to 1:1) to give the desired compound (330 mg, yield 69.59%) as a pale yellow solid. MS (ESI) RT = 2.116 min, m / z 203.0 [M+H] + Purity: 96.59% @ 254nm.

[0392] Step 4. Synthesize 2-(8-chloroquinoline-6-yl)acetic acid (GEN1-307-4)

[0393]

[0394] To a solution of 2-(8-chloroquinoline-6-yl)acetonitrile (330 mg, 1.628 mmol) in THF (5 mL) and H₂O (5 mL), NaOH (260.48 mg, 6.512 mmol) was added. The resulting mixture was stirred at 80 °C for 48 hours. The solution was adjusted to pH 4 and concentrated. The crude residue was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H₂O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (190 mg, yield 52.65%) was obtained as a white solid. MS (ESI) RT = 1.811 min, m / z 222.0 [M+H] + ,purity:41.9%@254nm,33.25%@214nm.

[0395] Step 5. Synthesis of 1-(1-(2-(8-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-307)

[0396]

[0397] DIPEA (51.66 mg, 0.4 mmol) was added to a DMF (2 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (57.05 mg, 0.2 mmol), 2-(8-chloroquinoline-6-yl)acetic acid (44.32 mg, 0.2 mmol), EDCI (57.51 mg, 0.3 mmol), and HOBT (40.54 mg, 0.3 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with water (30 mL). The mixture was extracted with EA (20 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated to give a brown solid. The crude product was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN:H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (20 mg, yield 20.45%) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.51(s,1H),9.02(dd,J=4.2,1.7Hz,1H),8.45(dd,J=8.4,1.7Hz,1H) ,7.87(dd,J=17.5,1.8Hz,2H),7.67(dd,J=8.3,4.2Hz,1H),7.39(dd,J=8.2,1.2Hz,1H),7.31( dd,J=7.8,1.2Hz,1H),7.19(t,J=7.9Hz,1H),4.65(d,J=12.0Hz,1H),4.37-4.17(m,2H),4.04( s,2H),3.11(t,J=13.2Hz,1H),2.72–2.54(m,3H),1.84-1.67(m,2H).MS(ESI)RT=2.639min,m / z 489.2[M+H] + ,purity:100%@254nm,99.53%@214nm.

[0398] Example 16

[0399] 7-(trifluoromethyl)-1-(1-(2-(2-(8-(trifluoromethyl)quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-308)

[0400]

[0401] Step 1. 8-(trifluoromethyl)quinoline-6-carboxylic acid methyl ester (GEN1-308-1)

[0402]

[0403] To a mixture of 4-amino-3-(trifluoromethyl)benzoic acid (1.8 g, 8.78 mmol), nitrobenzene (648 mg, 5.27 mmol), glycerol (3.22 g, 35 mmol), and FeSO4·7H2O (972 mg, 3.49 mmol), concentrated H2SO4 (3.44 g, 35 mmol) was added dropwise. The mixture was stirred at 140 °C for 3 hours. The mixture was then cooled to 60 °C, and MeOH (3 mL) was added, followed by stirring at 60 °C overnight. The mixture was concentrated and diluted with water (10 mL), then the pH was adjusted to 8 with saturated sodium bicarbonate solution at 0 °C, and extracted with EA (100 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated to give a yellow liquid. The crude product was purified by reversed-phase rapid chromatography (C2SO4). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded methyl 8-(trifluoromethyl)quinoline-6-carboxylate (460 mg, yield 21%) as a yellow solid. MS (ESI) RT = 2.84 min, m / z 256.1 [M+H] + ,purity:59.60%@254nm,73.06%@214nm.

[0404] Step 2. (8-(trifluoromethyl)quinoline-6-yl)methanol (GEN1-308-2)

[0405]

[0406] At 0 °C, LiAlH4 (29 mg, 0.76 mmol) was added to a THF (6 mL) solution of methyl 8-(trifluoromethyl)quinoline-6-carboxylate (240 mg, 0.94 mmol). The mixture was stirred at room temperature for 2 h. Then, Na2SO4·10H2O was added at 0 °C until no bubbles were observed, and the mixture was filtered. The filtrate was concentrated to give crude product (8-(trifluoromethyl)quinoline-6-yl)methanol (228 mg) as a yellow liquid. MS (ESI) RT = 2.15 min, m / z 228.1 [M+H] + ,purity:90.52%@254nm,78.64%@214nm.

[0407] Step 3. 6-(bromomethyl)-8-(trifluoromethyl)quinoline (GEN1-308-3)

[0408]

[0409] (8-(trifluoromethyl)quinoline-6-yl)methanol (228 mg, 1.0 mmol) was dissolved in 45% HBr in HOAc (4 mL), and the solution was heated to 70 °C and stirred for 1 h. The crude product was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 6-(bromomethyl)-8-(trifluoromethyl)quinoline (189 mg, yield 65%) as a white solid. MS (ESI) RT = 2.99 min, m / z 290.0 [M+H] + ,purity:86.75%@254nm,83.95%@214nm.

[0410] Step 4.2 - (8-(trifluoromethyl)quinoline-6-yl)acetonitrile (GEN1-308-4)

[0411]

[0412] At 0 °C, K₂CO₃ (144 mg, 1.04 mmol) and TMSCN (97 mg, 0.98 mmol) were added to a solution of 6-(bromomethyl)-8-(trifluoromethyl)quinoline (189 mg, 0.74 mmol) in ACN (10 mL). The mixture was stirred overnight at 60 °C. The reaction mixture was poured into water (10 mL) and extracted with EA (20 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na₂SO₄ and concentrated to give a crude product, which was purified by reversed-phase rapid chromatography (C18 reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) to give 2-(8-(trifluoromethyl)quinoline-6-yl)acetonitrile (105 mg, yield 68%) as a white solid. MS(ESI)RT=2.50min,m / z 237.1[M+H] + ,purity:76.07%@254nm,16.36%@214nm.

[0413] Step 5.2 - (8-(trifluoromethyl)quinoline-6-yl)acetic acid (GEN1-308-5)

[0414]

[0415] To a mixture of 2-(8-(trifluoromethyl)quinoline-6-yl)acetonitrile (105 mg, 0.44 mmol) in THF (1.6 mL) and H₂O (1.6 mL), NaOH (71 mg, 1.78 mmol) was added. The mixture was stirred at 80 °C for 18 hours, then diluted with H₂O (20 mL) and washed with EA (20 mL). The aqueous phase was adjusted to pH 4 with hydrochloric acid aqueous solution (2 M) and extracted with EA (20 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated to give crude 2-(8-(trifluoromethyl)quinoline-6-yl)acetic acid (118 mg) as a white solid. MS (ESI) RT = 2.22 min, m / z 256.1 [M + H₂] + ,purity:74.63%@254nm,76.86%@214nm.

[0416] Step 6.7-(trifluoromethyl)-1-(1-(2-(2-(8-(trifluoromethyl)quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-308)

[0417]

[0418] To a solution of 2-(8-(trifluoromethyl)quinolin-6-yl)acetic acid (50 mg, 0.2 mmol) in DMF (3 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (60 mg, 0.21 mmol), EDCI (45 mg, 0.23 mmol), HOBT (35 mg, 0.25 mmol), and DIEA (50 mg, 0.39 mmol) were added. The mixture was stirred at room temperature for 3 h. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 7-(trifluoromethyl)-1-(1-(2-(8-(trifluoromethyl)quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (46 mg, 45% yield) as a white solid. 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),9.02(dd,J=4.0,1.6Hz,1H),8.49(dd,J=8.4,2.0Hz,1H), 8.22–8.02(m,2H),7.68(dd,J=8.4,4.0Hz,1H),7.37(dd,J=8.0,1.2Hz,1H),7.28(d,J=7.6Hz,1 H),7.16(t,J=8.0Hz,1H),4.62(d,J=12.4Hz,1H),4.27(q,J=13.2,12.4Hz,2H),4.10(s,2H),3. 18–3.02(m,1H),2.78–2.54(m,3H),1.76(d,J=12.0Hz,2H).MS(ESI)RT=2.90min,m / z523.2[M+H] + ,purity:100%@254nm,99.76%@214nm.

[0419] Example 17

[0420] 1-(1-(2-(4-(4-hydroxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-309)

[0421]

[0422] Step 1.6-(4,4,5,5-Tetramethyl-1,3,2-dioxaboran-2-yl)quinoline-4-ol (GEN1-309-1)

[0423]

[0424] To a solution of 6-bromoquinoline-4-ol (3 g, 13.4 mmol) in 1,4-dioxane (30 mL), KOAc (3.9 g, 39.8 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborane) (5.1 g, 20.1 mmol), and Pd(dppf)Cl2.CH2Cl2 (1.095 g, 1.34 mmol) were added. The mixture was stirred at 100 °C for 2 hours. The reaction mixture was filtered, and the filter cake was dissolved in EA (300 mL), then filtered again, and the filtrate was concentrated to give 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-acyl)quinoline-4-ol (1.5 g, 41% yield) as a yellow solid. MS(ESI)RT=2.16min,m / z271.9[M+H] +,purity:88.43%@254nm,92.31%@214nm.

[0425] Step 2.2 - (4-hydroxyquinoline-6-yl)ethyl acetate (GEN1-309-2)

[0426]

[0427] To a solution of Pd(OAc)₂ (13 mg, 0.06 mmol) in THF (5 mL), K₃PO₄ (635 mg, 2.99 mmol), 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborborane-2-yl)quinoline-4-ol (243 mg, 0.9 mmol), ethyl 2-bromoacetate (100 mg, 0.6 mmol), and tris(1-naphthyl)phosphine (74 mg, 0.18 mmol) were added. The mixture was stirred overnight at 73 °C. Four identical reactions were carried out simultaneously. The four reactions were mixed and filtered, and the filtrate was concentrated to give the crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded ethyl 2-(4-hydroxyquinoline-6-yl)acetate (80 mg, yield 14%) as a white solid. MS (ESI) RT = 1.85 min, m / z 231.9 [M+H] + ,purity:17.58%@254nm,48.70%@214nm.

[0428] Step 3.2-(4-hydroxyquinoline-6-yl)acetic acid (GEN1-309-3)

[0429]

[0430] LiOH (40 mg, 1.67 mmol) was added to a mixture of ethyl 2-(4-hydroxyquinoline-6-yl)acetate (80 mg, 0.35 mmol) in MeOH (8 mL) and H₂O (2 mL). The mixture was stirred at room temperature for 1 h. The crude product was purified by reversed-phase rapid chromatography (C₂O₃). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 35% gradient; 30 min; detection, UV 214 nm) yielded 2-(4-hydroxyquinoline-6-yl)acetic acid (130 mg crude) as a white solid. MS (ESI) RT = 1.08 min, m / z 203.9 [M+H] + ,purity:17.35%@254nm,55.18%@214nm.

[0431] Step 4.1-(1-(2-(4-(4-hydroxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-309)

[0432]

[0433] To a solution of 100 mg crude 2-(4-hydroxyquinoline-6-yl)acetic acid in DMF (6 mL), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (93 mg, 0.33 mmol), EDCI (68 mg, 0.35 mmol), HOBT (48 mg, 0.35 mmol), and DIEA (77 mg, 0.60 mmol) were added. The mixture was stirred overnight at room temperature. It was then diluted with water (20 mL) and filtered to give a white solid crude product, which was purified by reversed-phase rapid chromatography (C1). 18 Reversed-phase silica column; mobile phase ACN, water, 10% to 85% gradient; 30 min; detection, UV 214 nm) yielded 1-(1-(2-(4-(4-hydroxyquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (60 mg, yield 26%) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ11.50(s,1H),8.18(dd,J=8.0,1.6Hz,1H),7.88(d,J=7.6Hz,1H),7.77-7.65(m,1H),7 .43(d,J=8.8Hz,1H),7.42-7.34(m,2H),7.30(d,J=7.6Hz,1H),7.18(t,J=8.0Hz,1H),6.07(d,J=7.6Hz,1H),5 .58-5.14(m,2H),4.49(d,J=12.4Hz,1H),4.40-4.28(m,1H),4.16(d,J=13.2Hz,1H),3.21(t,J=13.2Hz,1H),2 .94-2.74(m,1H),2.73-2.53(m,2H),1.86(d,J=12.4Hz,1H),1.76(d,J=12.0Hz,1H).MS(ESI)RT=2.32min,m / z 471.2[M+H] + ,purity:0%@254nm,99.77%@214nm.

[0434] Example 18

[0435] 6-(2-oxo-2-(4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)quinoline-8-nitrile (GEN1-310)

[0436]

[0437]

[0438] Step 1. Synthesis of 8-bromo-6-methylquinoline (GEN1-310-1)

[0439]

[0440] H₂SO₄ (4.6 mL, 86.16 mmol) was added to a mixture of 2-bromo-4-methylaniline (4.007 g, 21.54 mmol), propane-1,2,3-triol (8.16 g, 88.6 mmol), FeSO₄·7H₂O (2.417 g, 8.695 mmol), and nitrobenzene (1.624 g, 13.19 mmol) at room temperature. The mixture was then stirred at 128 °C for 3 h. The reaction mixture was quenched with ice water and the pH was adjusted to alkaline with sodium bicarbonate solution. The mixture was extracted with DCM (80 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated to give a brown solid. The crude product was purified by column chromatography on silica gel (PE:EA = 3:1) to give the desired compound (350 mg, yield 7.3%) as a yellow solid. MS(ESI)RT=2.704min,m / z222.0[M+H] + Purity: 94.38% @ 254nm.

[0441] Step 2. 8-Bromo-6-(Bromomethyl)quinoline (GEN1-310-2)

[0442]

[0443] Under nitrogen protection, NBS (294.5 mg, 1.655 mmol) was added to a CCl4 (10 mL) solution of 8-bromo-6-methylquinoline (350 mg, 1.576 mmol), AIBN (25.88 mg, 0.1576 mmol), and the resulting mixture was stirred at 70 °C for 8 h. The mixture was concentrated and purified by silica gel column chromatography, eluting with (PE:EA = 3:1) to give the desired compound (250 mg, yield 52.7%) as a white solid. MS (ESI) RT = 2.794 min, m / z 301.9 [M+H] +Purity: 88.77% @ 254nm.

[0444] Step 3. Synthesis of 2-(8-bromoquinoline-6-yl)acetonitrile (GEN1-310-3)

[0445]

[0446] TMS-CN (164.69 mg, 1.66 mmol) was added to a suspension of 8-bromo-6-(bromomethyl)quinoline (250 mg, 0.83 mmol), K₂CO₃ (131.92 mg, 0.955 mmol) in acetonitrile (5 mL) at room temperature and under nitrogen protection. The resulting mixture was stirred at 60 °C for 16 h. The mixture was filtered. The filtrate was concentrated and purified by silica gel column chromatography, eluting with PE:EA = 3:1 to 1:1, to give the desired compound (135 mg, yield 65.8%) as a pale yellow solid. MS (ESI) RT = 2.217 min, m / z 247.0 [M+H] + Purity: 67.39% @ 214nm.

[0447] Step 4. Synthesis of 2-(8-bromoquinoline-6-yl)acetic acid (GEN1-310-4)

[0448]

[0449] To a solution of 2-(8-bromoquinoline-6-yl)acetonitrile (135 mg, 0.546 mmol) in THF (10 mL) and H₂O (30 mL), NaOH (87.36 mg, 2.184 mmol) was added. The resulting mixture was stirred at 80 °C for 16 h. The solution was adjusted to pH 4 and concentrated. The residue was purified by reversed-phase chromatography using a crude column. Purification conditions: C18 silica gel column; mobile phase ACN: H₂O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (110 mg, yield 75.71%) was obtained as a white solid. MS (ESI) RT = 1.913 min, m / z 266.0 [M+H] + Purity: 93.87% @ 254nm.

