CN114634491B

CN114634491B - Polycyclic compound for inhibiting RNA helicase DHX33

Info

Publication number: CN114634491B
Application number: CN202011487589.4A
Authority: CN
Inventors: 张严冬; 李相鲁
Original assignee: Chengdu Kaiyue Life Technology Co ltd
Current assignee: Chengdu Kaiyue Life Technology Co ltd
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2024-03-19
Anticipated expiration: 2040-12-16
Also published as: CN114634491A

Abstract

The invention relates to a polycyclic compound for inhibiting RNA helicase DHX 33. In particular, the invention relates to a compound shown in a formula I or a pharmaceutically acceptable form thereof, a pharmaceutical composition containing the compound, a preparation method of the compound and medical application of the compound and the pharmaceutical composition in preventing and/or treating DHX33 related diseases.

Description

Polycyclic compound for inhibiting RNA helicase DHX33

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and relates to a small molecule inhibitor of DHX33, a pharmaceutical composition containing the small molecule inhibitor, a preparation method of the small molecule inhibitor and medical application of the small molecule inhibitor in preventing and/or treating DHX33 related diseases.

Background

DHX33 belongs to the family of RNA helicase proteins containing the DEAD/H cassette. Wherein DEAD/H represents the abbreviation Asp-Glu-Ala-Asp/His of an amino acid, which sequence, along with a number of other conserved amino acid sequences, is present in the protein sequence of an RNA helicase family member, is highly involved in nucleic acid substrate binding and ATP hydrolysis. Although these family members share these same sequences, each RNA helicase has its own specific and unique biological functions. The molecular weight of the DHX33 protein is 72kDa, and the DHX33 protein has the function of unwinding nucleic acid, and utilizes bioenergy released by ATP hydrolysis to drive and change the conformation of RNA and protein complexes, thereby participating in the metabolic activities of various RNAs, in particular a series of biological processes from RNA transcription, shearing, editing, translation, degradation and the like. The function of DHX33 is not limited to modification of RNA molecules, but studies have shown that DHX33 protein is involved in DNA metabolism in addition to unwinding RNA duplex. In particular, DHX33 protein can break the double-stranded structure of DNA and play an important role in gene expression.

The research shows that DHX33 influences the methylation state of DNA by being combined with gene promoters related to various cancers, so that the genome level regulates and controls the expression of various cancer genes and signal paths related to tumor development, and has a vital effect on various cell activities such as cell growth, proliferation, migration, apoptosis, carbohydrate metabolism and the like. In addition, DHX33 was found to be able to sense invasion of foreign double stranded RNA molecules and play an important role in the innate immunity of the cell. DHX33 is highly expressed as a very important cell growth regulatory gene in various cancers such as lung cancer, lymphoma, glioblastoma, breast cancer, colon cancer, liver cancer, etc. The development and progression of a variety of cancers is dependent on the high expression of DHX33 protein. The genetic knockout of DHX33 can obviously inhibit the occurrence and development of RAS oncogene-driven lung cancer; in vivo and in vitro experiments prove that after the DHX33 protein is inhibited, the occurrence and development of various cancers such as breast cancer, colon cancer, glioma, lymphoma and the like are obviously inhibited.

Studies have shown that DHX33 protein function is dependent on its helicase activity. The helicase activity deletion mutant of DHX33 does not have the function of DHX33 protein and cannot replace the function of wild-type DHX33 gene. At present, small molecule inhibitors aiming at DHX33 as targets are rare, so that development of a type of DHX33 inhibitor medicament with high activity and good patentability is urgently needed.

Disclosure of Invention

Through a great deal of research, the invention discovers a series of small molecule compounds with the activity of inhibiting RNA helicase of DHX33, and has potential value in preventing and/or treating DHX33 related diseases (such as cancers of brain glioblastoma and the like).

In a first aspect, the present invention provides a compound having the structure of formula I:

wherein,

each R ¹ Each independently selected from halogen, amino, -NH (C) _1-4 Alkyl), -N (C) _1-4 Alkyl group ₂ Nitro, C _1-4 Alkyl, C _1-4 Haloalkyl, C _1-4 Alkoxy, C _1-4 Haloalkoxy, C _1-4 Hydroxyalkyl, -O- (C) _1-4 Alkylene) -O- (C _1-4 Alkyl), -C (=o) -NH- (C) _1-4 Alkylene) -N (C) _1-4 Alkyl group ₂ or-C (=O) -O- (C) _1-4 Alkyl), or a plurality of R ¹ Forms a 5-7 membered ring with the atom to which it is attached;

R ² selected from hydrogen, C _1-4 Alkyl or- (C) _1-4 Alkylene) -O- (C _1-4 An alkyl group);

X ¹ selected from N or-CR ⁶ ；

R ⁶ Selected from hydrogen or C _1-4 An alkyl group;

R ³ selected from hydrogen or C _1-4 An alkyl group;

R ⁴ selected from hydrogen or C _1-4 An alkyl group;

X ² selected from N or-CR ⁷ ；

R ⁷ Selected from hydrogen or C _1-4 An alkyl group;

each R ⁵ Each independently selected from halogen, cyano, C _1-4 Alkyl, C _1-4 Haloalkyl, C _1-4 Alkoxy, C _1-4 Haloalkoxy, C _3-6 Cycloalkyl, -C (=o) -O- (C) _1-4 Alkyl), phenyl, benzyl, pyridyl, -C (=o) -NH ₂ or-NH-C (=o) - (C _1-4 Alkyl) optionally substituted with one or more groups selected from halogen, cyano, amino, hydroxy, C _1-4 Alkyl or C _1-4 Substitution of the substituent of the alkoxy group;

m is selected from 0, 1, 2, 3 or 4;

n is selected from 0, 1, 2, 3 or 4;

provided whenIs->When m is not 0;

the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, tautomers, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs.

In some embodiments, each R in a compound of formula I above or a pharmaceutically acceptable form thereof ¹ Each independently selected from fluorine, chlorine, bromine, methyl, ethyl, isopropyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, trifluoromethoxy, nitro, amino, -NH (CH) ₃ )、-NH(CH ₂ CH ₃ )、-N(CH ₃ ) ₂ 、-N(CH ₂ CH ₃ ) ₂ 、-CH ₂ OH、-CH ₂ CH ₂ OH、-O-(CH ₂ ) ₂ -OCH ₃ 、-C(＝O)-NH-(CH ₂ ) ₂ -N(CH ₃ ) ₂ 、-C(＝O)-NH-(CH ₂ ) ₃ -N(CH ₃ ) ₂ 、-C(＝O)-OCH ₃ or-C (=o) -OCH ₂ CH ₃ Or two R ¹ Forms a 6 membered ring with the atom to which it is attached;

in some preferred embodiments, each R in a compound of formula I above or a pharmaceutically acceptable form thereof ¹ Each independently selected from fluorine, chlorine, bromine, methyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, trifluoromethoxy, nitro, amino, -NH (CH) ₃ )、-N(CH ₃ ) ₂ 、-CH ₂ OH、-O-(CH ₂ ) ₂ -OCH ₃ 、-C(＝O)-NH-(CH ₂ ) ₂ -N(CH ₃ ) ₂ or-C (=o) -OCH ₂ CH ₃ Or two R ¹ With atoms to which it is attached

In some embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ² Selected from hydrogen, methyl, ethyl, isopropyl, - (CH) ₂ ) ₂ -OCH ₃ or-CH ₂ OCH ₃ 。

In some preferred embodiments, a compound of formula I above or a pharmaceutically acceptable form thereofR in (a) ² Selected from hydrogen, methyl, ethyl or-CH ₂ OCH ₃ 。

In some embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁶ Selected from hydrogen, methyl, ethyl or isopropyl.

