Phthalazinone derivative with PARP1 / CDK4 / 6 double-target inhibitory activity as well as preparation method and application of phthalazinone derivative

Abstract

The invention discloses a phthalazinone derivative with PARP1 / CDK4 / 6 double-target inhibitory activity as well as a preparation method and application of the phthalazinone derivative. The invention provides a phthalazinone derivative with PARP1 / CDK4 / 6 double-target inhibitory activity, a pharmaceutical composition containing the derivative shown in the formula (I), a hydrate, and isotope derivatives, chiral isomers, allosteric substances, different salts, prodrugs, preparations and the like of the compounds, wherein the phthalazinone derivative has the structure shown in the formula (I). The invention also provides a preparation method of the phthalazinone derivative with PARP1 / CDK4 / 6 double-target inhibitory activity and application of the compounds in treatment of solid tumors and hematoma.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to anticancer compounds derived from nuclear steroid receptor binders, products containing such compounds, and their use and preparation methods. Specifically, it relates to a phthalazinone derivative with dual PARP1 / CDK4 / 6 inhibitory activity, its preparation method, and its application. Background Technology

[0002] Poly(ADP-ribose)polymerase-1 (PARP1) is the most abundant and best-understood protein in the PARP family. As a DNA-dependent ribozyme, PARP1 plays a crucial role in DNA damage signaling and repair. When single-stranded DNA (ssDNA) breaks occur, PARP1 participates in the repair of single-strand breaks (SSBs). However, due to its low accuracy in repair, the tendency to perform erroneous SSB repair increases the likelihood of cell carcinogenesis, leading to PARP1 overexpression in cancer cells. In PARP1 deficiency, ssDNA damage is repaired by homologous recombination (HR). Due to its low accuracy in DNA damage repair and its high expression levels in cancer cells, PARP1 has been considered a major target for cancer therapy. PARP inhibitors were the first drugs to achieve clinical success using the concept of "synthetic lethality." "Synthetic lethality" refers to the phenomenon where mutations in two genes simultaneously lead to cell death, whereas mutations in a single gene alone do not cause cell death. PARP inhibitors work by blocking DNA repair, causing cell death, and thus inhibiting tumor growth. This mechanism of preventing cell growth results in significant antitumor activity in tumors with BRCA1, BRCA2, and PALB2 mutations because these proteins are important for repairing double-strand DNA breaks via the homologous recombination repair (HRR) pathway. Normal cells divide less rapidly than tumor cells and do not carry mutated BRCA1 or BRCA2, thus maintaining the integrity of the HRR pathway, which allows them to survive better when faced with PARP inhibition. PARP inhibitors, such as olaparib, rucaparib, niraparib, talazoparib, fluzoparib, and pamiparib, are approved for clinical treatment of patients with BRCA-mutated ovarian and breast cancer.

[0003] Cyclin-dependent kinases (CDKs) and cyclins are crucial factors in cell cycle regulation. CDKs can bind to cyclins to form heterodimers, where CDKs are the catalytic subunit and cyclins are the regulatory subunit, forming various cyclin-CDK complexes that phosphorylate different substrates, thus promoting and transitioning between different phases of the cell cycle. Over the past decade, CDK inhibitors have become a hot topic in the global pharmaceutical industry as novel anti-tumor drugs, with more than 20 CDK inhibitors entering clinical development. Although CDK inhibitors have shown significant preclinical pharmacodynamic results in anti-tumor studies, many early clinical trials yielded unsatisfactory results, mainly due to a lack of efficacy against solid tumors and significant toxicity. The clinical trials of CDK inhibitors AG-24322, ZK-304709, SNS-032, R547, Seliciclib, and AZD5438 were terminated due to efficacy and toxicity issues. Analysis of the causes of severe toxic side effects revealed that these drugs lacked selectivity in inhibiting CDK subtypes, thus leading to serious adverse reactions.

[0004] Studies have found that the Wnt signaling pathway is activated in acquired olaparib-resistant cells. Any activation of the Wnt signaling pathway, such as overexpression of β-catenin (a core molecule of the Wnt pathway) or its hyperphosphorylation at Ser675 (promoting its nuclear translocation and thus positively regulating Wnt), when combined with CDK4 / 6-PARP1 inhibitors, can mediate resistance to olaparib. However, this can be inhibited by the combined use of CDK4 / 6-PARP1 inhibitors like palbociclib. This research may pave the way for new treatment options for both primary and acquired olaparib resistance. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a phthalazinone derivative with dual-target inhibitory activity against PARP1 / CDK4 / 6.

[0006] The present invention adopts the following technical solution:

[0007] A phthalazinone derivative with dual PARP1 / CDK4 / 6 inhibitory activity, or its optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate, wherein the structure of the derivative is shown in formula (I):

[0008]

[0009] in:

[0010] R1 R 2 R 3 R 4 R 5 The group is selected from hydrogen atoms, halogen atoms, alkyl groups, hydroxyl groups, amino groups, nitro groups, aryl groups, cycloalkyl groups, heterocyclic groups, or electrophilic groups that can covalently react with amino acid residues of the target protein; wherein, the electrophilic group that can covalently react with amino acid residues of the target protein includes acrylamide, α-haloketone, cyano, acetamido, mercapto, epoxide, vinyl sulfone, or activated acetylene. Indicate Y and L 1 The connection site, L represents 1 With L 2 The connection site, L represents 2 With L 3 The connection site, L represents 3 and The connection site,

[0011] X is selected from C or N; when X is selected from N, there is no R. 6 replace;

[0012] Y is CH2 or carbonyl;

[0013] R 5 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, or C3-C6 cycloalkyl-substituted methyl groups, specifically the following structural fragments:

[0014]

[0015] Where n = 0, 1, 2;

[0016] R 6 Selected from one of H, F, CN, and CH3;

[0017] L 1 Selected from NH, chemical bonds, or the following structural fragments:

[0018]

[0019] Where m 1 m 3 =1, 2, 3, 4; m 2 =0, 1, 2, 3, 4, 5, 6; R 7 R 8 R 9 R 10 R 11The components are independently selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituents are H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, and C3-C6 cycloalkyl groups; wherein L represents 1 Connection site with Y, L represents 1 With L 2 Connection sites;

[0020] L 2 Selected from C2-C 12 Alkyl, cycloalkyl, chemical bond or the following structural fragments:

[0021]

[0022] Where m 1 m 2 =0, 1, 2, 3, 4, where R 7 R 8 R 9 R 10 R 11 The group is selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituent is H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, or C3-C6 cycloalkyl groups; wherein L represents 1 With L 2 The connection site, L represents 2 With L 3 Connection sites;

[0023] L 3 It is either NH or a chemical bond.

[0024] or L 1 -L 2 Together they form the following heterocyclic segments:

[0025]

[0026] Where m 1 m 3 =0, 1, 2, 3, 4; R 7 R 8 R 11 R 12The components are independently selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituents are H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, and C3-C6 cycloalkyl groups; wherein L represents 1 -L 2 Connection site with Y, L represents 1 -L 2 With L 3 Connection sites;

[0027] As a preferred option, R 1 R 2 R 3 R 4 R 5 Selected from halogen atoms, alkyl groups, or electrophilic groups that can covalently react with amino acid residues of the target protein; including acrylamide, α-haloketone, cyano, acetamido, mercapto, epoxide, vinyl sulfone, or activated acetylene; more preferably, R l R 2 R 3 For F, R 4 For methyl, R 5 It is isopropyl;

[0028] L 1 Selected from NH, chemical bonds, or the following structural fragments:

[0029]

[0030] L 2 Selected from C2-C 12 Alkyl, cycloalkyl, chemical bond, or selected from the following structural segments:

[0031]

[0032] L 3 It is either NH or a chemical bond.