[0450] Step 5. Synthesize 2-(8-cyanoquinoline-6-yl)acetic acid (GEN1-310-5)

[0451]

[0452] Pd(PPh3)4 (71.43 mg, 0.062 mmol) was added to an anhydrous DMF (5 mL) solution of 2-(8-bromoquinoline-6-yl)acetic acid (110 mg, 0.413 mmol) and Zn(CN)2 (48.49 mg, 0.413 mmol) at room temperature and under nitrogen protection. The resulting mixture was stirred at 90 °C for 16 h. The reaction solution was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (100 mg, 50% purity) was obtained as a white solid. MS (ESI) RT = 1.759 min, m / z 212.9 [M+H] + Purity: 54.22% @ 254nm.

[0453] Step 6. Synthesis of 6-(2-oxo-2-(4-(2-oxo-7-(trifluoromethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)quinoline-8-onitrile (GEN1-310)

[0454]

[0455] DIPEA (206.6 mg, 1.6 mmol) was added to a DMF (5 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (150 mg, crude, 0.4 mmol), 2-(8-bromoquinoline-6-yl)acetic acid (84.9 mg, 0.4 mmol), EDCI (115 mg, 0.6 mmol), and HOBT (81 mg, 0.6 mmol). The resulting mixture was stirred at room temperature for 24 h. The reaction solution was purified by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN: H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired product (30 mg, yield 15.64%) was obtained as a white solid. 1H NMR (400MHz, DMSO-d6) δ11.49(s,1H),9.08(dd,J=4.3,1.7Hz,1H),8.54(dd,J=8.4,1.6Hz,1H),8 .29(d,J=1.9Hz,1H),8.21(d,J=1.9Hz,1H),7.74(dd,J=8.3,4.2Hz,1H),7.39(dd,J=8.1,1.3Hz, 1H),7.30(d,J=7.6Hz,1H),7.19(t,J=7.9Hz,1H),4.64(d,J=12.2Hz,1H),4.35–4.22(m,2H),4.1 0(s,2H),3.13(t,J=13.0Hz,1H),2.71-2.54(m,3H),1.86-1.70(m,2H).MS(ESI)RT=2.597min,m / z 480.2[M+H] + Purity: 100% @ 254nm.

[0456] Example 19

[0457] 1-(1-(2-(7-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-311)

[0458]

[0459] Step 1.2-(7-methylquinoline-6-yl)tert-butyl acetate (GEN1-311-1)

[0460]

[0461] Under nitrogen protection, TMCSC1 (16 mg, 0.14 mmol) was added to a solution of Zn (192 mg, 3.00 mmol) in THF (0.8 mL). The resulting mixture was stirred at room temperature for 20 minutes. A solution of 2-bromoacetic acid tert-butyl ester (290 mg, 1.50 mmol) in THF (2.0 mL) was added dropwise to the solution. The resulting mixture was stirred at 45 °C for 20 minutes. 6-Bromo-7-methylquinoline (100 mg, 0.46 mmol) and Pd(PPh3)4 (208 mg, 0.18 mmol) were added to the solution. The mixture was reacted in a microwave reactor at 120 °C for 30 minutes (repeated 3 times). The reaction mixture was purified by reversed-phase rapid chromatography (C10). 18Reverse silica column chromatography; mobile phase CH3CN, water, 0% to 70% gradient, 50 min; detection, UV 214 nm) yielded 2-(7-methylquinoline-6-yl) tert-butyl acetate (57 mg, yield 48%) as a yellow solid. MS (ESI) RT = 2.932 min, m / z 258.1 [M+H] + ,purity:64%@254nm,82%@214nm.

[0462] Step 2.2 - Potassium (7-methylquinoline-6-yl)acetate (GEN1-311-2)

[0463]

[0464] To a solution of 2-(7-methylquinoline-6-yl)-tert-butyl acetate (55 mg, 0.21 mmol) in EtOH (1.0 mL), KOH (18 mg, 0.32 mmol) and water (1.0 mL) were added. The resulting mixture was stirred at 85 °C for 5 hours. The mixture was concentrated to obtain a crude product (78 mg), which could be used directly in the next step without further purification. MS (ESI) RT = 1.070 min, m / z 202.1 [M+H] + Purity: 89% @ 214nm.

[0465] Step 3.1 -(1-(2-(7-methylquinolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-311)

[0466]

[0467] DIEA (96 mg, 0.74 mmol) was added dropwise to a solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (71 mg, 0.25 mmol), 2-(7-methylquinoline-6-yl)acetyl ester (78 mg crude), EDCI (72 mg, 0.38 mmol), and HOBT (50 mg, 0.37 mmol) in DMF (2.0 mL). The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was added dropwise to water (20 mL). The precipitated solid was collected by filtration. The crude product was purified by slurrying with MeOH and THF to give 1-(1-(2-(7-methylquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (66 mg, 57% yield) as a white solid. 1H NMR(400MHz, DMSO-d6)δ:11.49(s,1H),8.82(s,1H),8.29(d,J=8.1Hz,1H),7.82(s,1H),7.68(s,1H),7.49–7.09(m,4H),4.65(s,1H) ),4.40–4.10(m,2H),4.08–3.83(m,2H),3.19–3.05(m,1H),2.79–2.54(m,3H),2.43(s,3H),1.75(s,2H).MS(ESI)RT=2.607min,m / z 469.2[M+H] + ,purity:100%@254nm,97%@214nm.

[0468] Example 20

[0469] 7-(trifluoromethyl)-1-(1-(2-(3-(3-(trifluoromethyl)quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-312)

[0470]

[0471] Step 1.3 - Iodoquinoline-6-carboxylic acid methyl ester (GEN1-312-1)

[0472]

[0473] Under nitrogen protection, NIS (1.68 g, 7.49 mmol) was added to a solution of 6-carboxylated quinoline methyl ester (1.0 g, 5.35 mmol) in AcOH (9.0 mL). The resulting mixture was stirred at 100 °C for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with (EA / (PE+EA) = 20%), to give 3-iodoquinoline-6-carboxylate methyl ester (800 mg, yield 48%) as a pale yellow oil. 1 H NMR(400MHz,Chloroform-d)δ:9.10(d,J=2.1Hz,1H),8.62(d,J=2.1Hz,1H),8.45(d,J=1.8Hz, 1H),8.30(dd,J=8.8,1.9Hz,1H),8.09(d,J=8.8Hz,1H),3.99(s,3H).MS(ESI)RT=2.953min,m / z 313.9[M+H] + ,purity:96%@254nm,89%@214nm.

[0474] Step 2.3-(trifluoromethyl)quinoline-6-carboxylic acid methyl ester (GEN1-312-2)

[0475]

[0476] Under argon protection, methyl 2-chloro-2,2-difluoroacetate (736 mg, 5.11 mmol), KF (148 mg, 2.55 mmol), and CuI (485 mg, 2.55 mmol) were added to a DMF (7.0 mL) solution of methyl 3-iodoquinoline-6-carboxylate (400 mg, 1.28 mmol). The resulting mixture was stirred at 120 °C for 16 hours under argon protection, cooled, and filtered. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography, eluting with (EA / (PE+EA) = 10%), to give methyl 3-(trifluoromethyl)quinoline-6-carboxylate (60 mg, yield 18%) as a pale yellow oil. 1 H NMR(400MHz, DMSO-d6)δ:9.32(d,J=2.3Hz,1H),9.19(d,J=2.4Hz,1H),8.92(d,J=1.9Hz,1H) ,8.39(dd,J=8.9,2.0Hz,1H),8.25(d,J=8.8Hz,1H),3.96(s,3H).MS(ESI)RT=2.901min,m / z 256.0[M+H] + ,purity:87%@254nm,92%@214nm.

[0477] Step 3.3-(trifluoromethyl)quinoline-6-carboxylic acid (GEN1-312-3)

[0478]

[0479] To a methanol (2.0 mL) solution of methyl 3-(trifluoromethyl)quinoline-6-carboxylic acid (55 mg, 0.21 mmol), KOH (15 mg, 0.27 mmol) and water (0.2 mL) were added. The resulting mixture was stirred at room temperature for 2 hours under nitrogen protection. The reaction mixture was purified by reversed-phase rapid chromatography (C1). 18 Reverse silica gel column chromatography; mobile phase CH3CN, water (0.1% formic acid), 0% to 70% gradient, 50 min; detection, UV 214 nm) yielded the desired compound (47 mg, 90% yield) as a white solid. MS (ESI) RT = 2.387 min, m / z 344.0 [M+H] + ,purity:99%@254nm,96%@214nm.

[0480] Step 4.2 - Diazo-1-(3-(trifluoromethyl)quinolin-6-yl)acet-1-one (GEN1-312-4)

[0481]

[0482] Under nitrogen protection, 3-(trifluoromethyl)quinoline-6-carboxylic acid (47 mg, 0.20 mmol) was dissolved in SOCl2 (2.0 mL) and stirred at 80 °C for 5 hours, then cooled and concentrated. The resulting residue was dissolved in THF / MeCN (1.0 / 1.0 mL), and a THF / MeCN (2.0 / 2.0 mL) solution of TMSCHN2 (2.0 M hexane solution, 0.24 mL) was added at 0 °C. The mixture was stirred for 10 minutes and then concentrated. The crude product was purified by silica gel column chromatography, eluting with (EA / (PE+EA) = 20%) to give the desired product (41.3 mg, yield 80%) as a pale yellow solid. MS (ESI) RT = 2.599 min, m / z 266.0 [M+H] + ,purity:100%@254nm,92%@214nm.

[0483] Step 5.2 - Ethyl (3-(trifluoromethyl)quinoline-6-yl)acetate (GEN1-312-5)

[0484]

[0485] Under nitrogen protection, silver benzoate (35 mg, 0.15 mmol) was added to an ethanol (4.0 mL) solution of 2-diazo-1-(3-(trifluoromethyl)quinolin-6-yl)ethyl-1-one (31 mg, 0.12 mmol). The resulting mixture was stirred at 50 °C for 3 hours under nitrogen protection. The reaction mixture was filtered and the filtrate was concentrated. The crude product was purified by silica gel column chromatography, eluting with (EA / (PE+EA) = 8%) to give the desired product (30 mg, yield 91%) as a white solid. MS (ESI) RT = 2.935 min, m / z 284.1 [M+H] + ,purity:63%@254nm,90%@214nm.

[0486] Step 6.2 - Potassium (3-(trifluoromethyl)quinoline-6-yl)acetate (GEN1-312-6)

[0487]

[0488] To a solution of 2-(3-(trifluoromethyl)quinoline-6-yl)ethyl acetate (30 mg, 0.11 mmol) in ethanol (2.0 mL), KOH (7.1 mg, 0.13 mmol) and water (0.2 mL) were added. The resulting mixture was stirred at room temperature for 2 hours under nitrogen protection. The mixture was then concentrated under vacuum to obtain a crude product (40 mg), which could be used directly in the next step without further purification.

[0489] Step 7.7-(trifluoromethyl)-1-(1-(2-(3-(3-(trifluoromethyl)quinolin-6-yl)acetyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-312)

[0490]

[0491] DIEA (27 mg, 0.21 mmol) was added dropwise to a solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (36 mg, 0.13 mmol), 2-(3-(trifluoromethyl)potassium 6-quinoline acetate quinoline (40 mg), EDCI (30 mg, 0.16 mmol), and HOBT (21 mg, 0.16 mmol) in DMF (1.0 mL). The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by reversed-phase rapid chromatography (C1). 18 Reverse silica column chromatography; mobile phase CH3CN, water, 0% to 70% gradient, 50 min; detection, UV 214 nm) yielded the desired compound (16 mg, 30% yield) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ:11.44(s,1H),9.15(d,J=2.3Hz,1H),8.90(s,1H),8.11(d,J=8.7H z,1H),8.03(d,J=1.9Hz,1H),7.86(dd,J=8.7,1.9Hz,1H),7.36(d,J=8.0Hz,1H),7.28(d,J =7.7Hz,1H),7.17(d,J=7.9Hz,1H),4.63(d,J=11.8Hz,1H),4.35–4.16(m,2H),4.13–3.98( m,2H),3.15–3.02(m,1H),2.69–2.50(m,3H),1.80–1.65(m,2H).MS(ESI)RT=2.927min,m / z 523.1[M+H] + ,purity:100%@254nm,99.5%@214nm.

[0492] Example 21

[0493] 1-(1-(2-(7-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-313)

[0494]

[0495]

[0496] Step 1. Synthesis of 7-chloro-6-methylquinoline (GEN1-313-1)

[0497]

[0498] H₂SO₄ (4.6 mL, 86.16 mmol) was added to a mixture of 3-chloro-4-methylaniline (3.05 g, 21.54 mmol), propane-1,2,3-triol (8.16 g, 88.6 mmol), FeSO₄·7H₂O (2.417 g, 8.695 mmol), and nitrobenzene (1.624 g, 13.19 mmol) at room temperature. The resulting mixture was stirred at 140 °C for 3 h. The reaction was then quenched with ice water, and the pH was adjusted to alkaline with sodium bicarbonate solution. The mixture was extracted with DCM (80 mL × 3). The combined organic phases were washed with brine (20 mL × 2), dried over Na₂SO₄, and concentrated to give a brown solid. The crude product was purified by column chromatography on silica gel (PE:EA = 4:1) to give the desired compound (1.8 g, yield 47.04%) as a yellow solid. MS(ESI)RT=2.49min,m / z 178.1[M+H] + Purity: 42.61% @ 214nm.

[0499] Step 2. 6-(bromomethyl)-7-chloroquinoline (GEN1-313-2)

[0500]

[0501] Under nitrogen protection, NBS (1.894 g, 10.64 mmol) was added to a mixture of 7-chloro-6-methylquinoline (1.8 g, 10.13 mmol), AIBN (1.894 g, 10.64 mmol), and CCl4 (50 mL). The resulting mixture was stirred at 75 °C for 6 hours. The reaction was concentrated and purified by column chromatography on silica gel (PE:EA = 3:1) to give the desired compound (1.6 g, yield 61.57%) as a yellow solid. MS (ESI) RT = 2.748 min, m / z 255.9 [M+H] +Purity: 92.51% @ 254nm.

[0502] Step 3. Synthesis of 2-(7-chloroquinoline-6-yl)acetonitrile (GEN1-313-3)

[0503]

[0504] 6-(bromomethyl)-7-chloroquinoline (600 mg, 2.34 mmol), K₂CO₃ (743.57 mg, 5.38 mmol), ACN (20 mL), and TMSCN (928.6 mg, 9.36 mmol) were added to a microwave tube at room temperature under nitrogen protection. The resulting mixture was stirred in a sealed container at 75 °C for 48 hours. The mixture was filtered. The filtrate was concentrated and purified by silica gel column chromatography (PE:EA = 3:1 to 1:1) to give the desired compound (145 mg, yield 30.58%) as a pale yellow solid. MS (ESI) RT = 2.146 min, m / z 203.1 [M+H] + Purity: 90.74% @ 254nm.