In some preferred embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁶ Selected from hydrogen or methyl.

In some embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ³ Selected from hydrogen, methyl, ethyl or isopropyl.

In some preferred embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ³ Selected from hydrogen or methyl.

In some embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁴ Selected from hydrogen, methyl, ethyl or isopropyl.

In some preferred embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁴ Selected from hydrogen or methyl.

In some embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁷ Selected from hydrogen, methyl, ethyl or isopropyl.

In some preferred embodiments, R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁷ Selected from hydrogen or methyl.

In some embodiments, each R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁵ Each independently selected from fluorine, chlorine, bromine, cyano, methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, isopropoxy, trifluoromethoxy, -C (=o) -OCH ₃ 、-C(＝O)-OCH ₂ CH ₃ Phenyl, benzyl, pyridinyl, -C (=o) -NH ₂ 、-NH-C(＝O)-CH ₃ or-NH-C (=O) -CH ₂ CH ₃ 。

In some preferred embodiments, the above formula IEach R in the compound or pharmaceutically acceptable form thereof ⁵ Each independently selected from fluorine, chlorine, bromine, cyano, methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, methoxy, trifluoromethoxy, -C (=o) -OCH ₃ 、-C(＝O)-OCH ₂ CH ₃ Phenyl, benzyl, pyridinyl, -C (=o) -NH ₂ or-NH-C (=O) -CH ₃ 。

In some more preferred embodiments, each R in a compound of formula I above or a pharmaceutically acceptable form thereof ⁵ Each independently selected from cyano, methyl or phenyl.

In some embodiments, the invention provides a compound having the structure of formula I:

wherein,

each R ¹ Each independently selected from fluorine, chlorine, bromine, methyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, trifluoromethoxy, nitro, amino, -NH (CH) ₃ )、-N(CH ₃ ) ₂ 、-CH ₂ OH、-O-(CH ₂ ) ₂ -OCH ₃ 、-C(＝O)-NH-(CH ₂ ) ₂ -N(CH ₃ ) ₂ or-C (=o) -OCH ₂ CH ₃ Or two R ¹ With atoms to which it is attached

R ² Selected from hydrogen, methyl, ethyl or-CH ₂ OCH ₃ ；

X ¹ Selected from N or-CR ⁶ ；

R ⁶ Selected from hydrogen or methyl;

R ³ selected from hydrogen or methyl;

R ⁴ selected from hydrogen or methyl;

X ² selected from N or-CR ⁷ ；

R ⁷ Selected from hydrogen or methyl;

each R ⁵ Each independently selected from cyano, methyl or phenyl;

m is selected from 0, 1, 2, 3 or 4;

n is selected from 0, 1, 2, 3 or 4;

provided whenIs->When m is not 0;

In some embodiments, the compound of formula I above or a pharmaceutically acceptable form thereof is a compound of formula Ia-1 or a pharmaceutically acceptable form thereof:

wherein R is ¹ 、R ² 、R ⁵ M and n are as defined in formula I.

In some preferred embodiments, the compound of formula I above or a pharmaceutically acceptable form thereof is a compound of formula Ib-1 or formula Ib-2 or a pharmaceutically acceptable form thereof:

wherein R is ¹ 、R ² And m is as defined in formula I.

Those skilled in the art will appreciate that the present invention encompasses compounds resulting from any combination of the various embodiments. Embodiments resulting from the combination of technical features or preferred technical features of one embodiment with technical features or preferred technical features of another embodiment are also included within the scope of the present invention.

In a second aspect, the invention also provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, solvate, nitroxide, isotopic label, metabolite, or prodrug thereof:

in a third aspect, the present invention provides a process for the preparation of a compound having the structure of formula Ia-1, comprising the steps of:

step 1-1: reacting a compound of formula S-1 with cyanoacetic acid to produce a compound of formula M-1;

step 1-2: performing a ring closure reaction on the compound of formula M-1 to produce a compound of formula M-2;

step 1-3: reacting a compound of formula S-2 with a compound of formula S-3 to produce a compound of formula M-3;

Step 1-4: carrying out an hydroformylation reaction on the compound of the formula M-3 to generate a compound of the formula M-4;

step 1-5: reacting a compound of formula M-2 with a compound of formula M-4 to produce a compound of formula Ia-1;

wherein R is ¹ 、R ² 、R ⁵ M and n are as defined in formula I.

In some embodiments, step 1-1 of the above preparation process is performed in a suitable organic solvent or a mixed solution of an organic solvent and water, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform, 1, 2-dichloroethane, etc.), methanol, ethanol, t-butanol, N-Dimethylformamide (DMF), or ethers (e.g., ethylene glycol dimethyl ether (DME), tetrahydrofuran (THF), dioxane, and any combination thereof, preferably N, N-dimethylformamide.

In some embodiments, step 1-1 of the above preparation process is carried out at a suitable temperature, said temperature being between 0 and 80 ℃, preferably 25 ℃.

In some embodiments, steps 1-2 in the above preparation process are performed in the presence of a suitable acid, which may be selected from hydrochloric acid, acetic acid, and any combination thereof, preferably acetic acid.

In some embodiments, step 1-2 in the above preparation process is performed at a suitable temperature, which is 0-150 ℃, preferably 110 ℃.

In some embodiments, steps 1-3 in the above preparation process are performed in a suitable organic solvent or a mixed solution of an organic solvent and water, which may be selected from methanol, ethanol, t-butanol, N-Dimethylformamide (DMF) or ethers (e.g., ethylene glycol dimethyl ether (DME), tetrahydrofuran (THF), dioxane), and any combination thereof, preferably tetrahydrofuran.

In some embodiments, steps 1-3 in the above preparation process are performed in the presence of a suitable acid, which may be selected from hydrochloric acid, acetic acid, p-toluene sulfonic acid, and any combination thereof, preferably p-toluene sulfonic acid.

In some embodiments, steps 1-3 of the above preparation process are carried out at a suitable temperature, which is 0-150 ℃, preferably 40-80 ℃.