[0033] or L 1 -L 2 Together they form the following heterocyclic segments:

[0034]

[0035] Preferably, the chemical structural formula of the phthalazinone derivative is any one of the following formulas 1 to 36:

[0036]

[0037]

[0038] The second objective of this invention is to provide a method for preparing the above-mentioned phthalazinone derivatives with dual PARP1 / CDK4 / 6 inhibitory activity, which is prepared using the general reaction process of Scheme 1 or Scheme 2 below:

[0039]

[0040] When Y is a carbonyl group, the synthetic route (I) of Scheme 1 is adopted. The starting material a1 and a group containing a protecting group... The a2 fragment undergoes substitution or Buchwald–Hartwig coupling reaction to give intermediate a3; after the protecting group of a3 is removed, intermediate a4 is given; a4 and a5 amide condensation reaction gives the compound shown in formula (I).

[0041]

[0042] When Y is CH2, the synthesis route (I) of scheme 2 is adopted: raw material b1 and a compound containing a protecting group The fragment b2 undergoes substitution or Buchwald–Hartwig coupling reaction to give intermediate b3; b3 is deprotected and reacted to give intermediate b4; b4 and b5 are substituted to give the compound shown in formula (I).

[0043] The preparation methods of compounds of general formula (I) of the present invention have been specifically described above, but these specific methods do not constitute any limitation on the present invention. The compounds of general formula (I) described above can be synthesized using standard synthetic techniques or known techniques combined with the methods described herein. Furthermore, the solvents, temperatures, and other reaction conditions mentioned herein can be varied. The starting materials used for the synthesis of the compounds can be obtained synthetically or from commercial sources. Meanwhile, the compounds described herein and other related compounds with different substituents can be synthesized using known techniques and starting materials.

[0044] On the other hand, the compounds described herein are prepared according to methods known in the art. However, the conditions of the method, such as reactants, solvents, bases, amounts of compounds used, reaction temperatures, and reaction times, are not limited to those explained below. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art.

[0045] A third objective of this invention is to provide the use of the phthalazinone derivatives having dual PARP1 / CDK4 / 6 inhibitory activity, or their optical isomers, racemates, single enantiomers, possible diastereomers, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, or solvates in the preparation of drugs for treating or preventing tumors.

[0046] A fourth objective of this invention is to provide an antitumor drug containing a safe and effective amount of the aforementioned phthalazinone derivative having dual PARP1 / CDK4 / 6 inhibitory activity, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

[0047] Preferably, the antitumor drug may further include a pharmacologically acceptable salt and a pharmacologically acceptable excipient or carrier.

[0048] Preferably, in the application and the antitumor drug, the tumor includes solid tumors and hematologic malignancies.

[0049] Because the compounds of the present invention have the activity of inhibiting the proliferation of various tumor cell lines, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate various diseases, including various cancers.

[0050] The "safe and effective amount" described in this invention refers to an amount of compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of this invention per dose, more preferably, 5-1000 mg of the compound of this invention per dose. Preferably, "one dose" refers to one capsule or tablet.

[0051] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0052] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and local administration.

[0053] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0054] Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active compound or compound in such compositions may be delayed at a specific site within the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0055] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0056] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0057] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

[0058] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0059] Dosage forms of the compounds of this invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

[0060] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0061] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–5000 mg, preferably 5–2000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0062] The beneficial effects of this invention are as follows:

[0063] Compared with existing technologies, this invention combines the PARP1 inhibitor olaparib and the CDK4 / 6 inhibitor abemaciclib using high-throughput screening, homologue synthesis, substrate structure design, and fragment assembly strategies to synthesize novel phthalazinone compounds with dual PARP1 / CDK4 / 6 inhibitory activity, thereby enhancing antitumor activity. This is groundbreaking compared to simple structural modifications. This invention also provides methods for preparing phthalazinone compounds with dual PARP1 / CDK4 / 6 inhibitory activity, their uses, and their inhibitory activity against the proliferation of various tumor cell lines. The phthalazinone compounds with dual PARP1 / CDK4 / 6 inhibitory activity described in this invention hold promise as antitumor drug candidates for treating various cancers, such as solid tumors and hematological malignancies. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0065] Example 1: Preparation of Compound 1

[0066]

[0067] Step 1: Preparation of Compound 1-1

[0068] 4-(6-aminopyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester (300 mg, 1.08 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (347 mg, 1.08 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (278 mg, 2.16 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (98.7 mg, 0.11 mmol) and XantPhos (62 mg, 0.11 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellowish-brown solid compound 1-1 (512 mg, 84% yield). LC-MS (ESI): m / z 565.3 [M+1] + .

[0069] Step 2: Preparation of Compounds 1-2

[0070] Compound 1-1 (512 mg, 0.91 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 1-2 (413 mg, 91.1% yield).

[0071] Step 3: Preparation of Compound 1

[0072] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compounds 1-2 (201 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 1 (173.0 mg, 68.7% yield) as a white solid. LC-MS (ESI): m / z 745.0 [M+1] + .

[0073] Example 2: Preparation of Compound 2

[0074]

[0075] Step 1: Preparation of Compound 2-1

[0076] 4-(6-aminonicotinyl)piperazine-1-carboxylic acid tert-butyl ester (285 mg, 0.93 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (300 mg, 0.93 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (240 mg, 1.86 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (85 mg, 0.09 mmol) and XantPhos (54 mg, 0.09 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a pale yellow solid compound 2-1 (491 mg, 76.7% yield). LC-MS (ESI): m / z 593.0 [M+1] + .

[0077] Step 2: Preparation of Compound 2-2

[0078] Compound 2-1 (491 mg, 0.91 mmol) was dissolved in MeOH (5 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 2-2 (384 mg, 87.5% yield).

[0079] Step 3: Preparation of Compound 2

[0080] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 2-2 (216 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 2 (143.0 mg, 54.6% yield) as a white solid. LC-MS (ESI): m / z 773.0 [M+1] + .

[0081] Example 3: Preparation of Compound 3

[0082]

[0083] Step 1: Preparation of Compound 3-1

[0084] 4-((6-aminopyridin-3-yl)methyl)piperazine-1-carboxylic acid tert-butyl ester (285 mg, 0.93 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (300 mg, 0.93 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (240 mg, 1.86 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (85 mg, 0.09 mmol) and XantPhos (54 mg, 0.09 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellowish-brown solid compound 3-1 (355 mg, 59.5% yield). LC-MS (ESI): m / z 581.0 [M+1] + .

[0085] Step 2: Preparation of compound 3-2

[0086] Compound 3-1 (355 mg, 0.91 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 3-2 (295 mg, 93.4% yield).

[0087] Step 3: Preparation of Compound 3

[0088] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 3-2 (193 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 3 (173.0 mg, 68.7% yield) as a white solid. LC-MS (ESI): m / z 759.0 [M+1] +.

[0089] Example 4: Preparation of Compound 4

[0090]

[0091] Step 1: Preparation of Compound 4-1

[0092] 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester (250 mg, 0.90 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (290 mg, 0.90 mmol) were dissolved in 1,4-dioxane (5 mL), and Cs₂CO₃ (438 mg, 1.35 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (82 mg, 0.09 mmol) and X-Phos (43 mg, 0.09 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellowish-brown solid compound 4-1 (381 mg, 75% yield). LC-MS (ESI): m / z 564.0 [M+1] + .

[0093] Step 2: Preparation of compound 4-2

[0094] Compound 4-1 (381 mg, 0.67 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 4-2 (264 mg, 78.8% yield).

[0095] Step 3: Preparation of Compound 4

[0096] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 4-2 (193 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 4 (184.0 mg, 74% yield) as a white solid. LC-MS (ESI): m / z 744.0 [M+1] + .