[0505] Step 4. Synthesize 2-(7-chloroquinoline-6-yl)acetic acid (GEN1-313-4)

[0506]

[0507] To a mixture of 2-(7-chloroquinoline-6-yl)acetonitrile (145 mg, 0.715 mmol) in THF (8 mL) and H₂O (8 mL), NaOH (114.49 mg, 2.862 mmol) was added. The resulting mixture was stirred at 80 °C for 16 h. The solution was adjusted to pH 4 and concentrated. The residue was purified by C18 column chromatography (ACN:H₂O = 0% to 50%) over 45 min to give the desired product (98 mg, yield 61.84%) as a white solid. MS (ESI) RT = 1.648 min, m / z 222.0 [M+H] + ,purity:55.48%@254nm.78.1%@214nm.

[0508] Step 5. Synthesis of 1-(1-(2-(7-chloroquinoline-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-313)

[0509]

[0510] DIPEA (114.43 mg, 0.886 mmol) was added to a DMF (5 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (126.37 mg, 0.443 mmol), 2-(7-chloroquinoline-6-yl)acetic acid (98 mg, 0.443 mmol), EDCI (127.48 mg, 0.665 mmol), and HOBT (89.86 mg, 0.665 mmol). The resulting mixture was stirred at room temperature for 24 h. The reaction solution was purified by silica gel column chromatography (EA: MeOH, 0% to 10%) to give the desired compound (20 mg, yield 9.23%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ11.48(s,1H),9.04-8.86(m,1H),8.39(d,J=8.3Hz,1H) ,8.24-7.90(m,2H),7.68-7.52(m,1H),7.33(dd,J=32.8,7.9Hz,3H),7.17(t,J =7.8Hz,1H),4.88-4.50(m,1H),4.39–4.20(m,2H),4.17–3.92(m,2H),3.16(t, J=13.2Hz,1H),2.88–2.53(m,3H),1.85-1.65(m,2H).MS(ESI)RT=2.636min,m / z 489.1[M+H] + ,purity:100%@254nm.95.78%@214nm.

[0511] Example 22

[0512] 1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one

[0513]

[0514] Step 1. Synthesis of 1-(1-(2-(quinolin-6-yl)acetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (GEN1-075)

[0515]

[0516] DIEA (387 mg, 2.99 mmol) was added to a DMF (4 mL) solution of 2-(6-quinolinyl)acetic acid (187 mg, 0.999 mmol), 3-(4-piperidinyl)-1H-benzimidazole-2-one (217 mg, 0.999 mmol), and HATU (494 mg, 1.30 mmol). After stirring at 25 °C for 6 hours, the mixture was purified by C18 column chromatography (CH3CN:water = 5% to 50%) to give the desired compound (100 mg, yield 25%) as a white solid. 1 H NMR (400MHz, CDCl3): δ9.54(s,1H),8.91(s,1H),8.15(t,J=8.4Hz,2H),7.78(s,1H),7.71(d,J=8.4Hz,1H) ,7.44-7.40(m,1H),7.05-6.98(m,2H),6.89(t,J=7.6Hz,1H),6.76(d,J=8.0Hz,1H),4.93(d,J=13.2Hz,1H) ,4.57-4.46(m,1H),4.13(d,J=13.6Hz,1H),4.02(s,2H),3.20(t,J=12.4Hz,1H),2.73(t,J=14.8Hz,1H),2 .33-2.23(m,1H),2.03-1.93(m,1H),1.89(d,J=12.8Hz,1H),1.78-1.73(m,1H).MS(ESI):Rt=2.639min,m / z 387.2,[M+H] + Purity: 99.84% @ 214nm, 96.55% @ 254nm.

[0517] Example 23

[0518] 3-[1-[2-(1H-indol-5-yl)acetyl]-4-piperidinyl]-1H-benzimidazol-2-one

[0519]

[0520]

[0521] Step 1. Synthesis of methyl 2-(4-amino-3-bromophenyl)acetate

[0522]

[0523] A solution of 1-bromopyrrolidine-2,5-dione (3.23 g, 18.2 mmol) in acetonitrile (10 mL) was added dropwise to a solution of methyl 2-(4-aminophenyl)acetate (3.00 g, 18.2 mmol) in acetonitrile (10 mL). The mixture was stirred overnight at room temperature. The resulting mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (petroleum ether:EtOAc = 5:1) to give the desired compound (4.15 g, 94% yield) as a pale yellow oil. MS (ESI) Rt = 1.43 min, m / z 245.0 [M+H] + Purity: 100% @ 254nm, 100% @ 214nm.

[0524] Step 2. Synthesis of methyl 2-(4-amino-3-((trimethylsilyl)ethynyl)phenyl)acetate

[0525]

[0526] Pd(PPh3)2Cl2 (575 mg, 0.819 mmol) was added to a solution of methyl 2-(4-amino-3-bromophenyl)acetate (2.00 g, 8.19 mmol), ethynyltrimethylsilane (8.05 g, 81.9 mmol), and DMAP (100 mg, 0.819 mmol) in Et3N (10 mL). The mixture was stirred overnight at 70 °C under nitrogen. The resulting mixture was concentrated and water (50 mL) was added. The desired compound was extracted by adding DCM (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:EtOAc = 20:1) to give the desired compound (1.16 g, yield 54%) as a yellow oil. MS(ESI)Rt=1.71min,m / z 262.2[M+H] + Purity: 100% @ 254nm, 100% @ 214nm.

[0527] Step 3.2 Synthesis of methyl (1H-indol-5-yl)acetate

[0528]

[0529] CuI (85 mg, 0.446 mmol) was added to a mixture of methyl 2-(4-amino-3-(((trimethylsilyl)ethynyl)phenyl)acetate (1.16 g, 4.44 mmol) and DMF (10 mL). The mixture was stirred at 90 °C for 24 hours under nitrogen. The resulting mixture was cooled to room temperature and poured into water (30 mL). DCM (50 mL × 3) was added to extract the desired compound. The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by RP-HPLC (45–65% B; A: H₂O B: CH₃CN) to give the desired compound (393 mg, 47% yield) as a yellow oil. MS (ESI) Rt = 1.43 min, m / z 188.2 [MH] - Purity: 100% @ 254nm, 100% @ 214nm.

[0530] Step 4.2 Synthesis of (1H-indole-5-yl)acetic acid

[0531]

[0532] To a solution of methyl 2-(1H-indol-5-yl)acetate (393 mg, 2.08 mmol) in THF (6 mL) and H₂O (3 mL), NaOH (166 mg, 4.15 mmol) was added. The mixture was stirred overnight at 50 °C. The resulting mixture was concentrated, and water (15 mL) was added. The mixture was acidified to pH 1 with an aqueous HCl solution (12 M), and DCM (50 mL × 3) was added to extract the desired compound. The combined organic solutions were washed with brine (50 mL), dried over anhydrous Na₂SO₄, and concentrated to give the desired compound (341 mg, 94% yield) as a light yellow solid. MS (ESI) Rt = 1.142 min, m / z 176.0 [M + H] + Purity: 100% @ 214nm.

[0533] Step 5. Synthesis of 3-[1-[2-(1H-indol-5-yl)acetyl]-4-piperidinyl]-1H-benzimidazol-2-one (GEN1-077)

[0534]

[0535] Add 2-(1H-indol-5-yl)acetic acid (100 mg, 0.571 mmol) to a DMF (3 mL) solution of 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 one 2,2,2-trifluoroacetate (254 mg, 0.767 mmol), EDCI (164 mg, 0.856 mmol), HOBT (116 mg, 0.859 mmol), and DIEA (369 mg, 2.86 mmol). Stir the mixture overnight at room temperature. Pour the resulting mixture into water (20 mL) and filter. Wash the precipitate with water (10 mL) and recrystallize from MeOH. Then completely dissolve the precipitate in DMSO and add it dropwise to hot water (2 mL). Filter the mixture and dry the residue to give the desired compound (35 mg, 16% yield) as a white solid. 1 HNMR (400MHz, DMSO-d6): δ11.02(s,1H),10.79(s,1H),7.48(s,1H),7.37(d,J=8.4Hz,1H),7.31(s,1H),7 .05(d,J=8.0Hz,1H),6.92-6.89(m,2H),6.85-6.81(m,1H),6.75-6.73(m,1H),6.41(s,1H),4.60(d,J=12. 8Hz,1H),4.40-4.34(m,1H),4.15-4.09(m,1H),3.89-3.75(m,2H),3.14-3.08(m,1H),2.66(t,J=12.0Hz,1 H),2.02-1.93(m,1H),1.78-1.70(m,1H),1.65-1.63(m,1H),1.50-1.47(m,1H).MS(ESI)Rt=3.835min,m / z 375.2[M+H] + Purity: 97.76%@254nm, 99.07%@214nm;

[0536] Example 24

[0537] 1-(1-(2-(4-(dimethylamino)phenyl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-118)

[0538]

[0539] Step 1.1-(1-(2-(4-(dimethylamino)phenyl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-118)

[0540]

[0541] Compounds 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (71 mg, 0.25 mmol), HATU (140 mg, 0.37 mmol), and DIEA (129 mg, 1.00 mmol) were dissolved in DMF (1.5 mL), and 2-(4-(dimethylamino)phenyl)acetic acid (46 mg, 0.25 mmol) was added. After the addition was complete, the resulting mixture was stirred at room temperature for 5 hours. The reaction mixture was added dropwise to water (50 mL), and filtered to give a white solid. The crude product was purified by Prep-TLC (MeOH / (MeOH+DCM) = 60%) to give the desired compound (21.1 mg, yield 19%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ11.45(s,1H),7.36(d,J=8.2,1H),7.27(d,J=7.8Hz,1H) ,7.16(t,J=7.9Hz,1H),7.06(d,J=8.6Hz,2H),6.67(d,J=8.4Hz,2H),4.67–4.54(m ,1H),4.29–4.07(m,2H),3.74–3.49(m,2H),2.93(t,J=13.1Hz,1H),2.86(s,6H),2 .56–2.51(m,1H),2.49–2.33(m,2H),1.75–1.59(m,2H).MS(ESI):Rt=1.62min,m / z 446.9 [M+H] + Purity: 100% @ 254nm, 100% @ 214nm.

[0542] Example 25

[0543] 1-(1-(2-(quinoxolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-140)

[0544]

[0545] Step 1.2-(Quinoroline-6-yl)potassium acetate (GEN1-140-1)

[0546]

[0547] A solution of 28 mg KOH in 0.50 mmol of water (0.2 mL) was added to a solution of methyl 2-(quinoxalo-6-yl)acetate (100 mg, 0.50 mmol) in MeOH (2 mL). The resulting mixture was stirred at room temperature for 5 hours. The mixture was then concentrated. The crude product was used directly in the next step without further purification. TLC: MeOH / (DCM+MeOH) = 5%, Rf = 0.6

[0548] Step 2.1-(1-(2-(quinoxolin-6-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-140)

[0549]

[0550] DIEA (125 mg, 0.98 mmol) was added to a solution of potassium 2-(quinoxalo-6-yl)acetate (102 mg crude product), 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (140 mg, 0.49 mmol), and HATU (280 mg, 0.74 mmol) in DMF (2.5 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was added dropwise to water (20 mL). The precipitated solid was collected by filtration. The crude product was purified by reversed-phase rapid chromatography (C1). 18 Reverse silica column chromatography; mobile phase CH3CN, water, 10% to 70% gradient, 40 min; detection, UV 214 nm) yielded the desired compound (94.3 mg, yield 42%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ11.45(s,1H),8.96–8.89(m,2H),8.05(d,J=8.6Hz,1H),8.01– 7.96(m,1H),7.81–7.72(m,1H),7.36(d,J=8.2,1H),7.30–7.24(m,1H),7.16(t,J=7.9Hz ,1H),4.62(d,J=12.5Hz,1H),4.35–4.20(m,2H),4.18–3.97(m,2H),3.06(t,J=13.2Hz, 1H),2.67-2.51(m,2H),2.48-2.43(m,1H),1.82–1.61(m,2H).MS(ESI):Rt=1.42min,m / z 456.1[M+H] + Purity: 100% @ 254nm, 100% @ 214nm.

[0551] Comparative Example 1

[0552] 1-(1-(2-Phenylacetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (X4)

[0553]

[0554] Step 12.1. Synthesis of 1-(1-(2-phenylacetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (X4)

[0555]

[0556] 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one hydrochloride (1.50 g, 5.90 mmol) and pyridine (1.87 g, 23.6 mmol) were added to DCM (40 mL), followed by dropwise addition of 2-phenylacetyl chloride (2.28 g, 14.8 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h, diluted with water, and extracted with DCM. The combined organic layers were dried over Na2SO4, concentrated under reduced pressure to give a crude product, which was purified by high performance liquid chromatography (10–95% CH3CN aqueous solution) to give the desired white solid (1.46 g, 63% yield). 1 H NMR (400MHz, CDCl3) δ10.82(s,1H),7.38-7.21(m,5H),6.96-6.95(m,4H),4.57(d,J=13.2Hz,1H),4.45-4.35(m,1H),4.09(d ,J=13.6Hz,1H),3.84-3.74(m,2H),3.19-3.08(m,1H),2.73-2.62(m,1H),2.11-1.88(m,2H),1.72-1.54(m,2H).MS(ESI)m / z 336.1[M+H] + Purity: 97.9%@254nm, 99.9%@214nm.

[0557] Comparative Example 2

[0558] 1-(1-(2-(4-methoxyphenyl)acetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one

[0559]

[0560]

[0561] 1.1. Synthesis of 2-(4-methoxyphenyl)acetic acid (GEN1-070-1)

[0562]

[0563] Dimethyl sulfate (2.07 g, 16.4 mmol) was added dropwise to an aqueous solution of 2-(4-hydroxyphenyl)acetic acid (1.00 g, 6.57 mmol) and NaOH (1.58 g, 39.4 mmol) in 15 mL. The resulting mixture was stirred at 45 °C for 16 hours. The mixture was then acidified with concentrated hydrochloric acid. HCl was added to pH 1-2 and the mixture was filtered. The filter cake was washed with water (5 mL × 2) to give the desired compound (401 mg, yield 37%) as a white solid. 1 HNMR (400MHz, DMSO-d6): δ12.15(br s,1H),7.16(d,J=8.8Hz,2H),6.86(d,J=8.4Hz,2H),3.73(s,3H),3.48(s,2H).

[0564] Step 2. Synthesis of 1-(1-(2-(4-methoxyphenyl)acetyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (GEN1-070)

[0565]

[0566] HATU (343 mg, 0.902 mmol) was added to a DMF (1.5 mL) solution of 3-(4-piperidinyl)-1H-benzimidazol-2-one (157 mg, 0.723 μmol), 2-(4-methoxyphenyl)acetic acid (100 mg, 0.602 mmol), and DIEA (233 mg, 1.80 mmol), and the mixture was stirred at room temperature for 16 hours. The resulting mixture was purified by preparative HPLC (waters-3sunfire C18 5 μm 19*150 mm, 20%–50% B; A: H2O (0.1% TFA), B: CH3CN; UV: 214 nm, flow rate: 15 mL / min). Saturated Na2CO3 was then added to alkalize the mixture to pH 8. The mixture was extracted with EtOAc (20 mL × 3), and the combined organic layers were concentrated to give the desired compound (111 mg, yield 50%) as a white solid. 1HNMR (400MHz, CDCl3): δ9.23-9.13(m,1H),7.26-7.24(m,2H),7.08-6.99(m,3H),6.9 1(d,J=8.8Hz,2H),6.83(d,J=7.6Hz,1H),4.88(d,J=13.2Hz,1H),4.56-4.48(m,1H), 4.06(d,J=14Hz,1H),3.78(s,3H),3.76(s,2H),3.18-3.11(m,1H),2.72-2.65(m,1H) ,2.27-2.16(m,1H),1.96-1.81(m,2H),1.72-1.67(m,1H).MS(ESI):Rt=3.095min,m / z 366.2[M+H] + Purity: 99.31%@254nm, 99.64%@214nm.