In some embodiments, steps 1-4 of the above preparation process are performed in a suitable organic solvent or a mixed solution of an organic solvent and water, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform, 1, 2-dichloroethane, etc.), methanol, ethanol, t-butanol, N-Dimethylformamide (DMF), or ethers (e.g., ethylene glycol dimethyl ether (DME), tetrahydrofuran (THF), dioxane, and any combination thereof, preferably N, N-dimethylformamide.

In some embodiments, steps 1-4 of the above preparation process are carried out at a suitable temperature, which is 0-150 ℃, preferably 60-100 ℃.

In some embodiments, steps 1-5 of the above preparation process are performed in a suitable organic solvent or a mixed solution of an organic solvent and water, which may be selected from methanol, ethanol, t-butanol, N-Dimethylformamide (DMF) or ethers (e.g., ethylene glycol dimethyl ether (DME), tetrahydrofuran (THF), dioxane), and any combination thereof, preferably ethanol.

In some embodiments, steps 1-5 in the above preparation process are performed in the presence of a suitable base, which may be selected from piperidine, pyridine, triethylamine, and any combination thereof, preferably piperidine.

In some embodiments, steps 1-5 of the above preparation process are carried out at a suitable temperature, which is 0-150 ℃, preferably 60-100 ℃.

The preparation method of the compound of the present invention is not limited to the above preparation method.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising at least one compound of formula I, formula Ia-1, formula Ib-1 or formula Ib-2, or a pharmaceutically acceptable form thereof, as described above, together with one or more pharmaceutically acceptable carriers.

In a fifth aspect, the present invention provides a compound of formula I, formula Ia-1, formula Ib-1 or formula Ib-2, or a pharmaceutically acceptable form thereof, as described above, or a pharmaceutical composition as described above, for use as a DHX33 inhibitor for the prevention and/or treatment of a disease or disorder mediated at least in part by DHX33 (e.g., cancer such as glioblastoma).

In a sixth aspect, the present invention provides the use of a compound of formula I, formula Ia-1, formula Ib-1 or formula Ib-2 as described above, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention and/or treatment of a disease or condition mediated at least in part by DHX33 (e.g. cancer such as glioblastoma).

In a seventh aspect, the present invention provides a method for preventing and/or treating a disease or disorder mediated at least in part by DHX33 (e.g. cancer such as glioblastoma), comprising the steps of: a prophylactically and/or therapeutically effective amount of a compound of formula I, formula Ia-1, formula Ib-1 or formula Ib-2 above or a pharmaceutically acceptable form thereof or a pharmaceutical composition as described above is administered to a subject in need thereof.

The present invention is not limited to the specific embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definition of terms

Unless otherwise indicated, the following terms have the following meanings in the present invention.

The terms "comprising," "including," "having," or "containing," or any other variation thereof, are intended to cover a non-exclusive or open-ended inclusion. For example, a composition, method, or apparatus that comprises a list of elements is not necessarily limited to only those elements explicitly listed, but may also include other elements not explicitly listed or inherent to such composition, method, or apparatus.

Where lower and upper limits of a range of values are disclosed, any numerical value or any subrange falling within the range is indicated as being specifically disclosed. In particular, each range of values for the parameters disclosed herein (e.g., in "about a to b", or equivalently "about a to b";or an equivalent "form of about a-b") should be understood to encompass each numerical value and subrange therein. For example, "C _1-4 "is understood to encompass any subrange therein as well as every point value, e.g. C _2-4 、C _3-4 、C _1-2 、C _1-3 、C _1-4 Etc. and C ₁ 、C ₂ 、C ₃ 、C ₄ Etc.

The term "pharmaceutical composition" refers to a composition that can be used as a medicament comprising a pharmaceutically active ingredient (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an adjuvant that is administered with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers that can be used in the present invention include, but are not limited to: a) A diluent; b) A lubricant; c) An adhesive; d) A disintegrant; e) Absorbents, colorants, flavors and/or sweeteners; f) Emulsifying or dispersing agents; and/or g) substances that enhance the absorption of the compound, etc.

The above pharmaceutical composition may act systematically and/or locally. For this purpose, they may be administered by a suitable route, for example by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular route or as an inhalant.

The above route of administration may be accomplished by suitable dosage forms. Dosage forms useful in the present invention include, but are not limited to: tablets, capsules, troches, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups and the like.

When administered orally, the above pharmaceutical compositions may be formulated into any orally acceptable dosage form, including, but not limited to, tablets, capsules, aqueous solutions, aqueous suspensions, and the like.

The above pharmaceutical compositions may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions, or sterile injectable aqueous or oleaginous solutions. Among the carriers that may be used include, but are not limited to: water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oils may also be used as solvents or suspending media, such as mono-or diglycerides.

The above pharmaceutical composition may comprise 0.01mg to 1000mg of at least one compound of formula I, formula Ia-1, formula Ib-1 or formula Ib-2 as described above or a pharmaceutically acceptable form thereof.

The term "disease or disorder mediated at least in part by DHX 33" refers to a disease in which the pathogenesis includes at least a portion of DHX 33-related factors, such as cancers such as glioblastoma.

The term "effective amount" refers to a dose capable of eliciting a biological or medical response from a cell, tissue, organ or organism (e.g., an individual) and sufficient to achieve a desired prophylactic and/or therapeutic effect.

The dosing regimen may be adjusted to provide the best desired response. For example, it may be administered in a single dose, it may be administered in divided doses over time, or it may be administered after a proportional decrease or increase in dose depending on the actual situation. It will be appreciated that the particular dosage regimen for any particular individual will be adjusted according to the needs and the discretion of the attendant administering or supervising the administration of the compositions.

The term "in need thereof" refers to a judgment of a physician or other caregiver as to the need of an individual or as to the impending benefit from the prevention and/or treatment process based on various factors of the physician or other caregiver in their area of expertise.

The term "individual" (or subject) refers to a human or non-human animal. The subject of the present invention includes subjects (patients) suffering from diseases and/or disorders and normal subjects. Non-human animals of the present invention include all vertebrates, such as non-mammals, e.g., birds, amphibians, reptiles, etc., and mammals, e.g., non-human primates, domestic animals, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

The term "treatment" refers to the alleviation or elimination of a disease or condition for which it is intended. A subject is indicated to have been successfully "treated" if the subject has received a therapeutic amount of a compound of the invention, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the invention, at least one indicator and symptom of which exhibits observable and/or detectable remission and/or improvement. It is understood that treatment includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant results. In particular, "treatment" means that a compound of the invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the invention may achieve at least one of the following effects, for example: (1) Preventing disease in animals that may be predisposed to the disease but have not undergone or displayed disease pathology or symptomology; (2) Inhibiting the disease (i.e., preventing further development of pathology and/or symptomology) in an animal experiencing or exhibiting disease pathology or symptomology; (3) Disease is ameliorated (i.e., pathology and/or symptomology is reversed) in an animal that is experiencing or exhibiting pathology or symptomology of the disease.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention which are substantially non-toxic to organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed from the compounds of the present invention by reaction with pharmaceutically acceptable inorganic/organic acids or inorganic/organic bases, such salts also being referred to as acid addition salts or base addition salts. For a review of suitable salts see, for example, jusiak, soczewinski, et al, remington's Pharmaceutical Sciences [ M ], mack Publishing Company,2005 and Stahl, wermuth, handbook of Pharmaceutical Salts: properties, selection, and Use [ M ], wiley-VCH,2002. Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The term "pharmaceutically acceptable ester" refers to an ester that is substantially non-toxic to an organism in which it is hydrolyzed to form a compound of the invention or a salt thereof. Pharmaceutically acceptable esters generally include, but are not limited to, esters of the compounds of the present invention with pharmaceutically acceptable carboxylic or sulfonic acids, such esters also being referred to as carboxylic or sulfonic acid esters.