[0097] Example 5: Preparation of Compound 5

[0098]

[0099] Step 1: Preparation of Compound 5-1

[0100] 4-(4-aminobenzoyl)piperazine-1-carboxylic acid tert-butyl ester (300 mg, 0.98 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (315 mg, 0.98 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (253 mg, 1.96 mmol) was added. The system was purged three times with nitrogen, and Pd2(dba)3 (90 mg, 0.1 mmol) and XantPhos (57 mg, 0.1 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a pale yellow solid compound 5-1 (238 mg, 41% yield). LC-MS (ESI): m / z 592.0 [M+1] + .

[0101] Step 2: Preparation of Compound 5-2

[0102] Compound 5-1 (238 mg, 0.40 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 5-2 (384 mg, 87.5% yield).

[0103] Step 3: Preparation of Compound 5

[0104] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 5-2 (215 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 5 (126.0 mg, 48% yield) as a white solid. LC-MS (ESI): m / z 772.0 [M+1] + .

[0105] Example 6: Preparation of Compound 6

[0106]

[0107] Step 1: Preparation of Compound 6-1

[0108] 4-(4-aminobenzyl)piperazine-1-carboxylic acid tert-butyl ester (120 mg, 0.41 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (132 mg, 0.41 mmol) were dissolved in 1,4-dioxane (5 mL), and cesium carbonate (200 mg, 0.62 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (38 mg, 0.04 mmol) and X-Phos (20 mg, 0.04 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellow solid compound 6-1 (141 mg, 59.7% yield). LC-MS (ESI): m / z 581.0 [M+1] + .

[0109] Compound 6-1 (355 mg, 0.91 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a white solid compound 6-2 (123 mg, 94.6% yield).

[0110] Step 3: Preparation of Compound 6

[0111] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (60 mg, 0.24 mmol), HOBt (36 mg, 0.26 mmol), EDCI (100.0 mg, 0.52 mmol), and DIPEA (104 mg, 0.80 mmol) were added sequentially, followed by the slow addition of compound 6-2 (123 mg, 0.24 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (30 mL) and extracted with DCM (3 × 30 mL). The combined organic phases were washed with saturated saline solution (30 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 6 (95.0 mg, 52.5% yield) as a white solid. LC-MS (ESI): m / z 759.0 [M+1] + .

[0112] Example 7: Preparation of Compound 7

[0113]

[0114] Step 1: Preparation of Compound 7-1

[0115] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (142 mg, 0.48 mmol), HOBt (97 mg, 0.72 mmol), EDCI (274 mg, 1.44 mmol), and DIPEA (248 mg, 1.92 mmol) were added sequentially, followed by the slow addition of tert-butyl (5-aminopyridin-2-yl)carbamate (100 mg, 0.48 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 7-1 as a white solid (151.0 mg, 64% yield). LC-MS (ESI): m / z 490.0 [M+1] + .

[0116] Step 2: Preparation of Compound 7-2

[0117] Compound 7-1 (151 mg, 0.30 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until complete, and a large amount of solid appeared. The solid was collected by filtration and dried under vacuum to give a yellow solid, compound 7-2 (120 mg, 91.6% yield). LC-MS (ESI): m / z 390.0 [M+1] + .

[0118] Step 3: Preparation of Compound 7

[0119] Compound 7-2 (120 mg, 0.28 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (90 mg, 0.28 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (144 mg, 1.13 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (26 mg, 0.03 mmol) and XantPhos (16 mg, 0.03 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (30 mL) and extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with saturated saline solution (30 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellow solid compound 7 (86 mg, 45.5% yield). LC-MS (ESI): m / z 581.0 [M+1] + .

[0120] Example 8: Preparation of Compound 8

[0121]

[0122] Step 1: Preparation of Compound 8-1

[0123] Piperazine-1-carboxylic acid tert-butyl ester (345 mg, 1.86 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (300 mg, 0.93 mmol) were dissolved in DMF (5 mL), and potassium carbonate (251 mg, 1.86 mmol) was added. The system was stirred overnight at 85 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 8-1 (362 mg, 82.5% yield). LC-MS (ESI): m / z 473.2 [M+1] + .

[0124] Step 2: Preparation of compound 8-2

[0125] Compound 8-1 (362 mg, 0.77 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 8-2 (288 mg, 92% yield).

[0126] Step 3: Preparation of Compound 8

[0127] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 8-2 (166 mg, 0.34 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 8 (125.0 mg, 57% yield) as a white solid. LC-MS (ESI): m / z 653.0 [M+1] + .

[0128] Example 9: Preparation of Compound 9

[0129]

[0130] Step 1: Preparation of Compound 9-1

[0131] (2-((6-aminopyridin-3-yl)(methyl)amino)ethyl)(methyl ester) tert-butyl carbamate (120 mg, 0.43 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (138 mg, 0.43 mmol) were dissolved in 1,4-dioxane (5 mL), and cesium carbonate (279 mg, 0.86 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (40 mg, 0.04 mmol) and XantPhos (20 mg, 0.04 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried with anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a yellowish-brown solid compound 9-1 (95 mg, 39% yield).

[0132] Step 2: Preparation of Compound 9-2

[0133] Compound 9-1 (95 mg, 0.17 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 9-2 (55 mg, 57.9% yield).

[0134] Step 3: Preparation of Compound 9

[0135] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (33 mg, 0.1096 mmol), HOBt (19 mg, 0.1425 mmol), EDCI (54.0 mg, 0.2850 mmol), and DIPEA (57 mg, 0.4384 mmol) were added sequentially, followed by the slow addition of compound 9-2 (55 mg, 0.1096 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 9 (53.0 mg, 59% yield) as a white solid. LC-MS (ESI): m / z 747.0 [M+l] + .

[0136] Example 10: Preparation of Compound 10

[0137]

[0138] Step 1: Preparation of Compound 10-1

[0139] tert-Butyl-7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylic acid (100 mg, 0.40 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (129 mg, 0.40 mmol) were dissolved in toluene (5 mL), and cesium carbonate (263 mg, 0.85 mmol) was added. The system was purged three times with nitrogen, and Pd2(dba)3 (37 mg, 0.04 mmol) and XantPhos (23 mg, 0.04 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a brown solid compound 10-1 (136 mg, 64% yield).

[0140] Step 2: Preparation of compound 10-2

[0141] Compound 10⁻¹ (136 mg, 0.25 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 10⁻² (100 mg, 84% yield).

[0142] Step 3: Preparation of Compound 10

[0143] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 10-2 (160 mg, 0.34 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 10 (165.0 mg, 68% yield) as a white solid. LC-MS (ESI): m / z 715.0 [M+] + .

[0144] Example 11: Preparation of Compound 11

[0145]

[0146] Step 1: Preparation of Compound 11-1

[0147] 5-Aminoisoindole-2-carboxylic acid tert-butyl ester (100 mg, 0.40 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (138 mg, 0.40 mmol) were dissolved in toluene (5 mL), and cesium carbonate (280 mg, 0.85 mmol) was added. The system was purged three times with nitrogen, and Pd2(dba)3 (39 mg, 0.04 mmol) and XantPhos (25 mg, 0.04 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 11-1 (184 mg, 82% yield).

[0148] Step 2: Preparation of compound 11-2

[0149] Compound 11-1 (95 mg, 0.17 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 11-2 (150 mg, 76.5% yield).

[0150] Step 3: Preparation of Compound 11

[0151] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 11-2 (150 mg, 0.33 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 11 (145.0 mg, 60% yield) as a white solid. LC-MS (ESI): m / z 701.8 [M+1] + .

[0152] Example 12: Preparation of Compound 12

[0153]

[0154] Step 1: Preparation of Compound 12-1

[0155] 6-Amino-3,4-dihydro-2,7-naphthyl-2(1H)-carboxylic acid tert-butyl ester (100 mg, 0.40 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (130 mg, 0.40 mmol) were dissolved in toluene (3 mL), and cesium carbonate (260 mg, 0.80 mmol) was added. The system was purged three times with nitrogen, and Pd2(dba)3 (37 mg, 0.04 mmol) and XantPhos (23 mg, 0.04 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 12-1 (144 mg, 67% yield).