[0567] Comparative Example 3

[0568] 1-(1-(1-(2-(1H-indol-3-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-169)

[0569]

[0570] Step 1. Synthesis of 2-(1H-indol-3-yl)acetic acid (GEN1-218-1)

[0571]

[0572] At room temperature, KOH (41.2 mg, 0.735 mmol) was added to a suspension of ethyl 2-(1H-indol-3-yl)acetate (100 mg, 0.49 mmol) in MeOH (5 mL) and H₂O (5 mL). The resulting mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (30 mL) and the pH was adjusted to 4 with 1 N HCl. The mixture was then extracted with EtOAc (30 mL × 3). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated to give the desired compound (45 mg, yield 52.4%) as a white solid. MS (ESI): Rt = 0.26 min, m / z 176.2, [M+H] + ;

[0573] Step 2. Synthesis of 1-(1-(1-(2-(1H-indol-3-yl)acetyl)piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (GEN1-169)

[0574]

[0575] Add EDCI (50.4 mg, 0.263 mmol) to a DMF (1 mL) solution of 1-(piperidin-4-yl)-7-(trifluoromethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (50 mg, 0.175 mmol, see GEN1-116-4 for synthetic steps), 2-(1H-indol-3-yl)acetic acid (44.8 mg, 0.21 mmol), HOBT (35.5 mg, 0.263 mmol), and DIEA (45.2 mg, 0.35 mmol). Stir the resulting mixture at room temperature for 1 h. Dilute the reaction mixture with water (30 mL) and extract with EtOAc (20 mL × 3). Wash the combined organic phases with brine (30 mL), dry over anhydrous Na₂SO₄, and filter. Concentrate the filtrate to give the crude product. Purify the crude product by reversed-phase chromatography. Purification conditions: C18 silica gel column; mobile phase ACN:H2O, gradient: 5% to 50%; 45 min; monitoring wavelength: 214 nm. The desired compound (28.6 mg, yield 36.9%) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.5-11.34(m,1H),10.91(s,1H),7.57(d,J=7.9Hz,1H),7.41-7.31 (m,2H),7.28(d,J=7.6Hz,1H),7.24-7.20(m,1H),7.19-7.12(m,1H),7.07(t,J=7.5Hz,1H),6 .97(t,J=7.4Hz,1H),4.70-4.54(m,1H),4.30-4.16(m,2H),3.92–3.70(m,2H),2.98(t,J=13. 0Hz,2H),2.71-2.56(m,1H),2.47-2.31(m,2H),1.74-1.64(m,2H).MS(ESI):Rt=1.50min,m / z 443.2,[M+H] + Purity: 100% @ 254nm.

[0576] Example 1: HTRF Experiment

[0577] Experimental materials:

[0578] Multifunctional microplate reader (TECAN; INFINITE F PLEX);

[0579] 96-well cell culture plate (Bioyong), HTRF 384 microplate (HTRF 384well low volume plates) (Cisbio; Cat#66PL384025);

[0580] Human interferon β (IFN-β) (GenScript; Cat#P01574), STAT5 Y694 phosphorylation detection kit (STAT5 phospho-Y694 kit) (Cisbio; Cat#64AT5PEG).

[0581] Experimental procedure:

[0582] HeLa cells (ATCC, CCL-2) were cultured at 3 x 10⁻⁶ cells per cell line. 4 Cultured at a density of cells / well in 96-well plates using DMEM medium containing 5% fetal bovine serum, incubated overnight at 37°C with 5% CO2. The next day, the original DMEM medium in the 96-well plates was aspirated, and an equal volume of medium containing different concentrations of the compound (a stock solution of the compound prepared with dimethyl sulfoxide diluted to the required concentration) was added for pretreatment for 2 hours, followed by incubation at 37°C. Then, human interferon β stimulation was added to a final concentration of 10 nM, gently shaken to mix thoroughly, and incubated at 37°C for 30 minutes.

[0583] Remove the culture medium, wash once with an appropriate volume of PBS, and then aspirate dry. Add 40 μL of 1x lysis buffer to each well and incubate at room temperature for 30 minutes (1 mL of 1x lysis buffer is prepared by mixing 740 μL of ddH2O with 250 μL of 4x lysis buffer and 10 μL of blocking reagent thoroughly). After 30 minutes, repeatedly pipette the cells until the lysis buffer is no longer viscous (avoid blowing out air bubbles) to achieve complete lysis.

[0584] Take an HTRF 384 microplate, add 4 μL of antibody premix (the antibody premix contains two antibodies, Cryptotate and d2: these two antibodies are diluted 20 times with detection buffer and then mixed in equal volumes to prepare the antibody premix) and 16 μL of cell lysis buffer to each well, seal with a sealing film and incubate overnight at room temperature in the dark.

[0585] After overnight incubation, the signals at 665 nm and 620 nm were detected using a multi-functional microplate reader. The formula for calculating STAT5 Y694 phosphorylation detection is: Ratio = [(signal 665nm) / (signal 620nm)] x 10 4The group treated with dimethyl sulfoxide alone served as a negative control (Ratio). Negative The group stimulated with human interferon β without the addition of the compound served as a positive control (Ratio). Positive The inhibition rate of STAT5 Y694 phosphorylation in different samples was normalized: inhibition rate = [1-(Ratio)] Experiment -Ratio Negative ) / (Ratio Positive -Ratio Negative )]*100. The test results are shown in Table 1.

[0586] Table 1. Quantitative data on inhibitory activity at the cellular biochemical level (HTRF experiment)

[0587] Compound numbering Inhibition rate @10μM <![CDATA[IC 50 / μM]]> GEN1-169 13.47％ - GEN1-077 34.25％ - GEN1-117 89.31％ - GEN1-118 27.57％ - GEN1-140 53.79％ - GEN1-284 80.21％ 3.9 GEN1-284salt 82.46％ 2.6 GEN1-298 51.98％ - GEN1-300 72.83％ 7.6 GEN1-301 80.70％ 8.8 GEN1-302 80.37％ 7.0 GEN1-303 91.91％ 5.5 GEN1-304 40.76％ - GEN1-305 63.05％ - GEN1-306 95.64％ - GEN1-312 55.50％ -

[0588] Note: "-" indicates that it was not measured.

[0589] Effect Example 2: PK Study Comparison

[0590] Methods: SD rats (N=3, female, 6-8 weeks old, weighing 200-230g, purchased from JHLaboratory Animal Co.LTD, SCXK(SH)2017-0012 20170012001153) or beagles (N=3, non-primary experimental animals, female, weighing 6.55-8.62 kg, purchased from Beijing Marshall Biotechnology Co., Ltd., quality inspection number SCXK(BJ)2016-0001110318201100068072) were administered the appropriate doses of the test compound GEN1-117 or GEN1-284s via intravenous injection or oral gavage (3 animals per route). (Intravenous injection samples were dissolved in 10% DMSO and 10% Soltol.) HS15, 80% PBS solution (oral samples dissolved in 0.5% methylcellulose aqueous solution), peripheral blood 150 μL was collected at predetermined time points (venous injection time points: 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 hours; oral injection time points: 0, 0.25, 0.5, 1, 2, 4, 8, 24 hours). The samples were collected in K2-EDTA anticoagulant tubes and centrifuged at 2000g, 4C, for 5 min over 15 minutes. Plasma was separated and analyzed by LC-MS (LC-MS / MS-37, triple Quad 6500) using standard methods, with Diclofenac as the internal standard. Mass spectrometry conditions: ESI cation, MRM detection:

[0591]

[0592] HPLC conditions: Mobile phase A: 0.1% FA aqueous solution; Mobile phase B: 0.1% FA, ACN solution

[0593]

[0594] Chromatographic column: Waters ACQUITY UPLC HSS T3 (2.1×50mm, 1.8μm)

[0595]

[0596] Standard curves for the analytes were prepared at concentrations of 1, 2, 10, 30, 100, 300, 1000, 2700, and 3000 ng / mL (50% ACN-water solution). 3 μL of the standard curve solution was mixed with 57 μL of blank plasma of the same species. 60 μL of the plasma sample was added to 6 μL of 10% FA aqueous solution and mixed thoroughly. 30 μL of this mixture was then mixed with 200 μL of the internal standard compound diclofenac (40 ng / mL in ACN), vortexed for 1 minute, and centrifuged at 5800 rpm for 10 minutes. 100 μL of the supernatant was transferred to a new test plate, and 1 μL of the solution was loaded onto the LC-MS / MS system.

[0597] Table 2. PK data of rats treated with GEN1-117:

[0598]

[0599] Table 3. Rat PK study data after administration of GEN1-284s:

[0600]

[0601]

[0602] Table 4. PK study data of beagle dogs after administration of GEN1-284s:

[0603]

[0604] Example 3: Evaluation of hERG ion channel inhibition ability

[0605] CHO cell lines stably transfected with cDNA and expressing the P15 hERG channel were used for the study. Cells were kept in a humidified incubator with 5% CO2 at 37°C in culture dishes or flasks and cultured in medium containing: Ham's F12, 10% (v / v) heat-inactivated FBS, 100 μg / ml hygromycin B (from Invitrogen), and 100 μg / ml genimycin B. Cells were allowed to grow and reach approximately 80-90% confluence under the above conditions. Before the experiment, cells were treated with Detachin (from Genlantis) for 3-5 minutes. At 37°C, the cells were then gently mixed with medium at room temperature, pipetted 15-20 times, and then resuspended in serum-free medium containing CHO-S-SFM II medium (from Invitrogen), buffered with HEPES (25 mM). Cells used in QPatch studies must meet the following criteria: under a microscope, the majority of cells in the suspension should be single and isolated; their viability should be greater than 95%, with only a small amount of debris and cell clumps (which may clog the pores in the QPlate during whole-cell clamp recording); and the final cell density in the suspension should be in the range of 3–8 × 10⁶ cells / ml before being applied to the QPatch mixing chamber. Cells under these conditions can only be used for recording within four hours of harvest.

[0606] Data acquisition was performed using an automated QPatch (Sophion Biosciences, Denmark). Cell voltage was clamped at a holding potential of -80 mV. The hERG current was activated by depolarization at +20 mV for 5 seconds, after which the current was restored to -50 mV for 2.5 seconds to remove inactivation and observe the outward tail current. The maximum amount of the tail current was used to determine the hERG current amplitude.

[0607] reagents External solution (mM) Internal solution (mM) CaCl2 2 5.374 MgCl2 1 1.75 KCl 4 120 NaCl 145 - Glucose 10 - HEPES 10 10 EGTA - 5 Na-ATP - 4 pH 7.4 (adjusted with NaOH), 7.25 (adjusted with KOH), osmotic pressure ~305mOsm ~295mOsm

[0608] Six doses (30, 10, 3, 1, 0.3, and 0.1 μM) were selected to obtain fitted curves and IC50 values. 50 Prior to QPatch testing, the compound was prepared as a serial dilution in 3, 1, 0.3, and 0.1 mM DMSO solvent to the final μM range. Sonication and vortexing were typically used for 5–10 minutes to aid complete dissolution of the solution. The final DMSO concentration was 0.1% or lower, except for the 30 μM point where the DMSO concentration was approximately 0.3%. The IC50 assessment doses for the positive control cisapride were 3, 1, 0.3, 0.1, 0.03, and 0.01 μM.

[0609] After achieving the insertion (whole-cell) configuration, cell data were recorded for 120 seconds to assess current stability. The aforementioned voltage settings were then applied to the cells, repeated every 15 seconds throughout the process. Only cells with stable recorded parameters exceeding the threshold were allowed to proceed to the drug evaluation procedure. All experiments were conducted at room temperature (approximately 25°C).

[0610] An external solution containing 0.1% DMSO (solvent) is applied to the cells to establish a baseline. The compound is added after the current has stabilized for 3 minutes. The compound solution is added, and the cells are left in the test solution until the compound reaches a steady state or for a maximum of 4 minutes. For dose-response assays, the compound is cumulatively applied to the cells from low to high concentrations. The cells are rinsed with the external solution after the compound test. A positive control is performed using cisapride in the same batch of cells used to test the compound to ensure normal cell response and good quality.

[0611] Table 5

[0612] 1.1 GEN1-117

[0613]

[0614]

[0615] *Sample testing was repeated in different cells to ensure that the standard error of a single concentration was below 10. Outliers were not included in the report.

[0616] Table 6

[0617] 1.2 GEN1-284S

[0618]

[0619] *Sample testing was repeated in different cells to ensure that the standard error of a single concentration was below 10. Outliers were not included in the report.

[0620] Conclusion: Neither GEN1-117 nor GEN1-284s showed significant hERG inhibitory activity.

[0621] Example 4: Evaluation of the therapeutic effect of GEN1-117 using a rat AIA model.

[0622] Abstract: Arthritis is a chronic inflammatory disease, and establishing arthritis disease models is particularly important for pathological and pharmacological research. AIA animal models can be easily induced in susceptible strains by a single subcutaneous injection of adjuvant antigen at the tail root, generating an immune response. Therefore, this method can be used to establish models for studying drug efficacy.

[0623] Objective: GEN1-117 has the function of inhibiting STAT5 activation. This experiment aims to evaluate the therapeutic effect of GEN1-117 on an in vivo rat model of adjuvant arthritis.

[0624] Methods: A tuberculosis-induced arthritis model was established in female Lewis rats (6–8 weeks old) using standard methods. Nine days later, during the symptom onset period, rats were administered tofacitinib or a control daily orally, and GEN1-117 compound was injected intraperitoneally daily. Body weight was measured daily, and paw swelling and right hind paw volume were measured every two days to evaluate the therapeutic effect of the test compound. All animals were euthanized at the end of the in vivo experiment.

[0625] Results: The disease model was successfully established, and GEN1-117 showed significant efficacy in reducing disease severity. Foot swelling and the volume of the right hind paw were significantly smaller than those in the control group.

[0626] Conclusion: GEN1-117 has a good in vivo therapeutic effect on arthritis in AIA rats.

[0627] 1. Experimental Objective

[0628] To evaluate the therapeutic effect of the test compound GEN1-117 on an in vivo rat model of adjuvant arthritis.

[0629] 2. Experimental Procedures

[0630] This project was conducted at Geneplus Biomedical Technology (Hangzhou) Co., Ltd. All animal experimental procedures involved in the process were carried out in accordance with Geneplus IACUC's standard laboratory animal operating procedures, laboratory animal husbandry and use guidelines, and animal welfare requirements stipulated by the Laboratory Animal Welfare Office. This experiment was a non-GLP experiment.

[0631] 3. Experimental Materials

[0632] 3.1 Instruments

[0633] Analytical balance, model: SQP, manufacturer: Beijing Sartorius Scientific Instruments Co., Ltd.

[0634] Electronic balance, model: MP5002, manufacturer: Changzhou Tianzhiping Instrument Equipment Co., Ltd.

[0635] Thermostatic magnetic stirrer, model: 85-2, manufacturer: Shanghai Sile Instruments Co., Ltd.

[0636] Miniature vortex mixer, model: XW-80A, manufacturer: Shanghai Huxi Analytical Instruments Co., Ltd.

[0637] High-frequency digitally controlled ultrasonic cleaner, model: KQ-50TDB, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.

[0638] Centrifuge, model: 5424R, manufacturer: Eppendorf.

[0639] Stereo microscope: Model: MZ6, Manufacturer: Leica.

[0640] Digital camera, model: EOS800D, manufacturer: Canon.

[0641] Olympus microscope, model: Olympus BX43, manufacturer: Japan.