The term "isomer" refers to a compound that has the same molecular weight due to the same number and type of atoms, but differs in the spatial arrangement or configuration of the atoms.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. The present invention also includes stereoisomers and mixtures thereof, as the compounds of the present invention present asymmetric centers and other chemical structures that may lead to stereoisomers. All stereoisomeric forms of the compounds of the invention are within the scope of the invention unless otherwise indicated.

The term "tautomer" (or "tautomeric form") refers to structural isomers having different energies that can be converted to each other by a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or proton transfer tautomers) include, but are not limited to, interconversions by proton transfer, such as keto-enol isomerisation, imine-enamine isomerisation, amide-imine alcohol isomerisation, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "solvate" refers to a substance formed by the association of a compound of the invention (or a pharmaceutically acceptable salt thereof) with at least one solvent molecule by non-covalent intermolecular forces. For example, solvates include, but are not limited to, hydrates (including hemihydrate, monohydrate, dihydrate, trihydrate, and the like), ethanolates, acetonates, and the like.

The term "nitroxide" refers to compounds formed by oxidation of nitrogen atoms in tertiary amines or nitrogen (aromatic) containing heterocyclic structures. For example, the nitrogen atom in the parent nucleus of the compound of formula I may form the corresponding nitroxide.

The term "isotopic label" refers to the replacement of a specific atom in a compound of the invention by itA derivative compound formed by isotopic atoms. Unless otherwise indicated, the compounds of the present invention include various isotopes of H, C, N, O, F, P, S, cl, such as but not limited to ² H(D)、 ³ H(T)、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O、 ¹⁸ O、 ¹⁸ F、 ³¹ P、 ³² P、 ³⁵ S、 ³⁶ S and ³⁷ Cl。

the term "metabolite" refers to a derivative compound of the present invention which is formed after metabolism. For further information on metabolism see Goodman and Gilman's The Pharmacological Basis of Therapeutics (9 ^th ed.)[M]McGraw-Hill International Editions,1996. The present invention encompasses all possible metabolite forms of the compounds of the invention, i.e. substances formed in the body of the individual to whom the compounds of the invention are administered. Metabolites of a compound may be identified by techniques well known in the art and their activity may be characterized by assays.

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly after administration to a subject. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to an individual, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and various prodrug forms are known in the art, for example, see T.Higuchi, V.Stella, pro-drugs as Novel Drug Delivery Systems [ J ], american Chemical Society, vol.14,1975. Furthermore, the present invention also covers compounds of the invention containing protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules of interest, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example those described in T.W.Greene, P.G.M.Wuts, protective Groups in Organic Synthesis [ M ], john Wiley & Sons, 2006. These protecting groups may be removed at a suitable subsequent stage using methods known in the art.

The term "independently" means that at least two groups (or ring systems) present in the structure that are the same or similar in value range may have the same or different meanings in the particular case. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl; similarly, when the substituent Y is hydrogen, the substituent X may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl.

The term "halogen" as used herein, alone or in combination with other groups, refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "alkyl" as used herein, alone or in combination with other groups, refers to a straight or branched aliphatic hydrocarbon group. For example, the term "C" as used in the present invention _1-4 Alkyl "refers to an alkyl group having 1 to 4 carbon atoms. For example, alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like. The alkyl group may be optionally substituted or unsubstituted.

The term "alkylene" as used herein, alone or in combination with other groups, refers to a straight or branched chain divalent saturated aliphatic hydrocarbon group to which two groups (or fragments) may be attached, either to the same carbon atom or to different carbon atoms. For example, the term "C" as used herein _1-4 Alkylene "refers to an alkylene group having 1 to 4 carbon atoms (e.g., methylene, 1-ethylene, 1, 2-propylene, 1, 3-butylene, etc.). The alkylene group may be optionally substituted or unsubstituted.

The term "haloalkyl" as used herein, alone or in combination with other groups, refers to an alkyl group substituted with one or more (such as 1 to 3) same or different halogen atoms. For example, the term "C" as used in the present invention _1-4 Haloalkyl "refers to haloalkyl groups having 1 to 4 carbon atoms. For example, haloalkyl includes, but is not limited toin-CH ₂ F、-CHF ₂ 、-CF ₃ 、-CH ₂ CF ₃ 、-CF ₂ CF ₃ 、-CH ₂ CH ₂ CF ₃ 、-CH ₂ Cl, and the like. Haloalkyl may be optionally substituted or unsubstituted.

The term "hydroxyalkyl" as used herein, alone or in combination with other groups, refers to an alkyl group substituted with one or more (such as 1 to 3) hydroxy groups. For example, the term "C" as used in the present invention _1-4 Hydroxyalkyl "refers to hydroxyalkyl groups having 1 to 4 carbon atoms. For example, hydroxyalkyl groups include, but are not limited to Etc. Hydroxyalkyl groups may be optionally substituted or unsubstituted.

The term "alkoxy" as used herein, alone or in combination with other groups, refers to an alkyl group attached to the remainder of the molecule through an oxygen atom. For example, alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like. Alkoxy groups may be optionally substituted or unsubstituted.

The term "haloalkoxy", as used herein, alone or in combination with other groups, refers to a monovalent, linear or branched haloalkyl-O-group substituted with at least one atom selected from fluorine, chlorine, bromine and iodine, may contain unsaturation, and is attached to the other groups by a single bond to an oxygen atom, e.g., C _1-4 Haloalkoxy groups. For example, haloalkoxy groups include, but are not limited to fluoromethoxy (-OCH) ₂ F) Difluoromethoxy (-OCHF) ₂ ) Trifluoromethoxy (-OCF) ₃ ) 1-fluoroethoxy (-OCHFCH) ₃ ) 2-fluoroethoxy (-OCH) ₂ CH ₂ F) 1, 2-difluoroethoxy (-OCHFCH) ₂ F) 2, 2-difluoroethoxy (-OCH) ₂ CHF ₂ ) 1, 2-trifluoroethoxy (-OCHFCHF) ₂ ) 2, 2-trifluoroethoxy (-OCH) ₂ CF ₃ ) Etc.