[0156] Step 2: Preparation of Compound 12-2

[0157] Compound 12-1 (144 mg, 0.27 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 12-2 (100 mg, 78% yield).

[0158] Step 3: Preparation of Compound 12-2

[0159] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 12-2 (160 mg, 0.34 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 12 (142.0 mg, 58% yield) as a white solid. LC-MS (ESI): m / z 716.8 [M+1] + .

[0160] Example 13: Preparation of Compound 13

[0161]

[0162] Step 1: Preparation of Compound 13-1

[0163] 4-Amino-1-tert-butoxycarbonylpiperidine (223 mg, 1.12 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (300 mg, 0.93 mmol) were dissolved in DMF (5 mL), and potassium carbonate (251 mg, 1.86 mmol) was added. The system was stirred overnight at 85 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 13-1 (376 mg, 83% yield).

[0164] Step 2: Preparation of Compound 13-2

[0165] Compound 13-1 (360 mg, 0.77 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 13-2 (298 mg, 94% yield).

[0166] Step 3: Preparation of Compound 13

[0167] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 13-2 (143 mg, 0.34 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 13 (144.0 mg, 64% yield) as a white solid.

[0168] Example 14: Preparation of Compound 14

[0169]

[0170] Step 1: Synthesis of Compound 14-1

[0171] p-Phenylenediamine (120 mg, 1.12 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (300 mg, 0.93 mmol) were dissolved in DMF (5 mL), and potassium carbonate (251 mg, 1.86 mmol) was added. The system was stirred overnight at 85 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 14-1 (100 mg, 28% yield).

[0172] Step 2: Synthesis of Compound 14

[0173] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (60 mg, 0.20 mmol), HOBt (35 mg, 0.26 mmol), EDCI (99.0 mg, 0.52 mmol), and DIPEA (103 mg, 0.80 mmol) were added sequentially, followed by the slow addition of compound 14-1 (95 mg, 0.24 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 14 (82.0 mg, 61% yield) as a white solid.

[0174] Example 15: Preparation of Compound 15

[0175]

[0176] Step 1: Synthesis of Compound 15-1

[0177] To a DMF (5 mL) solution of aminobenzoic acid (300 mg, 2.20 mmol), HOBt (386 mg, 2.86 mmol), EDCI (1.09 g, 5.72 mmol), and DIPEA (1.14 g, 8.80 mmol) were added sequentially, followed by the slow addition of tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (670 mg, 2.64 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 15-1 (685.0 mg, 71% yield) as a white solid.

[0178] Step 2: Synthesis of Compound 15-2

[0179] Compound 15-1 (231 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 15-2 (316 mg, 77% yield).

[0180] Step 3: Synthesis of Compound 15-3

[0181] Compound 15-2 (316 mg, 0.48 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 15-3 (259 mg, 96% yield).

[0182] Step 3: Synthesis of Compound 15

[0183] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 15-3 (243 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 15 as a white solid (184.0 mg, 65% yield).

[0184] Example 16: Preparation of Compound 16

[0185]

[0186] Step 1: Synthesis of Compound 16-1

[0187] To a DMF (5 mL) solution of aminobenzoic acid (300 mg, 2.20 mmol), HOBt (386 mg, 2.86 mmol), EDCI (1.09 g, 5.72 mmol), and DIPEA (1.14 g, 8.80 mmol) were added sequentially, followed by the slow addition of 2-Boc-2,7-diaza-spiro[4.4]nonane (596 mg, 2.64 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 16-1 (577.0 mg, 76% yield) as a white solid.

[0188] Step 2: Synthesis of Compound 16-2

[0189] Compound 16-1 (214 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 16-2 (272 mg, 69% yield).

[0190] Step 3: Synthesis of Compound 16-3

[0191] Compound 16-2 (272 mg, 0.43 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 16-3 (238 mg, 96% yield).

[0192] Step 4: Synthesis of Compound 16

[0193] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 16-3 (229 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 16 (173.0 mg, 62% yield) as a white solid.

[0194] Example 17: Preparation of Compound 17

[0195]

[0196] Step 1: Synthesis of Compound 17-1

[0197] To a DMF (5 mL) solution of aminobenzoic acid (300 mg, 2.20 mmol), HOBt (386 mg, 2.86 mmol), EDCI (1.09 g, 5.72 mmol), and DIPEA (1.14 g, 8.80 mmol) were added sequentially, followed by the slow addition of 2-Boc-octahydropyrrole[3,4-C]pyrrole (559 mg, 2.64 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 17-1 (535.0 mg, 73% yield) as a white solid.

[0198] Step 2: Synthesis of Compound 17-2

[0199] Compound 17-1 (205 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 17-2 (305 mg, 79% yield).

[0200] Step 3: Synthesis of Compound 17-3

[0201] Compound 17-2 (300 mg, 0.49 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 17-3 (248 mg, 98% yield).

[0202] Step 3: Synthesis of Compound 17

[0203] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 17-3 (223 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 17 (183.0 mg, 68% yield) as a white solid.

[0204] Example 18: Preparation of Compound 18

[0205]

[0206] Step 1: Synthesis of Compound 18-1

[0207] t-BuOK (378 mg, 3.38 mmol) was added to a DMA (10 mL) solution of 5-fluoro-2-nitropyridine (400 mg, 2.82 mmol). The system was stirred at 0 °C for 1 h, and then N-Boc-4-hydroxypiperidine (680 mg, 3.38 mmol) was slowly added. The system was stirred overnight at room temperature. The reaction was detected by TLC to indicate complete reaction. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 18-1 (664.0 mg, 73% yield).

[0208] Step 2: Synthesis of Compound 18-2

[0209] Compound 18-1 (650 mg, 2.00 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 18-2 (461 mg, 78% yield), which was used directly in the next step of the reaction without purification.

[0210] Step 3: Synthesis of Compound 18-3

[0211] 18-2 (182 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 18-3 (282 mg, 80% yield).

[0212] Step 4: Synthesis of Compound 18-4

[0213] Compound 18-3 (270 mg, 0.47 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 18-4 (219 mg, 95% yield).

[0214] Step 5: Synthesis of Compound 18

[0215] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 18-4 (210 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 18 (163.0 mg, 62% yield) as a white solid.

[0216] Example 19: Preparation of Compound 19

[0217]

[0218] Step 1: Synthesis of Compound 19-1

[0219] t-BuOK (378 mg, 2.55 mmol) was added to a DMA (10 mL) solution of 5-fluoro-2-nitrobenzene (300 mg, 2.13 mmol). The system was stirred at 0 °C for 1 h, and then N-Boc-4-hydroxypiperidine (513 mg, 2.55 mmol) was slowly added. The system was stirred overnight at room temperature. The reaction was detected by TLC to indicate complete reaction. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a pale yellow solid compound 19-1 (518.0 mg, 73% yield).

[0220] Step 2: Synthesis of Compound 19-2

[0221] Compound 19-1 (500 mg, 1.55 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 19-2 (411 mg, 90% yield), which was used directly in the next step of the reaction without purification.

[0222] Step 3: Synthesis of Compound 19-3

[0223] 19-2 (181 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 19-3 (288 mg, 80% yield).

[0224] Step 4: Synthesis of Compound 19-4

[0225] Compound 19-3 (280 mg, 0.48 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 19-4 (209 mg, 90% yield).

[0226] Step 5: Synthesis of Compound 19

[0227] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 19-4 (192 mg, 0.37 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 19 (163.0 mg, 62% yield) as a white solid.