[0642] 3.2 Test Drug

[0643] Test compound: GEN1-117, storage conditions: -20℃ (high concentration mother liquor, powder), working solution can be prepared fresh and stored at 2-8℃ for 1 day.

[0644] Tofacitinib, purchased from Meilunbio (batch: M0422A), storage conditions: 4℃.

[0645] 4. Preparation of solvents and compounds

[0646] Positive drug: Tofacitinib: Dissolve in PBS or saline.

[0647] Test compound: High-concentration DMSO stock solution, diluted to the appropriate concentration with PBS or physiological saline before use.

[0648] 5. Laboratory animals

[0649] 5.1 Use of Animals

[0650] Female Lewis rats, 7-8 weeks old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).

[0651] 5.2 Animal husbandry

[0652] All animals used in the following experiments were Lewis rats. After arriving at the facilities of Jianaishi Biomedical Technology Co., Ltd., the animals were housed in animal enclosures with strictly controlled environmental conditions. The temperature in the enclosures was maintained at 20–24°C, and the humidity at 30–70%. The lighting in the animal enclosures was controlled by an electronic timed lighting system, which turned the lights on for 12 hours and off for 12 hours each day (on at 6:00 AM and off at 6:00 PM). The animals had free access to feed and water.

[0653] 6. Experimental Methods

[0654] 6.1 Experimental Design

[0655] Arthritis was induced in rats by inoculation with Freund's complete adjuvant (CFA). On day 0, 0.1 mL of CFA (10 mg / mL) was injected intradermally into the left hind paw of rats into paraffin oil. On day 9, grouping was determined. Treatment was administered according to the following design. Treatment began with the first injection and continued for 14 days. Paw swelling and right hind paw volume were measured every two days; body weight was measured daily. After 14 days, all animals were euthanized.

[0656] 6.2 Grouping and Dosing

[0657] A total of 37 animals were screened in this experiment, and the results of each group are shown in Table 7:

[0658] Table 7 Animal grouping and treatment

[0659]

[0660] 6.3 Data Statistics

[0661] Data were collected and statistically plotted using Excel and GraphPad Prism software 5.0. Data are expressed as mean ± SEM. One-way ANOVA (Dennett's test) and t-test were used. P < 0.05 was considered statistically significant between groups.

[0662] 7. Research Results

[0663] Pharmacodynamic study of the test drug in an AIA rat model

[0664] In rats with a disease model of AIA arthritis treated with GEN1-117, the volume of the hind paw was significantly reduced compared with the control rats on days 17-23 after drug intervention (Table 8).

[0665] Table 8

[0666]

[0667]

[0668] Compared with the control group, the experimental group rats showed a significant decrease in body weight on day 9. Rats in the GEN1-117 group, after drug intervention, showed a significant increase in body weight (Table 9).

[0669] Table 9

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676] Compared with control rats, the clinical scores of rats in the GEN1-117 group decreased significantly from day 13 to 23, indicating that GEN1-117 can alleviate the AIA rat arthritis disease model, and its efficacy is similar to that of the positive control drug tofacitinib (Table 10).

[0677] Table 10

[0678]

[0679]

[0680]

[0681] Compared with the control rats, the data of the maximum arthritis score index of the GEN1-117 group rats showed that the GEN1-117 drug could effectively alleviate the AIA rat arthritis disease model, and the efficacy was similar to that of the positive drug tofacitinib (Table 11).

[0682] Table 11

[0683]

[0684]

[0685] 8. Conclusion

[0686] In this study, our results showed that daily intraperitoneal injection of the STAT5 inhibitor GEN1-117 dose-dependently suppressed the increase in hind paw volume and clinical scores in AIA rats. Based on this experiment, we can conclude that GEN1-117 can significantly alleviate disease progression in an AIA model.

[0687] Example 5: Efficacy Evaluation of GEN1-284s in a Mouse CIA Model

[0688] 1. Experimental Objective: This experiment aims to evaluate the in vivo efficacy of compound GEN1-284s in a collagen-induced arthritis model.

[0689] 2. Experimental Materials and Equipment 2.1 Experimental Materials

[0690] Bovine type II collagen (CII): Sichuan University, Product No.: 20190618

[0691] Acetic acid: Sigma (St. Louis, MO, USA), Product No.: A8976

[0692] Complete Freund's adjuvant: Sigma, catalog number: F5881

[0693] Tofacitinib: Dalian Meilun Biotechnology Co., Ltd., Product No.: MB3358-1g-MLNE

[0694] 2.2 Test Drug

[0695] (1) Test compound

[0696] Name: GEN1-284s

[0697] Source: Provided by the client

[0698] Batch number: E00144-18156-019P5

[0699] Appearance: White solid

[0700] Purity: 95.06%

[0701] Storage: Store at 4℃

[0702] Media: PBS

[0703] Preparation method: Prepare the compound every 3 days, and dissolve it directly in PBS.

[0704] (2) Control compound

[0705] Name: Tofacitinib

[0706] Source: Dalian Meilun Biotechnology Co., Ltd.

[0707] Storage: Store at -20℃

[0708] Solvent: 0.5% MC

[0709] Preparation method: Weigh the test sample, add 0.5% MC, and sonicate for 20-30 minutes.

[0710] 2.3 Experimental Apparatus

[0711] Anesthesia machine: Raymain, RM-HSIV-u

[0712] High-speed homogenizer: IKA, T10 basic

[0713] Compound weighing balance: Sartorius, CPA225D

[0714] Animal weighing balance: Changzhou Tianzhiping electronic balance, YH2000

[0715] 2.4 Laboratory animals and their housing environment

[0716] Animal lineage: DBA / 1 mice supplier: Beijing Vital River Laboratory Animal Co., Ltd. Certificate Number: 110011201107916352 Gender and weight: Male, 14-16g Breeding location: WuXi AppTec Animal Housing Center Adaptation period: 7 days temperature: 20～26℃ humidity: 40～70％ illumination: Fluorescent lamps, 12 hours on (08:00~20:00) and 12 hours off (20:00~08:00). Stocking density: 5 birds / cage food: Free access to feed (radiation-sterilized feed, Jiangsu Medison) Drinking water: Free drinking water (prepared by a mole (ultra)pure water system)

[0717] The animal procedures described in this experimental report were reviewed and approved by the Laboratory Animal Use and Management (Ethics) Committee (IACUC) of WuXi AppTec.

[0718] 3 Experimental Methods 3.1 Preparation of Experimental Drugs

[0719] Vehicle: PBS, store at 4°C for later use.

[0720] GEN1-284s: Weigh out compound GEN1-284s and prepare solutions with concentrations of 5, 15, and 30 mg / ml using vehicle as solvent.

[0721] 3.2 Arthritis Model Induction

[0722] Acetic acid preparation

[0723] Dilute 2N acetic acid to 100mM, filter through a 0.22-micron filter membrane, and store at 4°C.

[0724] Preparation of CII solution

[0725] Bovine type II collagen (CII) was dissolved in 100 mM acetic acid solution and stored overnight at 4°C. The collagen concentration was 8 mg / mL.

[0726] Emulsion preparation

[0727] The overnight CII solution was mixed with an equal volume of complete Freund's adjuvant and homogenized on ice at 30,000 rpm for about 60 minutes using a high-speed homogenizer until a stable emulsion was formed.

[0728] Arthritis Induction

[0729] Fifty DBA / 1 mice were all immunized. The day of the first immunization was designated as day 0, and subsequent days were numbered sequentially. After being anesthetized with isoflurane, DBA / 1 mice were injected subcutaneously into the tail (2-3 cm from the tail base) with 50 μL of prepared collagen emulsion (containing 200 μg CII). On day 21, the same volume of collagen emulsion was injected into the tail in the same manner. Mice in the control group did not require immunization.

[0730] 3.3 Administration

[0731] On day 28, when the average clinical score reached approximately 0.5, mice were grouped according to weight and score, as per the experimental protocol. This ensured that these two criteria were largely consistent across groups. The grouping details are shown in Table 12. Fifty mice were divided into five groups, excluding those with significant deviations in weight and score, resulting in eight mice per group.

[0732] The dosage for each group is shown in Table 12. The volume of administration by gavage was 10 ml / kg, and the administration was continued for 14 days.

[0733] Table 12 Grouping and Dosage Design

[0734]

[0735] 3.4 Determination of Indicators for Arthritis Incidence

[0736] Clinical observation: From before immunization to day 21 post-immunization, the basic health status and weight changes of DBA / 1 mice were observed daily (recorded once a week). After day 22, the health status, morbidity, and weight changes of the mice were observed daily (recorded at least three times a week) until the end of the experiment.

[0737] Clinical scoring: After day 22, observe the mice daily for disease progression. Once the mice show clinical symptoms of arthritis, score them according to the severity of the lesions (redness, swelling, joint deformity) on a scale of 0-4, with a maximum score of 4 points per limb and a maximum score of 16 points per animal. The scoring criteria are shown in Table 13. Scoring should be performed at least three times a week. [3] .

[0738] Table 13 Clinical scoring criteria for arthritis

[0739] Score Clinical symptoms 0 No erythema or swelling 1 Redness or mild swelling near the tarsal bone, ankle, or metatarsal bone; or redness and swelling on one toe. 2 Mild erythema and swelling of the ankle and metatarsals, with redness and erythema on two or more toes. 3 Moderate erythema and swelling of the ankle, wrist, and metatarsals. 4 The ankles, wrists, metatarsals, and toes are all severely swollen and red.

[0740] 3.5 Pathological Analysis

[0741] The experiment ended with the euthanasia of the mice. The right hind limb of the mice was removed, soaked in 10% formalin solution, decalcified with formic acid solution, embedded in paraffin, sectioned, stained with H&E, and observed under a microscope. The degree of joint damage was evaluated from four aspects: inflammatory cell infiltration, pannus formation, cartilage damage, and bone resorption, and scored according to a scale of 0-4. The scoring criteria are shown in Table 14.

[0742] Table 14. Pathological Scoring Criteria for Arthritis

[0743]

[0744] 4. Statistical analysis: Experimental data were expressed as mean ± standard error (Mean ± SEM). Clinical scores and weight were analyzed using two-way ANOVA, while AUC and pathological scores were analyzed using one-way ANOVA. A p < 0.05 was considered statistically significant.

[0745] 5. Experimental Results and Discussion 5.1 Clinical Scoring

[0746] This study evaluated the effect of compound GEN1-284s on improving clinical scores in a mouse model of colic (CIA). A mouse CIA model was established by subcutaneous injection of CII collagen emulsion into the tail of mice on days 0 and 21. Clinical symptoms of arthritis began to appear in mice on day 25 after the first immunization (day 4 after the second immunization). Grouping and drug administration began on day 28. The mean clinical score in the solvent control group gradually increased, reaching 7.5 points on day 41, indicating the successful establishment of the collagen-induced arthritis model. The positive control group treated with tofacitinib significantly inhibited arthritis scores from day 34. Compound GEN1-284s, at doses of 50 mg / kg and 150 mg / kg, consistently and significantly inhibited arthritis scores from day 32 and day 34, respectively. By the end of the experiment, compared with the solvent group's score of 7.5, the clinical scores of the low-dose and medium-dose GEN1-284s groups were as low as 3.1 (p<0.0001) and 4.4 (p<0.001), respectively.

[0747] Table 15 Statistical analysis of clinical score data in mice with collagen-induced arthritis

[0748]

[0749] By analyzing the clinical score curves of each animal in each group, the area under the curve (AUC) was calculated. Based on the average AUC of each group, the inhibition rate of each treatment group relative to the solvent control group was calculated. The GEN1-284s 50mg / kg treatment group significantly inhibited arthritis scores, with an inhibition rate of 56.9%, which was superior to the Tofacitinib group with an inhibition rate of 47.0%.

[0750] 5.2 Weight

[0751] Compared with the solvent group, there was no significant difference in average body weight among the drug administration groups after the onset of the disease (Table 16).

[0752] Table 16 Statistical Analysis of Weight Data

[0753]

[0754]

[0755] 5.3 Pathological Results

[0756] H&E staining revealed infiltration of monocytes, plasma cells, and lymphocytes in the joints of mice in the solvent control group, along with pannus proliferation and cartilage destruction on the cartilage surface. No significant joint tissue lesions were observed in the positive control group treated with tofacitinib. The total score of the GEN1-284s 50mg / kg group was lower than that of the solvent control group.

[0757] 6. Experimental Conclusion: The mouse CIA model was successfully established. Mice in the solvent control group developed severe clinical symptoms of arthritis under collagen induction. Subsequent joint tissue pathological analysis also showed cell infiltration and bone tissue damage. In contrast, the positive control group, Tofacitinib, showed significant improvement in arthritis symptoms and tissue lesions.

[0758] Compound GEN1-284s, at doses of 50 mg / kg and 150 mg / kg, sustained and significantly inhibited arthritis scores starting from Day 32 and Day 34, respectively. The AUC inhibition rate of the GEN1-284s 50 mg / kg group reached 56.9%, which was superior to the Tofacitinib group with an inhibition rate of 47.0%.

[0759] Mice tolerated compound GEN1-284s well at doses of 50 mg / kg, 150 mg / kg, and 300 mg / kg, with no significant decrease in body weight observed at the experimental endpoint. Mice died at a dose of GEN1-284s of 500 mg / kg.

[0760] From a histomorphological perspective, joint damage was induced in mice in the solvent control group under the influence of collagen. The 50 mg / kg dose of the tested compound GEN1-284s showed a tendency to alleviate arthritis in the animals.

[0761] Repeated trials, dose exploration

[0762] The method is the same as above.

[0763] The dosage was adjusted to GEN1-284s (10, 30, 50 mg / kg, qd, po).

[0764] in conclusion

[0765] The mouse CIA model was successfully established. The mice in the solvent control group developed severe clinical symptoms of arthritis under collagen induction. Subsequent joint tissue pathological analysis also showed cell infiltration and bone tissue damage. In contrast, the positive control group, Tofacitinib, showed a significant improvement in arthritis symptoms and tissue lesions.

[0766] Compound GEN1-284s, at doses of 10, 30, and 50 mg / k, consistently and significantly inhibited arthritis scores starting from Day 32 and Day 34, respectively, with no significant difference compared to the tofacitinib positive group.

[0767] Mice tolerated the compound GEN1-284s well at doses of 10, 30, and 50 mg / k, and no significant decrease in body weight was observed at the experimental endpoint.

[0768] Table 17 Clinical Scoring Table

[0769]

[0770] Table 18

[0771] Day 35 Day 38 Day 40 Day 42 G1 Vehicle vs.G2 Tofacitinib BID ** *** **** **** G1 Vehicle vs.G3 GEN1-284s 10mpk ns ns ns * G1 Vehicle vs.G4 GEN1-284s 30mpk ns ** * * G1 Vehicle vs.G5 GEN1-284s 50mpk * ** ** **

[0772] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.Comparing to Vehicle group,Two-way ANOVA.

[0773] Table 19 Weight Change Table

[0774]

[0775]

[0776] Example 6: Preliminary assessment of the safety of GEN1-284s in single-dose and multiple-dose acute toxicity experiments in mice.

[0777] summary

[0778] Objective: To evaluate the toxic dose of compound GEN1-284s in female BALB / c mice.

[0779] Materials and methods:

[0780] Part 1: Single Dosage. Six mice were randomly divided into two groups: a solvent control and GEN1-284s (1000 mg / kg). The mice were administered the drug orally via gavage once and observed for 6 days. Changes in body weight and activity were recorded.