The term "cycloalkyl" as used herein, alone or in combination with other groups, refers to saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon groups. For example, the term "C" as used in the present invention _3-6 Cycloalkyl "refers to cycloalkyl groups having 3 to 6 carbon atoms. For example, cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted.

The term "hydroxy" as used herein, alone or in combination with other groups, refers to-OH.

The term "cyano" when used herein, alone or in combination with other groups, refers to-CN.

The term "amino" as used herein, alone or in combination with other groups, refers to-NH ₂ 。

Drawings

FIG. 1 shows analysis of the results of SDS-PAGE separation of recombinant DHX33 protein prepared by the method of the present invention, after staining with Coomassie brilliant blue.

FIG. 2 shows the modulation of known DHX33 downstream genes by compounds of the present invention, wherein U251 cells were treated with AB24202 (0.5. Mu.M, 1. Mu.M, respectively) or AB24203 (0.5. Mu.M) for 16 hours, and control groups were treated with an equal volume of DMSO.

Detailed Description

In order to make the objects and technical solutions of the present invention more apparent, embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention.

The reagents or apparatus used in the examples are all conventional products commercially available. Those not specifying the specific conditions were carried out according to the conventional conditions or the conditions recommended by the manufacturer. The term "room temperature" as used herein refers to 20 ℃ ± 5 ℃. As used herein, the term "about" when used in reference to a particular value or range of values is intended to encompass the value or range of values as well as ranges of errors that are acceptable to those skilled in the art of the value or range of values, such as, for example, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

The structures of the compounds described in the examples below were determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS).

Nuclear Magnetic Resonance (NMR) measurement instrument Bruker 400MHz NMR was used, and the measurement solvent was deuterated methanol (CD) ₃ OD), deuterated chloroform (CDCl) ₃ ) Hexadeuterated dimethyl sulfoxide (DMSO-d) ₆ ) The internal standard substance is Tetramethylsilane (TMS).

Abbreviations in Nuclear Magnetic Resonance (NMR) data in the following examples represent the following meanings:

s: single peak, d: double peak, t: triplet, q: quadruplet, dd: double peak, qd: four doublets, ddd: double doublet, ddt: double triplet dddd: double peak, m: multiple peaks, br: broad peak, J: coupling constant, hz: hertz, delta: chemical shift.

All chemical shift (delta) values are given in parts per million (ppm).

The Mass Spectrum (MS) measuring instrument uses an Agilent 6120B mass spectrometer, and the ion source is an electrospray ion source (ESI).

HPLC was performed using Agilent 1200DAD high pressure liquid chromatography (Sunforc C18, 150X4.6mm,5 μm column) and Waters 2695-2996 high pressure liquid chromatography (Gimini C18, 150X4.6mm,5 μm column).

The thin layer chromatography silica gel plate uses Qingdao ocean GF254 silica gel plate, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15mm-0.2mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5mm silica gel plate.

Column chromatography generally uses 200-300 mesh silica gel of Qingdao ocean as carrier.

The progress of the reaction in the examples was monitored by Thin Layer Chromatography (TLC) using a system of developing agents of a: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate systems, and the volume ratio of the solvent is adjusted according to the polarity of the compounds.

The system of eluent for column chromatography and the system of developing agent for thin layer chromatography used for purifying the compound include a: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate system, the volume ratio of the solvent is regulated according to the polarity of the compound, and small amount of triethylamine, acidic or alkaline reagent and the like can be added for regulation.

Synthesis of Compounds

Example 1: synthesis of Compound (E) -2- (3- (2-cyano-2- (6-methoxy-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24202)

(1) Synthesis of Compound 3 (2- (2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

To a solution of compound 1 (2-amino-4, 5-dimethyl-3-furancarbonitrile) (2.5 g,18.36mmol,1.0 eq) in 50mL of Tetrahydrofuran (THF) was added 2 (2, 5-hexanedione) (3.3 g,29.38mmol,1.6 eq), 3A molecular sieve (50 g) and p-toluenesulfonic acid (TsOH-H) ₂ O,1.4g,7.44mmol,0.4 eq). The mixture was heated to reflux and stirred overnight. The solid was filtered and the filtrate was concentrated, and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate=100/1) to give compound 3 (2- (2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (2.8 g, yield: 71.2%) as a white solid. MS (ESI) m/z 215.3[ M+H ]] ⁺ . TLC: petroleum ether/ethyl acetate (10:1); r is R _f (compound 1) =0.1; r is R _f (compound 3) =0.7. ¹ H NMR(CDCl ₃ ,400Hz):δ5.87(s,2H),2.24(s,3H),2.11(s,6H),2.08(s,3H)。

(2) Synthesis of Compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

At 0℃and nitrogen (N) ₂ ) Under the conditions of (2) phosphorus oxychloride (POCl) ₃ 5.3g,35.04mmol,3.0 eq) was added dropwise to 30mL of Dimethylformamide (DMF), and the mixture was stirred at 0deg.C for 30 min and then warmed to room temperature. To this was added compound 3 (2- (2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (2.5 g,11.68mmol,1.0 eq) dissolved in 4mL DMF. The mixture was heated to 100 ℃ and at N ₂ Stirred for 2 hours. After cooling, the mixture was placed in ice water and basified with 30% aqueous NaOH to ph=10. The mixture was extracted with Ethyl Acetate (EA) and washed with brine. The organic layer was prepared by Na ₂ SO ₄ Drying, filtering and concentrating. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate=100/1) to give 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) as a yellow solid (2.2 g, yield: 78.5%). MS (ESI) m/z 243[ M+H ]] ⁺ . TLC: petroleum ether/ethyl acetate (20:1); r is R _f (compound 3) =0.7; r is R _f (compound 4) =0.5. ¹ H NMR(CDCl ₃ ,400Hz):δ9.86(s,1H),6.35(s,1H),2.38(s,3H),2.27(s,3H),2.10(s,6H)。

(3) Synthesis of Compound 7 (N- (2-amino-4-methoxyphenyl) -2-cyanoacetamide)

To a solution of compound 5 (4-methoxyphenylenediamine) (2.0 g,14.5mmol,1.1 eq) in dimethylformamide (20 mL) was added 6 (cyanoacetic acid) (1.36 g,15.95mmol,1.1 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDCI (3.34 g,17.4mmol,1.2 eq) and 1-hydroxybenzotriazole HOBT (1.96 g,14.5mmol,1.0 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=200/1-30/1) to obtain compound 7 (N- (2-amino)-4-methoxyphenyl) -2-cyanoacetamide) (1.3 g, yield: 43.7%). MS (ESI) m/z 206[ M+H ] ] ⁺ . TLC: dimethylformamide/methanol (10:1); r is R _f (compound 5) =0.2; r is R _f (compound 7) =0.4.