[0228] Example 20: Preparation of Compound 20

[0229]

[0230] Step 1: Synthesis of Compound 20-1

[0231] To a solution of 5-fluoro-2-nitropyridine (300 mg, 2.11 mmol) in DMA (10 mL), NaH (76 mg, 3.17 mmol) was added. The system was stirred at 0 °C for 1 h, and then N-Boc-4-piperidinemethanol (545 mg, 2.53 mmol) was slowly added. The system was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography to give a pale yellow solid compound 20⁻¹ (513.0 mg, 72% yield).

[0232] Step 2: Synthesis of Compound 20-2

[0233] Compound 20-1 (500 mg, 1.48 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 20-2 (389 mg, 85% yield), which was used directly in the next step of the reaction without purification.

[0234] Step 3: Synthesis of Compound 20-3

[0235] 20-2 (190 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 20-3 (320 mg, 87% yield).

[0236] Step 4: Synthesis of Compound 20-4

[0237] Compound 20-3 (310 mg, 0.52 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 20-4 (241 mg, 95% yield).

[0238] Step 5: Synthesis of Compound 20

[0239] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 20-4 (215 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 20 (128.0 mg, 49% yield) as a white solid.

[0240] Example 21: Preparation of compound 21

[0241]

[0242] Step 1: Synthesis of Compound 21-1

[0243] To a solution of 5-fluoro-2-nitrobenzene (300 mg, 2.11 mmol) in DMA (10 mL), NaH (76 mg, 3.17 mmol) was added. The system was stirred at 0 °C for 1 h, and then N-Boc-4-piperidinemethanol (548 mg, 2.53 mmol) was slowly added. The system was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography to give a pale yellow solid compound 21-1 (573.0 mg, 80% yield).

[0244] Step 2: Synthesis of Compound 21-2

[0245] Compound 21-1 (565 mg, 1.68 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 21-2 (441 mg, 86% yield), which was used directly in the next step of the reaction without purification.

[0246] Step 3: Synthesis of Compound 21-3

[0247] 20-2 (190 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 21-3 (311 mg, 85% yield).

[0248] Step 4: Synthesis of Compound 21-4

[0249] Compound 21-3 (300 mg, 0.51 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 21-4 (241 mg, 89% yield).

[0250] Step 5: Synthesis of Compound 21

[0251] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 21-4 (215 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 21 (172.0 mg, 66% yield) as a white solid.

[0252] Example 22: Preparation of compound 22

[0253]

[0254] Step 1: Preparation of Compound 22-1

[0255] 4-(3-aminobenzoyl)-piperazine-1-carboxylic acid tert-butyl ester (300 mg, 0.98 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (315 mg, 0.98 mmol) were dissolved in 1,4-dioxane (5 mL), and DIPEA (253 mg, 1.96 mmol) was added. The system was purged with nitrogen three times, and Pd2(dba)3 (90 mg, 0.1 mmol) and XantPhos (57 mg, 0.1 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellowish-brown solid compound 22-1 (238 mg, 41% yield). LC-MS (ESI): m / z 592.0 [M+1] + .

[0256] Step 2: Preparation of compound 22-2

[0257] Compound 22-1 (238 mg, 0.40 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 22-2 (384 mg, 87.5% yield).

[0258] Step 3: Preparation of Compound 22

[0259] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 22-2 (215 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 22 (126.0 mg, 48% yield) as a white solid. LC-MS (ESI): m / z 772.0 [M+1] + .

[0260] Example 23: Preparation of compound 23

[0261]

[0262] Step 1: Preparation of Compound 23-1

[0263] 4-(3-aminobenzyl)piperazine-1-carboxylic acid tert-butyl ester (120 mg, 0.41 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (132 mg, 0.41 mmol) were dissolved in toluene (5 mL), and cesium carbonate (200 mg, 0.62 mmol) was added. The system was purged three times with nitrogen, and Pd2(dba)3 (38 mg, 0.04 mmol) and X-Phos (20 mg, 0.04 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was then separated by silica gel column chromatography to give a yellowish-brown solid compound 23-1 (141 mg, 59.7% yield). LC-MS (ESI): m / z 581.0 [M+1] + .

[0264] Step 2: Preparation of compound 23-2

[0265] Compound 23-1 (355 mg, 0.91 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 23-2 (123 mg, 94.6% yield).

[0266] Step 3: Preparation of Compound 23

[0267] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (60 mg, 0.24 mmol), HOBt (36 mg, 0.26 mmol), EDCI (100.0 mg, 0.52 mmol), and DIPEA (104 mg, 0.80 mmol) were added sequentially, followed by the slow addition of compound 23-2 (123 mg, 0.24 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 23 (95.0 mg, 52.5% yield) as a white solid. LC-MS (ESI): m / z 759.0 [M+1] + .

[0268] Example 24: Preparation of compound 24

[0269]

[0270] Step 1: Preparation of Compound 24-1

[0271] To a THF (5 mL) solution of tert-butyl 3-oxo-1-piperazincarboxylate (500 mg, 2.5 mmol), NaH (90 mg, 3.75 mmol) was added. The system was stirred at 0 °C for 2 h, followed by slow addition of 5-fluoro-2-nitropyridine (282 mg, 2.00 mmol), and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 24-1 (614.0 mg, 76% yield) as a pale yellow solid.

[0272] Step 2: Synthesis of Compound 24-2

[0273] Compound 24-1 (600 mg, 1.68 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC until complete, filtered with diatomaceous earth, the filtrate was concentrated under reduced pressure to give a dark brown solid compound 24-2 (461 mg, 85% yield), which was used directly in the next step of the reaction without purification.

[0274] Step 3: Synthesis of Compound 24-3

[0275] Compound 24-2 (180 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 24-3 (247 mg, 68% yield).

[0276] Step 4: Synthesis of Compound 24-4

[0277] Compound 24-3 (240 mg, 0.41 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 24-4 (201 mg, 96% yield).

[0278] Step 5: Synthesis of Compound 24

[0279] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 24-4 (190 mg, 0.37 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 24 (122.0 mg, 47% yield) as a white solid.

[0280] Example 25: Preparation of compound 25

[0281]

[0282] Step 1: Preparation of Compound 25-1

[0283] DIPEA (2.39 g, 18.5 mmol) was added to a DMF (5 mL) solution of 1-BOC-3-hydroxymethylpiperazine (2.00 g, 9.25 mmol). The mixture was stirred at room temperature for 30 min, and then 3,4-difluoronitrobenzene (1.47 g, 2.53 mmol) was slowly added. The mixture was stirred overnight at 120 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography to give the target compound 25-1 (2.74 g, 86% yield) as a pale yellow solid.

[0284] Step 2: Synthesis of Compound 25-2

[0285] To a THF (5 mL) solution of compound 25-1 (2.7 g, 7.60 mmol), NaH (274 mg, 11.4 mmol) was added and the mixture was stirred at 0 °C for 2 h. The mixture was then transferred and stirred overnight at 80 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 25-2 (900 mg, 36% yield) as a pale yellow solid.

[0286] Step 3: Synthesis of Compound 25-3

[0287] Compound 25-2 (900 mg, 2.68 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 25-3 (461 mg, 85% yield), which was used directly in the next step of the reaction without purification.

[0288] Step 4: Synthesis of Compound 25-4

[0289] Compound 25-3 (190 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a yellowish-brown solid compound 25-4 (273 mg, 76% yield).

[0290] Step 5: Synthesis of Compound 25-5

[0291] Compound 25-4 (270 mg, 0.51 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 25-5 (222 mg, 95% yield).

[0292] Step Six: Synthesis of Compound 25

[0293] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 25-5 (205 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 25 (187.0 mg, 69% yield) as a white solid.

[0294] Example 26: Preparation of Compound 26

[0295]

[0296] Step 1: Preparation of Compound 26-1

[0297] At 0 °C, DMF (51 mg, 0.67 g) was added to a DCM (20 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (2 g, 6.71 mmol). The system was stirred for 5 min, followed by slow addition of oxaloyl chloride (8.5 g, 67.1 mmol), and the system was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC, and the system was concentrated under reduced pressure to give the target compound 26-1 (1.9 g, 80% yield) as a white solid, which was used directly in the next step without purification.