[0781] Part 2: Multiple Dosing. A total of 6 mice were used in this experiment, divided into two groups: a GEN1-284s-1000mg / kg group and a GEN1-284s-300mg / kg group, with 3 mice in each group. GEN1-284s was administered by gavage for 14 consecutive days, 200μl per mouse. The mice's condition and survival were observed daily, and their weight was recorded. Two mice died 2 hours after administration of GEN1-284s-1000mg / kg; the dosage was then changed to 600mg / kg, and the experiment continued.

[0782] result:

[0783] Part 1: A single dose of 1000 mg / kg GEN1-284s did not affect the body weight or activity level of BALB / c mice and was indistinguishable from the solvent control group, indicating that the mice tolerated this dose very well.

[0784] Part 2: Results of multiple administrations showed that GEN1-284s-1000mg / kg was extremely toxic to female BALB / c mice, with mice dying within 2 hours of administration; continuous administration of GEN1-284s-600mg / kg was also highly toxic to mice, with all mice dying within 5 days; however, continuous administration of GEN1-284s-300mg / kg did not affect the survival of mice, and there was no significant change in body weight.

[0785] Conclusion: Continuous administration of GEN1-284s at a dose of 300 mg / kg did not affect the survival of mice, nor did it significantly change their body weight. Therefore, this dose has low toxicity to female BALB / c mice, and continuous administration for 14 days did not affect their survival.

[0786] 1. Experimental Objective

[0787] Evaluation of the toxic dose of compound GEN1-284s in BALB / c mice

[0788] 2. Experimental Procedures

[0789] This project was conducted at Geneplus Biomedical Technology (Hangzhou) Co., Ltd. All animal experimental procedures involved in the process were carried out in accordance with Geneplus IACUC's standard laboratory animal operating procedures, laboratory animal husbandry and use guidelines, and animal welfare requirements stipulated by the Laboratory Animal Welfare Office. This experiment was a non-GLP experiment.

[0790] 3. Experimental Materials

[0791] 3.1 Instruments

[0792] Analytical balance, model: SQP, manufacturer: Beijing Sartorius Scientific Instruments Co., Ltd.

[0793] 3.2 Test Drugs and Reagents

[0794] Test compound:

[0795] GEN1-284s, storage conditions: -20℃ (high concentration mother liquor, powder), working solution can be prepared fresh, and stored at 2-8℃ for 1 day.

[0796] Reagents:

[0797] 1X PBS, Manufacturer: Hyclone, Lot: AE29449011, Storage conditions: 4℃.

[0798] 4. Experimental animals

[0799] 4.1 Use of animals

[0800] Female BALB / c mice, 6 weeks old, 12 in number, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0801] 4.2 Animal husbandry

[0802] All animals used in the following experiments were BALB / c mice. After arriving at the facilities of Jianaishi Biomedical Technology Co., Ltd., the animals were housed in animal housing with strictly controlled environmental conditions. The temperature in the housing was maintained at 20–24°C, and the humidity at 30–70%. The lighting in the animal housing was controlled by an electronic timed lighting system, which turned the lights on for 12 hours and off for 12 hours each day (on at 6:00 AM and off at 6:00 PM). The animals had free access to feed and water.

[0803] 5 Experimental Methods

[0804] 5.1 Experimental Design

[0805] Part One:

[0806] Six mice were used in this experiment and randomly divided into two groups: the solvent group and the GEN1-284s-1000mg / kg group, with three mice in each group. The mice were administered the drug by gavage once on day 0, 200μl per mouse. The mice's condition and survival were observed and their weight was recorded daily.

[0807] Part Two:

[0808] Six mice were used in this experiment and divided into two groups: a GEN1-284s-1000mg / kg group and a GEN1-284s-300mg / kg group, with three mice in each group. GEN1-284s was administered by gavage for 14 consecutive days, 200μl per mouse. The mice's condition and survival were observed daily, and their weight was recorded. Two mice died 2 hours after administration of GEN1-284s-1000mg / kg; the dosage was then changed to 600mg / kg, and the experiment continued.

[0809] 5.2 Grouping and Dosing

[0810] Part One

[0811] Grouping Animal number Administration method Dosage frequency Solvent comparison 3 PO 1, day 0 GEN1-284s-1000mg / kg 3 PO 1, day 0

[0812] Part Two

[0813]

[0814]

[0815] GEN1-284s was administered via gavage for 14 consecutive days, with each mouse receiving 200 μl / 20 g of the drug. The mice's condition and survival were observed daily, and their weight was recorded. Two mice died 2 hours after administration of GEN1-284s at 1000 mg / kg, and the dosage was changed to 600 mg / kg to continue the experiment.

[0816] 5.3 Detection Indicators

[0817] We observed changes in body weight and survival in mice 6 days after a single dose, as well as changes in body weight and survival in mice given a high dose of GEN1-284s for 14 consecutive days.

[0818] 5.4 Data Statistics

[0819] Data collection and statistical graphing were performed using Excel and GraphPad Prism software 5.0. Data are expressed as Mean ± SEM.

[0820] 6 Research Results

[0821] 6.1 Observation of mouse survival status

[0822] Part One:

[0823] No abnormalities were observed in the survival status of the mice after drug administration.

[0824] Part Two:

[0825] Mouse status:

[0826] Day 1: Mice in the GEN1-284s-300mg / kg group were normal, while 2 mice in the GEN1-284s-1000mg / kg group died 2 hours after administration.

[0827] Day 2: Mice in the GEN1-284s-300mg / kg group were normal. The GEN1-284s-1000mg / kg group was changed to GEN1-284s-600mg / kg for continued experimental administration, and the mice were in relatively good condition.

[0828] Day 3: Mice in the GEN1-284s-300mg / kg group were normal, while mice in the GEN1-284s-600mg / kg group were in poor condition.

[0829] Day 4: Mice in the GEN1-284s-300mg / kg group were normal, while one mouse in the GEN1-284s-600mg / kg group died, bringing the total number of mice to 1.

[0830] Days 5-14: Mice in the GEN1-284s-300mg / kg group were normal, and no significant change in body weight was observed.

[0831] 6.2 Mouse survival and weight changes

[0832] Part One:

[0833] Table 20: Body weight changes in GEN1-284s mice after a single dose of acute toxicity test.

[0834]

[0835]

[0836] Part Two:

[0837] Based on survival results, the 600 mg / kg group of GEN1-284s showed higher toxicity, with all mice dying within 5 days. However, continuous administration of GEN1-284s at a dose of 300 mg / kg did not affect mouse survival. As shown in Table 21 regarding body weight, continuous administration of GEN1-284s did not significantly affect mouse body weight.

[0838] Table 21

[0839]

[0840] 7. Conclusion

[0841] In summary:

[0842] A single dose of GEN1-284s 1000 mg / kg had no effect on survival or body weight in animals.

[0843] Under repeated administration, GEN1-284s-1000 mg / kg was extremely toxic to female BALB / c mice, with mice dying within 2 hours of administration. Continuous administration of GEN1-284s-600 mg / kg was also highly toxic to mice, resulting in the death of all mice within 5 days. However, continuous administration of GEN1-284s-300 mg / kg did not affect the survival of mice, and there was no significant change in body weight. Therefore, this dose was less toxic to female BALB / c mice, and continuous administration for 14 days did not affect their survival.

[0844] Example 7: Evaluation of GEN1-284s in an atherosclerosis model

[0845] summary

[0846] Atherosclerosis is a chronic inflammatory arterial disease. Because its pathogenesis remains unclear, successfully establishing an AS disease model is particularly important for pathological and pharmacological research. The apolipoprotein E gene knockout mouse model of atherosclerosis is caused by genetic damage, and its pathological characteristics are closer to those in humans. Apoe KO mice, after being fed a high-fat diet, spontaneously develop dyslipidemia symptoms and form intimal plaques similar to those in humans.

[0847] Objective: This experiment used a high-fat diet-induced Apoe gene knockout mouse model of atherosclerosis to evaluate the effects of compound GEN1-284s on blood lipids and pathological changes in arterial plaques in atherosclerotic model mice.

[0848] Methods: Baseline blood lipid levels were measured in Apoe KO mice, which were divided into a normal control group and a high-fat group. After 8 weeks of feeding, except for the normal control group, the mice were divided into 4 groups (approximately 8 mice per group): normal diet group (Chow), high-fat diet model group (HFD), atorvastatin positive group (HFD + Atorvastatin 5 mpk, QD), and GEN1-284s group (284-60 mpk, QD). The mice were administered the compound orally for 8 weeks. The effects of the compound on the following indicators in Apoe KO mice were observed: four lipid parameters (TC, TG, HDL-C, and LDL-C), aortic plaque en-face, and Oil Red staining of cardiac outflow tract pathology.

[0849] Results: The results showed that after 8 weeks of high-fat diet feeding, mice were orally administered the test compound for up to 16 weeks. Compared with the control group fed a normal diet, high-fat diet feeding caused dyslipidemia (increasing trend of TCHO and LDL-C, while decreasing trend of HDL-C) in mice, arterial plaque accumulation, and cardiac outflow tract lesions. Treatment with the GEN1-284s compound effectively reduced arterial plaque formation. It can partially alleviate dyslipidemia, reduce arterial plaque accumulation, and has a significant ameliorative effect on cardiac outflow tract plaque.

[0850] Conclusion: In summary, GEN1-284s can partially reduce blood lipids, reduce the formation of plaques in arteries and the left ventricular outflow tract, and improve cardiac outflow tract lesions, with effects similar to those of the positive control drug Atorvastatin.

[0851] 1. Experimental Objective

[0852] Using a high-fat diet-induced Apoe gene knockout mouse model of atherosclerosis, the effects of different doses of GEN1-284s on blood lipids, arterial plaques, and cardiac outflow tract lesions in the atherosclerosis model mice were evaluated.

[0853] 2 Experimental Specifications

[0854] This project was conducted at Geneplus Biomedical Technology (Hangzhou) Co., Ltd. All animal experimental procedures involved in the process were carried out in accordance with Geneplus IACUC's standard laboratory animal operating procedures, laboratory animal husbandry and use guidelines, and animal welfare requirements stipulated by the Laboratory Animal Welfare Office. This experiment was a non-GLP experiment.

[0855] 3 Experimental Materials

[0856] 3.1 Instruments

[0857] Analytical balance, model: SQP, manufacturer: Beijing Sartorius Scientific Instruments Co., Ltd.

[0858] Electronic balance, model: MP5002, manufacturer: Changzhou Tianzhiping Instrument Equipment Co., Ltd.

[0859] Thermostatic magnetic stirrer, model: 85-2, manufacturer: Shanghai Sile Instruments Co., Ltd.

[0860] Miniature vortex mixer, model: XW-80A, manufacturer: Shanghai Huxi Analytical Instruments Co., Ltd.

[0861] High-frequency digitally controlled ultrasonic cleaner, model: KQ-50TDB, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.

[0862] Centrifuge, model: 5424R, manufacturer: Eppendorf.

[0863] Stereo microscope: Model: MZ6, Manufacturer: Leica.

[0864] Hitachi 7180 Automated Biochemical Analyzer, Model: 7180, Manufacturer: Japan.

[0865] Digital camera, model: EOS800D, manufacturer: Canon.

[0866] Freezing slicer, model: Leica CM1950, manufacturer: Germany.

[0867] Olympus microscope, model: Olympus BX43, manufacturer: Japan.

[0868] 3.2 Test Drugs and Reagents

[0869] Test compound: GEN1-284s, storage conditions: -20℃ (high concentration mother liquor, powder), working solution can be prepared fresh and stored at 2-8℃ for 1 day.

[0870] Atorvastatin, Manufacturer: Sigma, Lot#: LRAA9204, Storage conditions: 4℃.

[0871] Reagent: DMSO; Storage conditions: room temperature.

[0872] PEG300, Manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., Storage conditions: Room temperature.

[0873] 0.9% Sodium Chloride Injection: Chenxin Pharmaceutical Co., Ltd.

[0874] 4% paraformaldehyde, manufacturer: Beyotime, storage conditions: -20℃.

[0875] Isopropyl alcohol, manufacturer: Gitai, storage conditions: room temperature.

[0876] Oil red O solution, manufacturer: Sigma, storage conditions: 4℃.

[0877] Total cholesterol test kit, manufacturer: Pulilai Biomedical. Storage conditions: 4℃.

[0878] Triglyceride assay kit, manufacturer: Pulilai Biomedical. Storage conditions: 4℃.

[0879] HDL-C Detection Kit, Manufacturer: Pulilai Biomedical. Storage conditions: 4℃.

[0880] LDL-C Detection Kit, Manufacturer: Pulilai Biomedical. Storage conditions: 4℃.

[0881] 4. Preparation of solvents and compounds

[0882] Positive control drug: Atorvastatin calcium: Prepare a suspension with physiological saline, mix well before administration, and administer at a volume of 10 ml / kg.

[0883] 5. Laboratory animals

[0884] 5.1 Use of Animals

[0885] Fifty-six male C57BL / 6Apoe KO mice, aged 7-8 weeks, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).

[0886] 5.2 Animal husbandry

[0887] All animals used in the following experiments were C57BL / 6. After arriving at the facilities of Jianaishi Biomedical Technology Co., Ltd., the animals were housed in animal housing with strictly controlled environmental conditions. The temperature in the housing was maintained at 20–24℃, and the humidity at 30–70%. The temperature and humidity in the housing were monitored in real time using thermometers and hygrometers, and recorded twice a day (once in the morning and once in the afternoon). The lighting in the animal housing was controlled by an electronic timed lighting system, with the lights on for 12 hours and off for 12 hours a day (on at 6:00 AM and off at 6:00 PM). The animals had free access to food and water. Except for the normal control group, which was fed a regular diet, the other groups of mice were fed a high-fat diet (D12079Bi, Research Diets Inc., NJ, US).

[0888] 6 Experimental Methods

[0889] 6.1 Experimental Design

[0890] Except for the control group (Chow), all other groups of mice were fed a high-fat diet daily for 16 weeks. Starting from week 9, each group was given a solvent or the corresponding test compound orally, and body weight was recorded daily for 8 weeks. After the first 8 weeks of high-fat diet, blood samples were collected, serum was separated by centrifugation, and blood lipid indicators (TCHO, TG, LDL-C, and HDL-C) were measured. Mice were randomly assigned to groups based on TCHO results and body weight. During the subsequent 8 weeks of drug administration, blood lipid indicators (TCHO, TG, LDL-C, and HDL-C) were measured every 4 weeks.

[0891] During the experiment, the animals' living conditions were observed, and any abnormalities were recorded. After the experiment, endpoint processing and sample collection were carried out.

[0892] 6.2 Grouping and Dosing

[0893] A total of 56 animals were screened for this experiment. Excluding the normal control group, they were divided into 7 groups of 8 animals each. The details of each group are shown in Table 22.

[0894] Table 22 Animal grouping and treatment

[0895]

[0896] 6.3 Endpoint detection indicators

[0897] 6.3.1 Blood lipid testing:

[0898] Mice were fasted overnight, and blood (150 μl) was collected via mandibular sampling or enucleation (limited to the endpoint). The blood was allowed to stand at room temperature for 2 hours. The blood was centrifuged at 7000 rpm for 10 minutes at 4°C, and the serum was collected, rapidly frozen on dry ice, and stored at -80°C for analysis. The biochemical indicators TCHO, TG, LDL-C, and HDL-C in the serum were detected using a kit and ELISA reader.