(4) Synthesis of Compound 8 (2- (6-methoxy-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 7 (N- (2-amino-4-methoxyphenyl) -2-cyanoacetamide) (490 mg,2.38mmol,1.0 eq) was dissolved in acetic acid (5 mL) and stirred overnight at 110 ℃. The mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate=50/1-1/1) to give black compound 8 (2- (6-methoxy-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (290 mg, yield: 64.8%). MS (ESI) m/z 188[ M+H ]] ⁺ . TLC: petroleum ether/ethyl acetate (10:1); r is R _f (compound 7) =0.7; r is R _f (compound 8) =0.75. ¹ H NMR(DMSO-d ₆ ，400Hz):δ12.35(s,1H),7.44-7.31(m,1H),7.01(s,1H),6.80-6.74(m,1H),4.28(s,2H),3.73(s,3H)。

(5) Synthesis of Compound AB24202 ((E) -2- (3- (2-cyano-2- (6-methoxy-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

To compound 8 (2- (6-methoxy-1H-benzo [ d ]) dissolved in ethanol (1 mL)]To a solution of imidazol-2-yl) acetonitrile (46 mg,0.24mmol,1.0 eq) was added compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (60 mg,0.24mmol,1.0 eq) and piperidine (20 mg,0.24mmol,1.0 eq). The mixture was heated to reflux and stirred for 1 hour, after which time the mixture was cooled to room temperature and filtered. Collecting The solid was dried to give the yellow solid compound AB24202 (E) -2- (3- (2-cyano-2- (6-methoxy-1H-benzo [ d ])]Imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (30 mg, yield: 30.3%). MS (ESI) m/z 412.3[ M+H ]] ⁺ 。 ¹ H NMR(DMSO-d6,400Hz):δ12.72(s,1H),8.04(s,1H),7.45(s,1H),7.01(s,1H),6.94(s,1H),6.82(d,J＝8.4Hz,1H),3.77(s,3H),2.32(s,3H),2.29(s,3H),2.13(s,3H),2.08(s,3H)。

Example 2: synthesis of Compound (E) -2- (3- (2- (6-chloro-1H-benzo [ d ] imidazol-2-yl) -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24201)

Compound AB24201 (yield: 58.3%) was obtained in the same manner as in example 1, ¹ H NMR(CDCl ₃ ,400Hz):δ9.49(s,1H),8.32(d,J＝4.4Hz,1H),7.68-7.60(m,1H),7.36-7.24(m,1H),6.99(s,1H),2.32(s,3H),2.28(s,3H),2.15(s,3H),2.11(s,3H)。

example 3: synthesis of Compound (E) -2- (3- (2-cyano-2- (6-methyl-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24203)

Compound AB24203 (yield: 30.6%) was obtained in the same manner as in example 1, ¹ H NMR(DMSO-d ₆ ,400Hz):δ12.70(s,1H),8.07(s,1H),7.39-7.26(m,2H),7.00-6.94(m,2H),2.40(s,3H),2.33(s,3H),2.29(s,3H),2.13(s,3H),2.08(s,3H)。

example 4: synthesis of Compound (E) -2- (3- (2-cyano-2- (6-fluoro-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24205)

Compound AB24205 (yield: 66.7%) was obtained in the same manner as in example 1, ¹ H NMR(DMSO-d6,400Hz):δ12.97(s,1H),8.11(s,1H),7.54(s,1H),7.35(s,1H),7.06-7.04(m,1H),6.94(s,1H),2.33(s,3H),2.29(s,3H),2.13(s,3H),2.08(s,3H)。

example 5: synthesis of the Compound (E) -2- (1-cyano-2- (1- (3-cyano-4, 5-dimethylfuran-2-yl) -2, 5-dimethyl-1H-pyrrol-3-yl) vinyl) -N- (2- (dimethylamino) ethyl) -1H-benzo [ d ] imidazole-6-carboxamide (AB 24206)

Compound AB24206 (yield: 15%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,DMSO)δ9.40(s,1H),8.71(t,J＝5.8Hz,1H),8.21(s,1H),8.13(s,1H),7.78(d,J＝8.5Hz,1H),7.64(d,J＝8.5Hz,1H),6.99(s,1H),3.63(t,J＝2.4Hz,2H),3.30(t,J＝6.0Hz,2H),2.87(d,J＝4.4Hz,6H),2.38(s,3H),2.33(s,3H),2.17(s,3H),2.11(s,3H)。

example 6: synthesis of Compound (E) -2- (1-cyano-2- (1- (3-cyano-4, 5-dimethylfuran-2-yl) -2, 5-dimethyl-1H-pyrrol-3-yl) vinyl) -1H-benzo [ d ] imidazole-6-carboxylic acid ethyl ester (AB 24208)

Compound AB24208 (yield: 35%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,DMSO)δ13.22(s,1H),8.14(d,J＝17.1Hz,2H),7.81(d,J＝8.5Hz,1H),7.73–7.50(m,1H),6.95(s,1H),4.29(q,J＝7.1Hz,2H),2.33(s,3H),2.28(s,3H),2.12(s,3H),2.07(s,3H),1.31(t,J＝7.1Hz,3H)。

example 7: synthesis of Compound (E) -2- (3- (2- (6-bromo-1H-benzo [ d ] imidazol-2-yl) -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24213)

Compound AB24213 (yield: 52.6%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,dmso)δ13.03(s,1H),8.13(s,1H),7.74(s,1H),7.50(s,1H),7.32(d,J＝8.4Hz,1H),6.95(s,1H),2.33(s,3H),2.29(s,3H),2.12(s,3H),2.07(s,3H)。

example 8: synthesis of Compound (E) -2- (3- (2-cyano-2- (6-nitro-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24217)

Compound AB24217 (yield: 56.8%) was obtained in the same manner as in example 1, ¹ H NMR(399MHz,DMSO-d6)δ8.42(s,1H),8.22(s,1H),8.10(d,J＝8.9Hz,1H),7.71(d,J＝8.9Hz,1H),6.96(s,1H),2.36(s,3H),2.31(s,3H),2.14(s,3H),2.09(s,3H)。

example 9: synthesis of Compound (E) -2- (3- (2- (6-amino-1H-benzo [ d ] imidazol-2-yl) -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24221)

To and from the embodiment1 in the same manner, compound AB24221 (yield: 7.8%), ¹ H NMR(399MHz,DMSO-d6)δ8.16(s,1H),7.49(d,J＝8.4Hz,1H),7.18(s,1H),7.04-6.98(m,2H),2.36(s,3H),2.31(s,3H),2.15(s,3H),2.12(s,3H)。

Example 10: synthesis of Compound (E) -2- (3- (2-cyano-2- (6- (dimethylamino) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24222)

Compound AB24222 (yield: 39.3%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,DMSO-d6)δ12.44(s,1H),7.94(s,1H),7.41(d,J＝8.9Hz,1H),6.92(s,1H),6.74(d,J＝8.7Hz,1H),6.61(s,1H),2.90(s,6H),2.30(d,J＝8.3Hz,6H),2.10(d,J＝18.4Hz,6H)。

example 11: synthesis of Compound (E) -2- (3- (2-cyano-2- (6- (2-methoxyethoxy) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24230)