[0298] Step 2: Synthesis of Compound 26-2

[0299] At 0 °C, NaBH4 (340 mg, 9.0 mmol) was added to an anhydrous THF (20 mL) solution of compound 26-1 (1.9 g, 6.0 mmol). The system was stirred for 2 h, and the reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 26-2 (600.0 mg, 80% yield) as a white solid.

[0300] Step 3: Synthesis of Compound 26-3

[0301] Compound 26-2 (250 mg, 0.88 mmol) and PPH3 (277 mg, 1.06 mmol) were dissolved in DCM. CBr4 (438 mg, 1.32 mmol) was slowly added at 0 °C, and the mixture was stirred for 2 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 26-3 (600.0 mg, 80% yield) as a white solid.

[0302] Step 4: Synthesis of Compound 26-4

[0303] 1-Boc-4-(4-aminophenyl)piperazine (172 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 26-4 (293 mg, 84% yield).

[0304] Step 4: Synthesis of Compound 26-5

[0305] Compound 26-4 (290 mg, 0.52 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 26-5 (212 mg, 89% yield).

[0306] Step 5: Synthesis of Compound 26

[0307] To a DMF (5 mL) solution of compound 26-5 (60 mg, 0.17 mmol), K₂CO₃ (72 mg, 0.52 mmol) was added, and the mixture was stirred at room temperature for 5 min. Then, compound 26-3 (83 mg, 1.34 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 26 (62.0 mg, 50% yield) as a white solid.

[0308] Example 27: Preparation of Compound 27

[0309]

[0310] Step 1: Preparation of Compound 27-1

[0311] Methyl 4-fluoro-3-nitrobenzoate (5 g, 25.1 mmol) and tert-butyl 3-methoxycarbonyl-piperazine-1-carboxylic acid (6.2 g, 25 mmol) were dissolved in acetonitrile solution (50 mL). Triethylamine (5.08 g, 50 mmol) was added at room temperature, and the mixture was stirred overnight at 80 °C. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography to give a yellow oily compound 27-1 (8.0 g, 78% yield).

[0312] Step 2: Preparation of Compound 27-2

[0313] Compound 27-1 (8.0 g, 19.5 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, and the system was stirred overnight at room temperature. The reaction was monitored by TLC until complete. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 27-2 (5.8 g, 82.1% yield), which was used directly in the next step of the reaction without purification.

[0314] Step 3: Preparation of compound 27-3

[0315] Compound 27-2 (5.8 g, 16 mmol) was dissolved in a methanol / water solution (3:1) (50 mL), and sodium hydroxide (2.57 g, 64 mmol) was added. The mixture was stirred at 40 °C for 12 h. The mixture was concentrated under reduced pressure and diluted with water. The pH was adjusted to 4-5 by adding 1 N hydrochloric acid aqueous solution at room temperature, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a yellow solid, target compound 27-3 (5.4 g, 97% yield). No further purification was required for use.

[0316] Step 4: Preparation of Compound 27-4

[0317] Compound 27-3 (2 g, 5.76 mmol) was dissolved in toluene (20 mL), and diphenyl azidophosphate (4.76 g, 17.3 mmol) and triethylamine (1.75 g, 17.3 mmol) were added. The mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. Benzyl alcohol (1.24 g, 11.52 mmol) was then added, and the mixture was stirred at 110 °C for 2 h under a nitrogen atmosphere. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give a yellow solid compound 27-4 (1.24 g, 48% yield).

[0318] Step 5: Preparation of compound 27-5

[0319] Compound 27-4 (500 mg, 1.10 mmol) was dissolved in MeOH (10 mL), Pd / C (10%, 100 mg) was added, hydrogen gas was introduced, the system was stirred overnight at room temperature, the reaction was monitored by TLC to ensure completion, the mixture was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a dark brown solid compound 27-5 (280 mg, 80% yield), which was used directly in the next step of the reaction without purification.

[0320] Step Six: Synthesis of Compound 27-6

[0321] 27-5 (197 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 27-6 (325 mg, 86% yield).

[0322] Step 7: Synthesis of Compound 27-7

[0323] Compound 27-6 (320 mg, 0.53 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 27-7 (278 mg, 97% yield).

[0324] Step 8: Synthesis of Compound 27

[0325] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 27-7 (220 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 27 (187.0 mg, 69% yield) as a white solid.

[0326] Example 28: Preparation of Compound 28

[0327]

[0328] Step 1: Preparation of Compound 28-1

[0329] 4-(5-aminopyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester (172 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 28-1 (296 mg, 84% yield).

[0330] Step 2: Preparation of Compound 28-2

[0331] Compound 28-1 (290 mg, 0.51 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 28-2 (239 mg, 95% yield).

[0332] Step 3: Preparation of Compound 28

[0333] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 28-2 (204 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 28 (156.0 mg, 61% yield) as a white solid.

[0334] Example 29: Preparation of compound 29

[0335]

[0336] Step 1: Preparation of Compound 29-1

[0337] 5-Aminoisoindole-2-carboxylic acid tert-butyl ester (145 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged with nitrogen three times, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging with nitrogen three more times. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 29-1 (257 mg, 80% yield).

[0338] Step 2: Preparation of compound 29-2

[0339] Compound 29-1 (250 mg, 0.48 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 29-2 (208 mg, 95% yield).

[0340] Step 3: Preparation of Compound 29

[0341] To a DMF (5 mL) solution of compound 29-2 (126 mg, 0.28 mmol), K₂CO₃ (95 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 5 min. Then, compound 26-3 (80 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 29 (92.0 mg, 58% yield) as a white solid.

[0342] Example 30: Preparation of compound 30

[0343]

[0344] Step 1: Preparation of Compound 30-1

[0345] 4-(4-aminobenzyl)piperazine-1-carboxylic acid tert-butyl ester (180 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 30-1 (297 mg, 83% yield).

[0346] Step 2: Preparation of compound 30-2

[0347] Compound 30-1 (290 mg, 0.50 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 30-2 (212 mg, 83% yield).

[0348] Step 3: Preparation of Compound 30

[0349] To a DMF (5 mL) solution of compound 30-2 (141 mg, 0.28 mmol), K₂CO₃ (95 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 5 min. Then, compound 26-3 (80 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 30 (102.0 mg, 60% yield) as a white solid.

[0350] Example 31: Preparation of compound 31

[0351]

[0352] Step 1: Synthesis of Compound 31-1

[0353] 6-(2-chloro-5-fluoro-pyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (322 mg, 1 mmol) and trans-(4-aminocyclohexyl)carbamate tert-butyl ester (257 mg, 1.2 mmol) were dissolved in NMP (6 mL), and the mixture was stirred overnight at 110 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 31-1 (416 mg, 83% yield) as a dark brown solid.

[0354] Step 2: Preparation of compound 31-2

[0355] Compound 31-1 (410 mg, 0.82 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 31-2 (324 mg, 90% yield).

[0356] Step 3: Synthesis of Compound 31

[0357] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 31-2 (178 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 31 (116.0 mg, 50% yield) as a white solid.

[0358] Example 32: Preparation of compound 32

[0359]

[0360] Step 1: Preparation of Compound 32-1

[0361] 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester (171 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 32-1 (274 mg, 79% yield).

[0362] Step 2: Preparation of compound 32-2

[0363] Compound 32-1 (270 mg, 0.48 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 32-2 (234 mg, 98% yield).

[0364] Step 3: Synthesis of Compound 32

[0365] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 32-2 (193 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 32 (144.0 mg, 57% yield) as a white solid.

[0366] Example 33: Preparation of compound 33

[0367]

[0368] Step 1: Preparation of compound 33-1

[0369] 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester (176 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 33-1 (284 mg, 80% yield).