[0899] 6.3.2 Sample Testing:

[0900] 6.3.2.1 En-face staining and plaque area analysis of aortic plaques:

[0901] At the experimental endpoint, mice were euthanized via CO2 inhalation, and their hearts and aortas (from the aortic arch to the iliac arteries) were isolated. The isolated arteries were longitudinally dissected from the aortic arch to the iliac arteries, fixed in 4% paraformaldehyde for 30 minutes, synchronized with 60% isopropanol for 10 minutes, stained in 60% Oil Red for 30 minutes, and washed three times with 60% isopropanol for 5 minutes each time. Finally, the stained aorta was washed with deionized water and photographed. The proportion of red patch areas was measured using Image Pro Plus 6.0 (MediaCybernetics, MD, US).

[0902] 6.3.2.2 Pathological examination of atherosclerotic plaque areas in animals (pathological sections) HE staining:

[0903] Mouse heart samples were fixed overnight in 4% paraformaldehyde solution and then dehydrated in sucrose solution. They were then embedded using OCT (Sakura, Japan) under a stereomicroscope and prepared into 10 μm frozen sections. After Oil Red ORO staining, the sections were used for routine pathological examination. Outflow tract images were acquired under a 4x optical microscope, and high-quality morphological observation was performed using a 20x eyepiece. The percentage of intimal lesions in the cardiac outflow tract was calculated. The detection criteria were based on the histological classification of human atherosclerotic diseases, as described below:

[0904] Table 23 Histological classification of atherosclerotic diseases

[0905]

[0906] *The last column is based on human clinical studies.

[0907] Type V injuries are classified into three types:

[0908] Type Va: Contains a large lipid nucleus and fibrotic tissue;

[0909] Type Vb: In addition to lipid nuclei and fibrosis, calcification is also present;

[0910] Vc type: lipid nuclei are significantly reduced, and fibrotic tissue is present.

[0911] Type VI injury is divided into 3 types:

[0912] Type VIa: The lesion surface is ruptured;

[0913] Type VIb: Bleeding;

[0914] Type VIc: Thrombosis.

[0915] We used a microscope to measure the plaque area in the left ventricular outflow tract and calculated the plaque ratio in the left ventricular outflow tract, which reflects the degree of stenosis of the blood vessels in the cardiac outflow tract.

[0916] 6.4 Data Statistics

[0917] Data were collected and statistically plotted using Excel and GraphPad Prism software 5.0. Data are expressed as mean ± SEM. One-way ANOVA (Dennett's test) and t-test were used. P < 0.05 was considered statistically significant between groups.

[0918] 7 Research Results

[0919] 7.1 Effects of the test drug on blood lipids in atherosclerotic mice

[0920] Long-term consumption of a high-fat diet can lead to dyslipidemia. Blood lipid levels can be assessed by regularly monitoring serum TCHO, TG, LDL-C, and HDL-C. As shown in Tables 24-27, except for the normal group, all other groups of mice showed an increasing trend in TCHO, TG, and LDL-C, and a decreasing trend in HDL-C after being fed a high-fat diet. Oral administration of Atorvastatin alleviated this trend of dyslipidemia. Mice were given GEN1-284s orally starting at week 8. Compared with the HFD group, the trend of dyslipidemia caused by the high-fat diet was partially improved. This indicates that oral administration of GEN1-284s can partially improve the dyslipidemia (TCHO, TG, HDL-C, and LDL-C) caused by a high-fat diet in mice.

[0921] Table 24

[0922]

[0923]

[0924]

[0925]

[0926] Table 25

[0927]

[0928]

[0929]

[0930]

[0931]

[0932] Table 26

[0933]

[0934]

[0935]

[0936]

[0937]

[0938] Table 27

[0939]

[0940]

[0941]

[0942]

[0943]

[0944] 7.2 Effects of the test drug on arterial plaques in atherosclerotic mice

[0945] The lesions of atherosclerosis are closely related to the formation of thrombi. (See Table 28 and...) Figure 1 As shown, the arterial plaque area in the HFD group was significantly larger than that in the normal control group after being fed a high-fat diet. Oral administration of atorvastatin significantly reduced the proportion of arterial plaque area in mice, far lower than that in the HFD group. The tested compound GEN1-284s significantly reduced the formation of atherosclerotic plaques, with effects comparable to those of atorvastatin.

[0946] Table 28

[0947]

[0948] 7.3 Effects of the test compound on cardiac outflow tract plaque

[0949] Abnormal blood lipids can cause plaque accumulation in the left ventricular outflow tract, leading to apoptosis. We evaluated the morphological changes in the left ventricular outflow tract and the proportion of outflow tract plaques in the outflow tract cross-section. As shown in Table 29 and Figure 2 as follows. By measuring the cross-sectional area of the left ventricular outflow tract, the outflow tract plaques in the HFD group were higher than those in the normal control group. Compared with the HFD group, the plaques in the Atorvastatin group were significantly reduced. GEN1-284s had a certain degree of improvement effect on the formation of outflow tract plaques.

[0950] Table 29

[0951]

[0952]

[0953] 8. Conclusion

[0954] In summary: GEN1-284s can reduce the formation of atherosclerotic plaques to a certain extent, similar to the positive drug Atorvastatin.

[0955] Effect Example 8 Evaluation of GEN1-284s on the IMQ-induced Psoriasis Model

[0956] Abstract

[0957] Psoriasis, commonly known as psoriasis vulgaris, is a chronic inflammatory skin disease with a long course and a tendency to relapse easily. Some cases are almost incurable for life. The disease mainly affects young and middle-aged people and has a greater impact on the physical and mental health of patients. The clinical manifestations are mainly erythema and scales, and the whole body can be affected, with the scalp and the extensor sides of the limbs being more common, and it usually worsens in winter. IMQ was continuously applied to the bare skin of the mouse back for six days to simulate the pathogenesis of psoriasis.

[0958] Objective: In this experiment, an IMQ-induced psoriasis model of Balb / c mice was used to evaluate the effects of the compound GEN1-284s on the pathological changes of skin redness, swelling, and scales in psoriasis mice.

[0959] Method: A total of 19 mice were used in this experiment. Natural skin was observed as a control (n = 1). 30 mice had their back shaved at 2 cm on day 0 2The fur was removed with a mild depilatory cream, and the original skin was photographed. Each mouse was treated with 62.5 mg of IMQ. The mice were divided into three groups: a mock group (n=6), a DEX group (3 mg / kg, PO., qD) group (n=6), and a GEN1-284s group (30 mg / kg, PO., qD) group (n=6). IMQ was administered 4 hours after application on the day of modeling, at a dose of 100 μl / 20 g body weight. The PASI scoring method was used to assess the erythema, swelling, and scaling of the mouse skin. The experiment lasted for 6 days, with day 0 being the first day and day 5 being the second day where only scoring was performed without IMQ application or administration. Mice were euthanized with carbon dioxide at the end of the experiment.

[0960] Results: The results showed that after 5 days of IMQ induction, mice were orally administered the test compound up to day 6. Compared with normal mice, the CTR group mice showed significantly more swollen, red, and scaly skin. Skin redness and scaling were effectively reduced after treatment with the GEN1-284s compound.

[0961] Conclusion: In summary, GEN1-284s can reduce the symptoms of redness, swelling, and scaling in psoriatic skin to a certain extent, with effects similar to those of the positive control drug Dexamethasone.

[0962] 1. Experimental Objective

[0963] Using an IMQ-induced Balb / c mouse model of psoriasis, we evaluated the effects of different doses of GEN1-284s on improving skin swelling, redness, and scaling lesions in the psoriasis model mice.

[0964] 2. Experimental Procedures

[0965] This project was conducted at Geneplus Biomedical Technology (Hangzhou) Co., Ltd. All animal experimental procedures involved in the process were carried out in accordance with Geneplus IACUC's standard laboratory animal operating procedures, laboratory animal husbandry and use guidelines, and animal welfare requirements stipulated by the Laboratory Animal Welfare Office. This experiment was a non-GLP experiment.

[0966] 3. Experimental Materials

[0967] 3.1 Instruments

[0968] Analytical balance, model: SQP, manufacturer: Beijing Sartorius Scientific Instruments Co., Ltd.

[0969] Electronic balance, model: MP5002, manufacturer: Changzhou Tianzhiping Instrument Equipment Co., Ltd.

[0970] Thermostatic magnetic stirrer, model: 85-2, manufacturer: Shanghai Sile Instruments Co., Ltd.

[0971] Digital camera, model: EOS800D, manufacturer: Canon.

[0972] 3.2 Test Drugs and Reagents

[0973] Test compound: GEN1-284s, storage conditions: -20℃ (high concentration mother liquor, powder), working solution can be prepared fresh and stored at 2-8℃ for 1 day.

[0974] Dexamethasone, Manufacturer: Sigma Lot: WXBC8102V, Storage conditions: 4℃.

[0975] Reagent: DMSO; Storage conditions: room temperature.

[0976] IMQ Imiquimod Cream (Idalor 250mg: 12.5mg*12 sachets)

[0977] MC, Manufacturer: Shanghai Sangon Biotech Co., Ltd., Storage conditions: Room temperature.

[0978] 4. Preparation of solvents and compounds

[0979] Solvent: Preparation of 0.5% MC: Accurately weigh 2g of MC into a 500ml screw-top bottle, add the rotor and place it on a magnetic stirrer, add a small amount of hot water, turn on the magnetic stirrer, and when the MC dissolves, add water in 3 portions to make 400ml, adjust the speed to 1200 rpm, and keep it for 15 minutes.

[0980] Positive reagent: Dexamethasone (3mg / kg) Preparation: Accurately weigh 3mg of the compound into a 15mL centrifuge tube, add 0.25mL of DMSO, add 4.75mL of 0.5% MC, dispense the solution into 1mL / tube, and freeze at -20℃. Before each use, take out one portion, let it thaw and mix well before use. The concentration is 0.6mg / mL.

[0981] Preparation of GEN1-284 (30mg / kg): Accurately weigh 30mg of the compound into a 15mL centrifuge tube, add 0.25mL of DMSO and 4.75mL of 0.5% MC, dispense the solution into 1mL / tube portions, and freeze at -20℃. Before each use, take out one portion and let it thaw before use. The concentration is 6mg / mL.

[0982] 5. Laboratory animals

[0983] 5.1 Use of Animals

[0984] Balb / c mice, female, 19 mice, 7-8 weeks old, weighing 18-20g, were purchased from Shanghai Slack Laboratory Animal Technology Co., Ltd.

[0985] 5.2 Animal husbandry

[0986] All animals used in the following experiments were Balb / c animals. After arriving at the facilities of Jianaishi Biomedical Technology Co., Ltd., the animals were housed in animal enclosures with strictly controlled environmental conditions. The temperature in the enclosures was maintained at 20–24°C, and the humidity at 30–70%. The temperature and humidity in the enclosures were monitored in real time using thermometers and hygrometers, and recorded twice daily (once in the morning and once in the afternoon). The lighting in the animal enclosures was controlled by an electronic timed lighting system, with the lights on for 12 hours and off for 12 hours daily (on at 6:00 AM and off at 6:00 PM). The animals had free access to feed and water.

[0987] 6. Experimental Methods

[0988] 6.1 Experimental Design

[0989] Model building

[0990] The experimental animals were anesthetized with isoflurane. A 2cm section was shaved off the back of the mice. 2 Hair was removed, and hair follicles were removed with depilatory cream. IMQ was applied for a total of 5 days, from day 0 to day 4. IMQ was applied each morning for assessment, and the medication was administered in the afternoon.

[0991] During the experiment, the animals' living conditions were observed, and any abnormalities were recorded. After the experiment, endpoint processing and sample collection were carried out.

[0992] 6.2 Grouping and Dosing

[0993] A total of 19 animals were selected for this experiment. Excluding the normal control group, they were divided into 7 groups of 8 animals each. The details of each group are shown in Table 30.

[0994] Table 30 Animal grouping and treatment

[0995]

[0996] 6.3 Endpoint detection indicators

[0997] Experimental indicators:

[0998] Desquamation (D): refers to the shedding of large patches of epidermal cells. The severity of skin lesions is scored as follows:

[0999] 0 = None; this sign cannot be confirmed upon careful observation.

[1000] 1 = Mild; this sign can be confirmed but requires careful observation.

[1001] In case of 2, this sign is quite obvious and can be confirmed immediately.

[1002] 3 = Heavy, this sign is very obvious.

[1003] 4 = Extremely heavy; this sign is very obvious.

[1004] Plaque thickening degree I: 0 - The lesion is level with the normal skin;

[1005] 1. The lesion is slightly raised above the normal skin surface;

[1006] 2- Moderate elevation, with rounded or sloping edges on the patches;

[1007] 3- The lesions are thickened and prominently raised;

[1008] 4. The skin lesions are highly thickened and the elevation is extremely obvious.

[1009] Erythema E:

[1010] 0 - No visible erythema;

[1011] 1- Appears as a pale red color;

[1012] 2-Red;

[1013] 3-Dark red;

[1014] 4-Extremely deep red

[1015] 6.4 Data Statistics

[1016] Data were collected and statistically plotted using Excel and GraphPad Prism software 5.0. Data are expressed as mean ± SEM. One-way ANOVA (Dennett's test) and t-test were used. P < 0.05 was considered statistically significant between groups.

[1017] 7. Research Results

[1018] 7.1 Effects of the test drug on the skin of psoriatic mice

[1019] Long-term application of IMQ caused severe skin swelling in mice, as shown in Table 31. Oral administration of DEX alleviated the skin swelling. From the start of modeling, mice were given oral GEN1-284s. Compared to the CTR group, the tendency for IMQ-induced skin swelling was improved, indicating that GEN1-284s can partially improve IMQ-induced psoriatic swelling in mice.

[1020] Table 31

[1021]

[1022]

[1023]

[1024]

[1025] As shown in Table 32, the redness of the mouse skin caused by applying IMQ could not be eliminated in a short time, and there was no significant difference.

[1026] Table 32

[1027]

[1028]

[1029]

[1030] As shown in Table 33, because the mice produced more scales in the later stages, this experiment only evaluated the quantity of scales and not their size. The scale score may not be of any guiding significance for this experiment.

[1031] Table 33

[1032]

[1033]

[1034]

[1035] As shown in Table 34, the overall therapeutic effect of GEN1-284s is similar to that of DEX, and it has a certain therapeutic effect on IMQ-induced psoriasis.

[1036] Table 34

[1037]

[1038]

[1039]

[1040]

[1041] 8. Conclusion

[1042] In conclusion, GEN1-284s can reduce skin swelling to some extent, similar to the positive control drug DEX.

[1043] Effect Example 9: Effect of GEN1-284s on LPS-induced TNFα production

[1044] summary

[1045] Inflammation is involved in the pathogenesis of many diseases, including aging, cancer, and cardiovascular dysfunction. Lipopolysaccharide (LPS)-induced acute inflammation in mice can lead to sepsis, causing a systemic, uncontrolled inflammatory response that can even result in death. This study used a LPS-induced sepsis mouse model and pretreated mice with GEN1-284s, using dexamethasone as a positive control, to investigate the protective effect of GEN1-284s and its possible mechanism of action.

[1046] Objective: This study evaluated the therapeutic efficacy of GEN1-284s in inhibiting LPS-induced TNF-α production in mice.

[1047] Materials and Methods: A total of 18 mice were used in this experiment. The mice were divided into three groups: the MOCK group (n=6), the Dexamethasone group (n=6), and the GEN1-284s 15mpk group (n=6). LPS (1mpk) was injected intraperitoneally 0.5 h after drug administration. Two hours after LPS injection, the mice were sacrificed and blood was collected. The blood was placed in EDTA-K2 anticoagulant tubes and incubated at room temperature for 20 minutes, then centrifuged at 8000 rpm for 10 minutes. The separated plasma was frozen at -80℃ for later analysis.