Compound AB24230 (yield: 30%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,DMSO-d6)δ8.04(s,0H),7.43(s,0H),7.03(s,0H),6.93(s,1H),6.82(d,J＝8.6Hz,1H),4.09(s,1H),3.65(s,1H),2.30(d,J＝10.7Hz,3H),2.10(d,J＝19.0Hz,3H)。

example 12: synthesis of Compound (E) -2- (3- (2-cyano-2- (6- (trifluoromethyl) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24243)

Compound AB24243 (yield: 80.7%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,dmso)δ8.20(s,1H),7.91(s,1H),7.73(d,J＝8.1Hz,1H),7.52(d,J＝8.0Hz,1H),6.96(s,1H),2.32(d,J＝20.5Hz,6H),2.11(d,J＝21.6Hz,6H)。

example 13: synthesis of Compound (E) -2- (3- (2-cyano-2- (6- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24255)

Compound AB24255 (yield: 57.6%) was obtained in the same manner as in example 1, ¹ H NMR(400MHz,dmso)δ8.16(s,1H),7.63(d,J＝8.7Hz,1H),7.56(s,1H),7.19(d,J＝8.7Hz,1H),6.95(s,1H),2.34(s,3H),2.29(s,3H),2.13(s,3H),2.08(s,3H)。

example 14: synthesis of the Compound ((E) -2- (3- (2-cyano-2- (6- (hydroxymethyl) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (AB 24225)

(1) Synthesis of Compound 10 (2- (6- (hydroxymethyl) -1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 9 (2- (cyanomethyl) -1H-benzo [ d ]]Imidazole-6-carboxylic acid) (157 mg,0.78mmol,1.0 eq) was dissolved in 6mL Tetrahydrofuran (THF), and after slow addition of Carbonyldiimidazole (CDI) (152 mg,0.94mmol,1.2 eq) was gently mixed, then a further 5mL sodium borohydride in water (NaBH) was slowly added ₄ ) (88 mg,2.34mmol,3.0 eq) and the mixture was stirred at room temperature for 16 hours. The mixture was extracted with ethyl acetate, and the organic layer was dried, filtered and concentrated. The resulting residue was purified by preparative TLC to give compound 10 (2- (6- (hydroxymethyl) -1H-benzo [ d ])]Imidazol-2-yl) acetonitrile) (59 mg, yield: 40.4%) of a yellow oily liquid. TLC: dichloromethane/methanol (10:1); r is R _f (compound 9) =0.1; r is R _f (compound 10) =0.3; ¹ H NMR(400MHz,CD ₃ OD-d4)δ7.86(s,1H),7.57(s,1H),7.53(d,J＝8.1Hz,1H),7.28(d,J＝8.1Hz,1H),7.13(s,1H),4.71(s,2H),3.34(s,2H)。

(2) Synthesis of Compound AB24225 ((E) -2- (3- (2-cyano-2- (6- (hydroxymethyl) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

To a solution of compound 10 in 1mL of ethanol was added compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (75 mg,0.31mmol,1.0 eq) and piperidine (26 mg,0.31mmol,1.0 eq). The mixture was heated under reflux and stirred for 1 hour, after completion the mixture was concentrated and purified by preparative high performance liquid chromatography to give AB24225 ((E) -2- (3- (2-cyano-2- (6- (hydroxymethyl) -1H-benzo [ d ])]Imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3 carbonitrile (25 mg, yield: 19.7%) of a yellow solid. MS (ESI) m/z 412[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d6)δ8.16(s,1H),7.55-7.51(m,2H),7.22(d,J＝8.4Hz,1H),6.96(s,1H),4.59(s,2H),2.28(d,J＝11.2Hz,6H),2.07(d,J＝12Hz,6H)。

Example 15: synthesis of the Compound ((E) -2- (3- (2-cyano-2- (5, 7-dichloro-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)) (AB 24239)

(1) Synthesis of Compound 12 ((2-amino-3, 5-dichlorobenzene) amine

A mixture of compound 11 (2, 4-dichloro-6-nitroaniline) (2.5 g,12.1mmol,1.0 eq) and zinc (6.3 g,96.6mmol,8.0 eq) was dissolved in methanol/acetic acid (10/1, 30 mL) and stirred at 45℃for 16 hours. After cooling, the solvent was removed. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate=100/1 to 5/1) to obtain compound 12 (2-amino-3, 5-dichloroaniline) as a brown solid (1.56 g, yield: 72.9%). MS (ESI) m/z 177[ M+H ]] ⁺ . TLC: petroleum ether, ethyl acetate= (5:1); r is R _f (compound 11) =0.7; r is R _f (compound 12) =0.4.

(2) Synthesis of Compound 14 (N- (2-amino-4, 6-dichlorophenyl) -2-cyanoacetamide)

To compound 12 (2-amino-3, 5-dichloroaniline) (1.56 g,8.8mmol,1.0 eq) dissolved in 20mL of dimethylformamide was added compound 6 (cyanoacetic acid) (0.9 g,106mmol,1.2 eq), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.0 g,10.6mmol,1.2 eq) and 1-hydroxybenzotriazole (1.2 g,8.8mmol,1.0 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with water and extracted with dichloromethane. After washing the organic layer with brine, it was dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=100/1 to 50/1) to give compound 13 (N- (2-amino-4, 6-dichlorophenyl) -2-cyanoacetamide) (1.0 g, yield: 46.6%) as a black solid powder. MS (ESI) m/z 244[ M+H ]] ⁺ 。TLC：DCM/MeOH(10:1)；R _f (compound 12) =0.5; r is R _f (compound 13) =0.7.

(3) Synthesis of Compound 14 (2- (5, 7-dichloro-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 13 (N- (2-amino-4, 6-dichlorophenyl) -2-cyanoacetamide) (1.0 g,4.10mmol,1.0 eq) was dissolved in 20mL acetic acid and stirred overnight at 110 ℃. The mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=100/1 to 20/1) to give compound 14 (2- (5, 7-dichloro-1H-benzo [ d ] as a yellow solid powder ]Imidazol-2-yl) acetonitrile) (520 mg, yield: 56.2%). MS (ESI) m/z 226[ M+H ]] ⁺ 。TLC：DCM/MeOH(20:1)；R _f (compound 13) =0.6; r is R _f (compound 14) =0.7.