[0370] Step 2: Preparation of compound 33-2

[0371] Compound 33-1 (280 mg, 0.49 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 33-2 (226 mg, 91% yield).

[0372] Step 3: Preparation of Compound 33

[0373] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 33-2 (206 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 33 (132.0 mg, 52% yield) as a white solid.

[0374] Example 34: Preparation of compound 34

[0375]

[0376] Step 1: Preparation of Compound 34-1

[0377] 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester (171 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 34-1 (274 mg, 79% yield).

[0378] Step 2: Preparation of compound 34-2

[0379] Compound 34-1 (270 mg, 0.48 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 34-2 (234 mg, 98% yield).

[0380] Step 3: Preparation of Compound 34

[0381] To a DMF (5 mL) solution of compound 34-2 (138 mg, 0.28 mmol), K₂CO₃ (95 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 5 min. Then, compound 26-3 (80 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 34 (90.0 mg, 52% yield) as a white solid.

[0382] Example 35: Preparation of compound 35

[0383]

[0384] Step 1: Preparation of Compound 35-1

[0385] 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester (176 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 35-1 (284 mg, 80% yield).

[0386] Step 2: Preparation of compound 35-2

[0387] Compound 35-1 (280 mg, 0.49 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 35-2 (226 mg, 91% yield).

[0388] Step 3: Preparation of Compound 35

[0389] To a DMF (5 mL) solution of compound 35-2 (141 mg, 0.28 mmol), K₂CO₃ (95 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 5 min. Then, compound 26-3 (80 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give the target compound 35 (84.0 mg, 49% yield) as a white solid.

[0390] Example 36: Preparation of compound 36

[0391]

[0392] Step 1: Preparation of Compound 36-1

[0393] 4-((4-aminophenyl)amino)piperidin-1-carboxylic acid tert-butyl ester (180 mg, 0.62 mmol) and 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (200 mg, 0.62 mmol) were dissolved in toluene (5 mL), and Cs₂CO₃ (403 mg, 1.24 mmol) was added. The system was purged three times with nitrogen, and Pd₂(dba)₃ (57 mg, 0.06 mmol) and XantPhos (36 mg, 0.06 mmol) were added, followed by purging three more times with nitrogen. The system was stirred overnight at 100 °C. The reaction was monitored by TLC until complete. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a yellowish-brown solid compound 36-1 (302 mg, 86% yield).

[0394] Step 2: Preparation of compound 36-2

[0395] Compound 36-1 (295 mg, 0.51 mmol) was dissolved in MeOH (6 mL), and HCl·in dioxane (2 mL, 4 M) was slowly added dropwise. The system was stirred at room temperature for 12 h. The reaction was monitored by TLC until it was complete, and a large amount of solid appeared in the system. The solid was collected by filtration and dried under vacuum to give a yellow solid compound 36-2 (248 mg, 96% yield).

[0396] Step 3: Preparation of Compound 36

[0397] To a DMF (5 mL) solution of 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (100 mg, 0.34 mmol), HOBt (59 mg, 0.44 mmol), EDCI (169.0 mg, 0.88 mmol), and DIPEA (219 mg, 1.70 mmol) were added sequentially, followed by the slow addition of compound 36-2 (206 mg, 0.41 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC until complete. The reaction solution was quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with saturated saline solution (50 mL) and dried over anhydrous Na₂SO₄. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography to give compound 36 (162.0 mg, 63% yield) as a white solid.

[0398] Example 37: Test of the inhibitory activity of the compound against PARP1 enzyme

[0399] 1. Experimental materials and instruments

[0400] Experimental materials: PARP1 (BPS, Cat. No. 80527), histone (Active Motif, Cat. No. 81167), SuperSignal ELISA Femto Substrate (THERMO PIERCE, Cat. No. 37074), Biotin-NAD+ (R&D, Cat. No. 6573), Strep-HRP (Thermo Pierce, Cat. No. 21127)

[0401] Instrument: SpectraMax Paradigm multi-functional microplate detector;

[0402] 2. Experimental Methods

[0403] Prepare histone-coated 384-well plates, adding 25 μL of histone solution to each well and incubating overnight at 4°C. Prepare PBST buffer, blocking buffer, and detection buffer. Wash the histone-coated 384-well plates three times with PBST buffer. Block the reaction with 50 μL of blocking buffer over 1 hour at room temperature. Wash the plates three times with PBST buffer. Prepare a 2000x compound in the source plate. Transfer 50 nmol of the compound from the source plate to a 96-well intermediate plate using 19.95 μL of detection buffer. Incubate at 1000 rpm for 1 min. Transfer 5 μL of DMSO / compound to each well. Enzyme reaction: Incubate the enzyme mixture at 25°C for 10 min. Add 10 μL of enzyme mixture (desired minimum control), incubate the compound at room temperature for 10 min, and add 10 μL of detection buffer. Add 10 μL of 2.5x biotin-NAD+ to each well and incubate at 25°C for 90 min. Wash the plates three times with PBST buffer.

[0404] Probe: Add 25 μL of Stre-HRP. Incubate at room temperature for 1 h, then wash the plate three times with PBS buffer. Add 25 μL of the quantitative enhancer mixture. Incubate for 10 min. Add 2.5 μL of quantitative red stop solution and shake the plate for 10–30 s to stop peroxidase activity.

[0405] Data processing:

[0406] Use equation (1) to fit the data in Excel to obtain the suppression value.

[0407] Equation (1): Inhibition% = (Max-Signal) / (Max-Min)*100.

[0408] Use equation (2) to fit the data in XL-Fit to obtain IC. 50 value.

[0409] Equation (2): Y = Bottom + (Top - Bottom) / (1 + (IC) 50 / X)*HillSlope)

[0410] Where Y is the inhibition percentage and X is the compound concentration.

[0411] 3. Experimental Results

[0412] The inhibitory effects of some target compounds on PARP1 enzyme were determined using the experimental methods described above, and the results are shown in Table 1.

[0413] Table 1. Half-maximal inhibitory concentrations of the target compounds against PARP1 enzyme.

[0414]

[0415] Note: + indicates compound IC. 50 ≥20.0nM

[0416] ++ indicates that the compound 20.0 nM > IC 50 >5nM

[0417] +++ indicates compound IC 50 ≤5nM

[0418] Conclusion: The representative compound of this invention can effectively inhibit PARP1 enzyme activity.

[0419] Example 38: Test of the inhibitory activity of the compound against CDK4 enzyme

[0420] 1. Experimental materials and instruments

[0421] Experimental materials: CDK4 / CycD1 (ProQinase, Cat. No. 0142-0143-1); HEPES, pH 7.5 (Gibco, Cat. No. 11344-041); MgCl2 (Sigma, Cat. No. M2670-500 g); DTT (Sigma, Cat. No. D0632-10G); ATP (Sigma, Cat. No. A7699-1G); 96-well plate (Corning, Cat. No. 3365); DMSO (Sigma, Cat. No. 34869-4L);

[0422] Instrument: SpectraMax Paradigm multi-functional microplate detector;

[0423] 2. Experimental Methods

[0424] Preparation of 1x kinase base buffer and stop buffer: 1x enzyme base buffer; 50mM HEPES; pH 7.5; 10mM magnesium chloride; 0.01% Tween-20; 0.01% 10% BSA; 2mM DTT

[0425] Compound preparation: The compound was diluted 100-fold to its final concentration and reacted with 100% dimethyl sulfoxide. 100 nL of each well was transferred from a 384-well Echo plate to a 384-well analytical plate via echo.

[0426] Enzyme reaction and detection: Except for the control wells without enzyme, add 5 μL of kinase solution to each well (replace with 10 μL of 1X kinase buffer); add substrate and adenosine triphosphate to the 1X substrate buffer to prepare 2X substrate solution; add 5 μL of 2X substrate solution to each well of the reagent plate to start the reaction; incubate the reaction at 28°C; prepare a detection solution with a final concentration of 2x in antibody dilution buffer; add 10 μL of detection solution to each well of the test plate to stop the reaction; incubate at room temperature for 60 minutes.