[1048] Results: In LPS-stimulated mice, GEN1-284s-15mpk inhibited TNF-α by 36%, showing a significant difference.

[1049] Conclusion: GEN1-284s inhibits LPS-induced TNF-α production in mice.

[1050] 1. Experimental Objective

[1051] To evaluate the therapeutic efficacy of GEN1-284s in inhibiting LPS-induced TNF-α production in mice.

[1052] 2. Experimental Procedures

[1053] This project was conducted at Geneplus Biomedical Technology (Hangzhou) Co., Ltd. All animal experimental procedures involved in the process were carried out in accordance with Geneplus IACUC's standard laboratory animal operating procedures, laboratory animal husbandry and use guidelines, and animal welfare requirements stipulated by the Laboratory Animal Welfare Office. This experiment was a non-GLP experiment.

[1054] 3. Experimental Materials

[1055] 3.1 Instruments

[1056] Analytical balance, model: SQP, manufacturer: Beijing Sartorius Scientific Instruments Co., Ltd.

[1057] Electronic balance, model: MP5002, manufacturer: Changzhou Tianzhiping Instrument Equipment Co., Ltd.

[1058] Miniature vortex mixer, model: XW-80A, manufacturer: Shanghai Huxi Analytical Instruments Co., Ltd.

[1059] Centrifuge, model: 5424R, manufacturer: Eppendorf.

[1060] 3.2 Test Drugs and Reagents

[1061] Test compound:

[1062] GEN1-284s, storage conditions: -20℃ (high concentration mother liquor, powder), working solution can be prepared fresh, and stored at 2-8℃ for 1 day.

[1063] Dexamethasone manufacturer: Sigma, Lot: WXBC8102V, Storage conditions: 4℃.

[1064] Reagents:

[1065] DMSO, Manufacturer: Solarbio, Lot: 1012D033, Storage conditions: Room temperature.

[1066] PEG300, Manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., Storage conditions: Room temperature.

[1067] 1×PBS, Manufacturer: Hyclone, Lot: AE29449011, Storage conditions: 4℃.

[1068] EDTA-K2, Manufacturer: Shanghai Sangon, A600075-0100, Storage conditions: 4℃.

[1069] LPS, Manufacturer: Sigma L2880, Storage conditions: 4℃.

[1070] Mouse TNF-α ELISA Kit: Manufacturer: Allison, Lot: #AA24, Storage conditions: 4℃.

[1071] 4. Preparation of solvents and compounds

[1072] Positive drug:

[1073] Dexamethasone: Dissolve in DMSO and PEG300, then dilute with PBS to the required concentration. Dosage concentration: 3 mg / kg.

[1074] 5. Laboratory animals

[1075] 5.1 Use of Animals

[1076] Eighteen female Balb / c mice, weighing 20-22 grams, were purchased from Shanghai Slack Laboratory Animal Technology Co., Ltd.

[1077] 5.2 Animal husbandry

[1078] All animals used in the following experiments were Balb / c animals. After arriving at the facilities of Jianaishi Biomedical Technology Co., Ltd., the animals were housed in animal housing with strictly controlled environmental conditions. The temperature in the housing was maintained at 20-24℃, and the humidity at 30-70%. The lighting in the animal housing was controlled by an electronic timed lighting system, which turned the lights on for 12 hours and off for 12 hours each day (on at 6:00 AM and off at 6:00 PM). The animals had free access to feed and water.

[1079] 6. Experimental Methods

[1080] 6.1 Experimental Design

[1081] Model building

[1082] 0.5 h after administration, mice were intraperitoneally injected with LPS (1 mpk). Two hours after LPS injection, mice were sacrificed and blood was collected. The blood was placed in EDTA-K2 anticoagulant tubes and left at room temperature for 20 minutes, then centrifuged at 8000 rpm for 10 minutes. The plasma was separated and frozen at -80°C for later analysis.

[1083] 6.2 Grouping and Dosing

[1084] Grouping Animal number MOCK 6 Dexamethasone (3mpk) 6 GEN1-284S(15mpk) 6

[1085] The test compound was administered by gavage, with each animal receiving 100 μl / 20 g of the compound.

[1086] Grouping Animal number Administration method frequency Start time End time MOCK 6 PO qD 13:20 15:50 Dexamethasone (3mpk) 6 PO qD 13:20 15:50 GEN1-284S(15mpk) 6 PO qD 13:30 16:00

[1087] 6.3 Endpoint detection indicators

[1088] The ELISA kit was used to detect the level of TNF-α in mice and to evaluate the inhibitory effect of GEN1-284s on it.

[1089] 6.4 Data Statistics

[1090] Data were collected and statistically plotted using Excel and GraphPad Prism software 5.0. Data are expressed as mean ± SEM. One-way ANOVA Dennett's test and t-test were used. P < 0.05 was considered statistically significant between groups.

[1091] 7. Research Results

[1092] 7.1 Detection of TNF-α concentration in mice

[1093] Following the instructions, the standard curve formula was obtained (y = 0.0038x + 0.1206, R0). 2 =0.9956), which can be used to calculate the detection value.

[1094] The results showed that the positive control drug dexamethasone significantly inhibited the production of TNF-α in LPS-induced mice, with an inhibition rate of 81% (p<0.0001, vs CTR, Two-tailed), while the test drug GEN1-284s also had a significant inhibitory effect, with an inhibition rate of 36% (p<0.01, vs CTR, Two-tailed), and the difference was statistically significant.

[1095] 8. Conclusion

[1096] In summary, in LPS-stimulated mice, GEN1-284s-15mpk significantly inhibited TNF-α production by 36%. GEN1-284s also inhibited LPS-induced TNF-α production in mice.

Claims

1. A fused ring-containing compound of Formula I or a pharmaceutically acceptable salt thereof, wherein: ###0001### Formula I m is 0, 1, 2, 3 or 4; t is 0, 1, 2, 3 or 4; with the proviso that the fused ring-containing compound of Formula I is not: ###0002### and / or, m is 0 or 1; and / or, t is 0 or 1; and wherein the definitions of the substituents are as described in any one of the following options: ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### ###0161### ###0162### ###0163### ###0164### ###0165### ###0166### ###0167### ###0168### ###0169### ###0170### ###0171### ###0172### ###0173### ###0174### ###0175### ###0176### ###0177### ###0178### ###0179### ###0180### ###0181### ###0182### ###0183### ###0184### ###0185### ###0186### ###0187### ###0188### ###0189### ###0190### ###0191### ###0192### ###0193### ###0194### ###0195### ###0196### ###0197### ###0198### ###0199### ###0200### ###0201### ###0202### ###0203### ###0204### ###0205### ###0206### ###0207### ###0208### ###0209### ###0210### ###0211### ###0212### ###0213### ###0214### ###0215### ###0216### ###0217### ###0218### ###0219### ###0220### ###0221### ###0222### ###0223### ###0224### ###0225### ###0226### ###0227### ###0228### ###0229### ###0230### ###0231### ###0232### ###0233### ###0234### ###0235### ###0236### ###0237### ###0238### ###0239### ###0240### ###0241### ###0242### ###0243### ###0244### ###0245### ###0246### ###0247### ###0248### ###0249### ###0250### ###0251### ###0252### ###0253### ###0254### ###0255### ###0256### ###0257### ###0258### ###0259### ###0260### ###0261### ###0262### ###0263### ###0264### ###0265### ###0266### ###0267### ###0268### ###0269### ###0270### ###0271### ###0272### ###0273### ###0274### ###0275### ###0276### ###0277### ###0278### ###0279### ###0280### ###0281### ###0282### ###0283### ###0284### ###0285### ###0286### ###0287### ###0288### ###0289### ###0290### ###0291### ###0292### ###0293### ###0294### ###0295### ###0296### ###0297### ###0298### ###0299### ###0300### ###0301### ###0302### ###0303### ###0304### ###0305### ###0306### ###0307### ###0308### ###0309### ###0310### ###0311### ###0312### ###0313### ###0314### ###0315### ###0316### ###0317### ###0318### ###0319### ###0320### ###0321### ###0322### ###0323### ###0324### ###0325### ###0326### ###0327### ###0328### ###0329### ###0330### ###0331### ###0332### ###0333### ###0334### ###0335### ###0336### ###0337### ###0338### ###0339### ###0340### ###0341### ###0342### ###0343### ###0344### ###0345### ###0346### ###0347### ###0348### ###0349### ###0350### ###0351### ###0352### ###0353### ###0354### ###0355### ###0356### ###0357### ###0358### ###0359### ###0360### ###0361### ###0362### ###0363### ###0364### ###0365### ###0366### ###0367### ###0368### ###0369### ###0370### ###0371### ###0372### ###0373### ###0374### ###0375### ###0376### ###0377### ###0378### ###0379### ###0380### ###0381### ###0382### ###0383### ###0384### ###0385### ###0386### ###0387### ###0388### ###0389### ###0390### ###0391### ###0392### ###0393### ###0394### ###0395### ###0396### ###0397### ###0398### ###0399### ###0400### ###0401### ###0402### ###0403### ###0404### ###0405### ###0406### ###0407### ###0408### ###0409### ###0410### ###0411### ###0412### ###0413### ###0414### ###0415### ###0416### ###0417### ###0418### ###0419### ###0420### ###0421### ###0422### ###0423### ###0424### ###0425### ###0426### ###0427### ###0428### ###0429### ###0430### ###0431### ###0432### ###0433### ###0434### ###0435### ###0436### ###0437### ###0438### ###0439### ###0440### ###0441### ###0442### ###0443### ###0444### ###0445### ###0446### ###0447### ###0448### ###0449### ###0450### ###0451### ###0452### ###0453### ###0454### ###0455### ###0456### ###0457### ###0458### ###0459### ###0460### ###0461### ###0462### ###0463### ###0464### ###0465### ###0466### ###0467### ###0468### ###0469### ###0470### ###0471### ###0472### ###0473### ###0474### ###0475### ###0476### ###0477### ###0478### ###0479### ###0480### ###0481### ###0482### ###0483### ### wherein R is hydrogen or 1 ​ R 2 is hydrogen or chloro; R 3 is hydrogen or C1-C3alkyl substituted by one or more halogen; R 4 For ​ R 4-4 independently cyano, halogen, C1-C3alkyl or C1-C3alkyl substituted with one or more halogen; ​ R 4-5 independently cyano, hydroxy, halogen, C1-C3alkyl, C1-C3alkoxy, or C1-C3alkyl substituted with one or more halogen; ​ 2. The fused ring-containing compound of claim 1, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein R 2 is hydrogen; and / or, R 1 is and / or, R 3 Ci-C3alkyl substituted by one or more halogen; ​ and / or, R 4-4 independently halogen or C1-C3alkyl; ​ and / or, R 4-5 independently cyano, halogen, C1-C3alkyl, C1-C3alkoxy, or C1-C3alkyl substituted with one or more halogen; and / or, when said R 1 is hydrogen, m+t is greater than or equal to 1.

3. The fused ring-containing compound of claim 2, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein R 4-5 independently halogen; and / or, when said R 1 m + t = 1 when said R is hydrogen.

4. The fused ring-containing compound of claim 1, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein ​ Option 1: R 1 For hydrogen or R 2 It is hydrogen; R 3 R is a C1-C3 alkyl group substituted with one or more halogens; 4 for m is 0 or 1; R 4-4 It is a halogen or a C1-C3 alkyl group; t is 0 or 1; R 4-5 It is a cyano group, a halogen, a C1-C3 alkyl group, a C1-C3 alkoxy group, or a C1-C3 alkyl group substituted with one or more halogens; however, the compound containing a fused ring as shown in Formula I is not one of the following compounds: Scheme 2: R 1 is hydrogen or R 2 is hydrogen; R 3 is C1-C3alkyl substituted by one or more halogen; R 4 is m is 0 or 1; R 4-4 is halogen or C1-C3alkyl; t is 0 or 1; R 4-5 is halogen; but the compounds containing fused rings as shown in formula I are not Scheme 3: R 1 is R 2 is hydrogen; R 3 is C1-C3alkyl substituted with one or more halogen; R 4 is m is 0 or 1; R 4-4 is halogen or C1-C3alkyl; t is 0 or 1; R 4-5 is cyano, halogen, C1-C3alkyl, C1-C3alkoxy or C1-C3alkyl substituted with one or more halogen; Scheme 4: R 1 is R 2 is hydrogen; R 3 is C1-C3 alkyl substituted with one or more halogen; R 4 is m is 0 or 1; R 4-4 is halogen or C1-C3 alkyl; t is 0 or 1; R 4-5 is halogen.

5. The fused ring-containing compound of Formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein when said R 3 when said R is C1-C3alkyl substituted with one or more halogen, said number is 2 or 3; and / or, when said R 3 is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine or chlorine; and / or, when said R 3 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl, ethyl, n-propyl or isopropyl; and / or, when said R 4-4 is halogen, said halogen is fluorine or chlorine; and / or, when said R 4-4 when said C1-C3alkyl is methyl, ethyl, n-propyl or isopropyl; and / or, when said R 4-4 when said R is C1-C3alkyl substituted with one or more halogens, said number is 2 or 3; and / or, when said R 4-4 when said R is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine or chlorine; and / or, when said R 4-4 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl, ethyl, n-propyl or isopropyl; and / or, when said R 4-5 is halogen, said halogen is fluorine or chlorine; and / or, when said R 4-5 is C1-C3alkyl, said C1-C3alkyl is methyl, ethyl, n-propyl or i-propyl; and / or, when said R 4-5 when said C1-C3alkoxy is C1-C3alkoxy, said C1-C3alkoxy is methoxy, ethoxy, n-propoxy or isopropoxy; and / or, when said R 4-5 is C1-C3alkyl substituted with one or more halogen, said number is 2 or 3; and / or, when said R 4-5 is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine or chlorine; and / or, when said R 4-5 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl, ethyl, n-propyl or isopropyl; and / or, when said R 1 is a pharmaceutically acceptable salt is a sodium salt.

6. The fused ring-containing compound of claim 5, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein when said R 3 when said R is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine; and / or, when said R 3 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl; and / or, when said R 4-4 is halogen, said halogen is chloro; and / or, when said R 4-4 is C1-C3alkyl, said C1-C3alkyl is methyl; and / or, when said R 4-4 is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine; and / or, when said R 4-4 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl; and / or, when said R 4-5 is halogen, said halogen is chloro; and / or, when said R 4-5 is C1-C3alkyl, said C1-C3alkyl is methyl; and / or, when said R 4-5 is C1-C3alkoxy, said C1-C3alkoxy is methoxy; and / or, when said R 4-5 is C1-C3alkyl substituted by one or more halogen, said halogen is fluorine; and / or, when said R 4-5 is C1-C3alkyl substituted by one or more halogen, said C1-C3alkyl is methyl; and / or, when said R 1 is sodium, one or both hydrogens in the -NH2group are replaced by a sodium ion.

7. The fused ring-containing compound of claim 6, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein when said R 3 when said R is a C1-C3alkyl substituted with multiple halogens, said "C1-C3alkyl substituted with multiple halogens" is trifluoromethyl; and / or, when said R 4-4 is a C1-C3alkyl substituted with multiple halogens, said "C1-C3alkyl substituted with multiple halogens" is trifluoromethyl; and / or, when said R 4-5 when said R is a C1-C3alkyl substituted with multiple halogens, said "C1-C3alkyl substituted with multiple halogens" is trifluoromethyl.

8. The fused ring-containing compound of claim 1, or a pharmaceutically acceptable salt thereof, according to Formula I, wherein ​ and / or, the pharmaceutically acceptable salt of the compound containing a fused ring as shown in Formula I is ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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