[0427] Curve fitting: Use equation (1) to fit the data in Excel to obtain the inhibition value. Equation (1): Inh% = (Max-Signal) / (Max-Min)*100. Use equation (2) to fit the data in XL-Fit to obtain the IC. 50 value

[0428] Equation (2): Y = bottom + (top - bottom) / (1 + (IC) 50 / X)*HillSlope). Where Y is the inhibition percentage and X is the compound concentration.

[0429] 3. Experimental Results

[0430] The inhibitory effects of some target compounds on CDK4 enzyme were determined using the experimental methods described above. The results are shown in Table 2.

[0431] Table 2. Half-maximal inhibitory concentrations of the target compounds against CDK4 enzyme.

[0432]

[0433] Note: + indicates compound IC. 50 ≥50.0nM

[0434] ++ indicates that the compound's 50.0 nM > IC value. 50 >10.0nM

[0435] +++ indicates compound IC 50 ≤10nM

[0436] Conclusion: The representative compound of this invention can effectively inhibit CDK4 enzyme activity.

[0437] Example 39: Test of the inhibitory activity of some compounds against tumor cells

[0438] 1. Cell lines and cell culture.

[0439] Human triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Cells were cultured in modified RPMI medium supplemented with 10% (vol / vol) fetal bovine serum, 50 μg / mL penicillin, and 50 μg / mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0440] 2. Cell viability assay.

[0441] Cells were seeded at the previously optimized seeding density into two 384-well white-walled tissue culture plates and incubated overnight in a 5% CO2 incubator. Cell viability was assessed using a CellTiter-Glo luminescence activity assay (Promega) after 5 days of drug treatment. Cell viability was measured immediately after drug administration (day 0) and after 6 days of incubation using Cell TiterGlo (CTG, Promega, G7573). Relative viability was calculated by normalizing the raw luminescence counts to those of cells treated with DMSO control (Ctrl.). The half-maximal inhibitory concentration (IC50) was calculated using Graphpad / Prism8 software. 50 The dose-response curve was fitted to the sigmoid dose-response value.

[0442] Table 3. Half-maximal inhibitory concentrations (IC50) of the target compounds against tumor cells.

[0443]

[0444]

[0445] Note: + indicates compound IC. 50 ≥10.0μM

[0446] ++ indicates that the compound is more potent than IC50 at 10.0 μM. 50 >1.0μM

[0447] +++ indicates compound IC 50 ≤1μM

[0448] Conclusion: The representative compound of this invention can effectively inhibit triple-negative breast cancer.

Claims

1. A phthalazinone derivative having dual PARP1 / CDK4 / 6 inhibitory activity, or its optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate, characterized in that, The structure of the derivative is shown in formula (I): in: R 1 R 2 R 3 R 4 R 5 The group is selected from hydrogen atoms, halogen atoms, alkyl groups, hydroxyl groups, amino groups, nitro groups, aryl groups, cycloalkyl groups, heterocyclic groups, or electrophilic groups that can covalently react with amino acid residues of the target protein; wherein, the electrophilic group that can covalently react with amino acid residues of the target protein includes acrylamide, α-haloketone, cyano, acetamido, mercapto, epoxide, vinyl sulfone, or activated acetylene. Indicate Y and L 1 The connection site, L represents 1 With L 2 The connection site, L represents 2 With L 3 The connection site, L represents 3 and The connection site, X is C or N; when X is N, there is no R. 6 replace; Y is CH2 or carbonyl; R 5 The methyl group is selected from C1-C6 alkyl, C3-C8 cycloalkyl, or C3-C6 cycloalkyl-substituted methyl groups; R 6 Selected from one of H, F, CN, and CH3; L 1 Selected from NH, chemical bonds, or the following structural fragments: Where m 1 m 3 =1, 2, 3, 4; m 2 =0, 1, 2, 3, 4, 5, 6; R 7 R 8 R 9 R 10 R 11 The components are independently selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituents are H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, and C3-C6 cycloalkyl groups; wherein L represents 1 Connection site with Y, L represents 1 With L 2 Connection sites; L 2 Selected from C2-C 12 Alkyl, cycloalkyl, chemical bond or the following structural fragments: Where m 1 m 2 =0, 1, 2, 3, 4, where R 7 R 8 R 9 R 10 R 11 The group is selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituent is H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, or C3-C6 cycloalkyl groups; wherein L represents 1 With L 2 The connection site, L represents 2 With L 3 Connection sites; L 3 It is either NH or a chemical bond; or L 1 -L 2 Together they form the following heterocyclic segments: Where m 1 m 3 =0, 1, 2, 3, 4; R 7 R 8 R 11 R 12 The components are independently selected from H, substituted C1-C6 alkyl groups, substituted C3-C6 cycloalkyl groups, CF3, CF2H, CN, CO2H, CONHCH3, CONH2, and COCH3, wherein the substitution is mono- or poly-substituted, and the substituents are H, F, Cl, Br, OH, NH2, C1-C3 alkyl groups, and C3-C6 cycloalkyl groups; wherein L represents 1 -L 2 Connection site with Y, L represents 1 -L 2 With L 3 The connection site.

2. The phthalazinone derivative with dual PARP1 / CDK4 / 6 inhibitory activity according to claim 1, characterized in that, R 1 R 2 R 3 R 4 R 5 Selected from halogen atoms, alkyl groups, or electrophilic groups that can covalently react with amino acid residues of the target protein; L 1 Selected from NH, chemical bonds, or the following structural fragments: L 2 Selected from C2-C 12 Alkyl, cycloalkyl, chemical bond or the following structural fragments: or L 1 -L 2 Together they form the following heterocyclic segments:

3. The phthalazinone derivative with dual PARP1 / CDK4 / 6 inhibitory activity according to claim 2, characterized in that, R l R 2 R 3 For F, R 4 It is methyl, R 5 It is isopropyl.

4. The phthalazinone derivative with dual PARP1 / CDK4 / 6 inhibitory activity according to claim 3, characterized in that, The chemical structural formula of the phthalazinone derivative is any one of the following formulas 1 to 36:

5. A method for preparing a phthalazinone derivative having dual-target inhibitory activity against PARP1 / CDK4 / 6 as described in any one of claims 1-4, characterized in that, Includes the following steps: Raw material a1 and containing a protecting group The a2 fragment undergoes substitution or Buchwald–Hartwig coupling to give intermediate a3; after the protecting group is removed from a3, intermediate a4 is given; a4 reacts with a5 via an acid-ammonia condensation to give the compound shown in formula (I); where Y is a carbonyl group.

6. A method for preparing a phthalazinone derivative having dual-target inhibitory activity against PARP1 / CDK4 / 6 as described in any one of claims 1-4, characterized in that, Includes the following steps: Raw material b1 and containing a protective group The fragment b2 undergoes substitution or Buchwald–Hartwig coupling to give intermediate b3; b3 is deprotected to give intermediate b4; b4 and b5 are substituted to give the compound shown in formula (I); where Y is CH.

7. The use of the phthalazinone derivatives with PARP1 / CDK4 / 6 dual-target inhibitory activity as described in any one of claims 1-4, or their optical isomers, racemates, single enantiomers, possible diastereomers, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, or solvates in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumors include solid tumors and hematologic tumors.

9. An antitumor drug, characterized in that, Contains a safe and effective amount of the phthalazinone derivative with PARP1 / CDK4 / 6 dual-target inhibitory activity as described in any one of claims 1-4, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

10. The antitumor drug according to claim 9, characterized in that, It also includes pharmacologically acceptable salts and pharmacologically acceptable excipients or carriers.

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