(4) Synthesis of Compound AB24239 ((E) -2- (3- (2-cyano-2- (5, 7-dichloro-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

Compound 14 (2- (5, 7-dichloro-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (56 mg,0.25mmol,10 eq) was dissolved in ethanol (1.0 mL) and compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (60 mg,0.25mmol,1.0 eq) and piperidine (21 mg,0.25mmol,1.0 eq) were added. The mixture was heated to reflux and stirred for 1 hour. After the reaction was completed, the reaction mixture was cooled to room temperature and filtered. The solid was collected and dried to give AB24239 ((E) -2- (3- (2-cyano-2- (5, 7-dichloro-1H-benzo [ d)) as a yellow powder]Imidazol-2-yl]Vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (74 mg, yield: 66.7%). MS (ESI) m/z 450[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d6)δ13.33(s,1H),8.20(s,1H),7.57(s,1H),7.38(s,1H),6.97(s,1H),2.36(s,3H),2.31(s,3H),2.14(s,3H),2.09(s,3H)。

Example 16: synthesis of Compound (E) -2- (3- (2-cyano-2- (5-fluoro-6-methoxy-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24246)

(1) Synthesis of Compound 16 (2-cyano-N- (5-fluoro-4-methoxy-2-nitrophenyl) acetamide)

To a solution of Compound 6 (cyanoacetic acid) (1.1 g,12.9mmol,1.2 eq) in 20mL of Dichloromethane (DCM) at 0deg.C was added (COCl) ₂ (1.36 g,16.12mmol,1.2 eq) the mixture was stirred at 0deg.C for 30 min, then compound 15 (5-fluoro-4-methoxy-2-nitroaniline) (2.0 g,10.75mmol,1.0 eq) was added and the mixture stirred at room temperature for 2 h, then the mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=200/1-30/1) to give 16 (2-cyano-N- (5-fluoro-4-methoxy-2-nitrophenyl) acetamide) (1.3 g, yield: 47.7%) as a yellow solid. MS (ESI) m/z 254[ M+H ]] ⁺ 。TLC：PE/EA(5:1)；R _f (compound 15) =0.6; r is R _f (compound 16) =0.4.

(2) Synthesis of Compound 17 (2- (5-fluoro-6-methoxy-1H-benzo [ d ] imidazol-2-yl)

To a solution of compound 16 (2-cyano-N- (5-fluoro-4-methoxy-2-nitrophenyl) acetamide) (500 mg,1.97mmol,1.0 eq) in 5mL of isopropanol (iPrOH) was added zinc (1.0 g,15.76mmol,8 eq) and acetic acid (5 mL) and stirred at 110℃overnight. The mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. Purification of the residue by flash column chromatography (dichloromethane/methanol=50/1-1/1) afforded compound 17 (2- (5-fluoro-6-methoxy-1H-benzo [ d ]) as a grey solid ]Imidazol-2-yl) acetonitrile) (100 mg, yield: 24.5%). MS (ESI) m/z 206[ M+H ]] ⁺ 。TLC：DCM/MeOH＝50/1；R _f (compound 16) =0.7; r is R _f (compound 17) =0.3. ¹ H NMR(DMSO-d ₆ ,400Hz):δ12.35(s,1H),7.44-7.31(m,1H),7.01(s,1H),6.80-6.74(m,1H),4.28(s,2H),3.73(s,3H)。

(3) Synthesis of Compound AB24246 ((E) -2- (3- (2-cyano-2- (5-fluoro-6-methoxy-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

To compound 17 (2- (5-fluoro-6-methoxy-1H-benzo [ d) dissolved in 1mL ethanol]To a solution of imidazol-2-yl) acetonitrile (50 mg,0.24mmol,1.0 eq) was added compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 4-dimethylfuran-3-carbonitrile) (60 mg,0.24mmol,1.0 eq) and piperidine (20 mg,0.24mmol,1.0 eq). The mixture was heated to reflux and stirred for 1h, after which time the mixture was cooled to room temperature and filtered. The solid was collected and dried to give compound AB24246 ((E) -2- (3- (2-cyano-2- (5-fluoro-6-methoxy-1H-benzo [ d)) as a yellow solid]-imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (85 mg, yield: 81%). MS (ESI) m/z 412.3[ M+H] ⁺ 。 ¹ H NMR(399MHz,DMSO-d6)δ8.06(s,1H),7.42(d,J＝11.2Hz,0H),7.22(s,1H),6.94(s,0H),3.86(s,2H),2.32(d,J＝10.2Hz,3H),2.11(d,J＝18.9Hz,3H)。

Example 17: synthesis of Compound (E) -2- (3- (2- (5-bromo-6-methoxy-1H-benzo [ d ] imidazol-2-yl ] -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24266)

Compound AB24266 (yield: 82%) was obtained in the same manner as in example 16, ¹ H NMR(399MHz,DMSO-d6)δ8.08(s,1H),7.76(s,1H),7.18(s,1H),6.94(s,1H),3.86(s,3H),2.31(d,J＝12.0Hz,6H),2.11(d,J＝18.9Hz,6H)。

example 18: synthesis of Compound (E) -2- (3- (2- (1H-benzo [ d ] imidazol-2-yl) -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -5-phenylfuran-3-carbonitrile (AB 24283)

Compound AB24283 (yield: 63.5%) was obtained in the same manner as in example 1, ¹ H NMR(DMSO-d6,400Hz):δ12.91(s,1H),8.16(s,1H),7.80(d,J＝7.6Hz,2H),7.72(s,1H),7.53–7.44(m,5H),7.20(s,2H),7.03(s,1H),2.44(s,3H),2.23(s,3H)。

example 19: synthesis of Compound (E) -2- (3- (2-cyano-2- (5, 6-dichloro-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24214)

(1) Synthesis of Compound 19 (N- (2-amino-4, 5-dichlorophenyl) -2-cyanoacetamide)

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Compound 18 (4, 5-dichloro-1, 2-phenylenediamine) (2.0 g,11.3mmol,1.0 eq) was dissolved in dimethylformamide (20 mL), and Compound 6 (cyanoacetic acid) (1.2 g,13.6mmol,1.2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.61 g,13.6mmol,1.2 eq) and 1-hydroxybenzotriazole (1.53 g,11.3mmol,1.0 eq) were added. The mixture was stirred at room temperature overnight. Then diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated.The residue was purified by flash column chromatography (dichloromethane/methanol=100/1 to 30/1) to obtain compound 19 (N- (2-amino-4, 5-dichlorophenyl) -2-cyanoacetamide) (470 mg, yield: 17.2%) as a black solid powder. TLC: DCM/MeOH (10:1); r is R _f (compound 18) =0.4; r is R _f (compound 19) =0.5.

(2) Synthesis of Compound 20 (2- (5, 6-dichloro-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 19 (N- (2-amino-4, 5-dichlorophenyl) -2-cyanoacetamide) (470 mg,1.93mmol,1.0 eq) was dissolved in acetic acid (10 mL), heated to 110℃and stirred overnight. The mixture was diluted with water and then extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate=100/1 to 20/1) to obtain a black powder compound 20 (2- (5, 6-dichloro-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (300 mg, yield: 22.4%). TLC: EA; r is R _f (compound 19) =0.7; r is R _f (compound 20) =0.75.

(3) Synthesis of Compound AB24214 ((E) -2- (3- (2-cyano-2- (5, 6-dichloro-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

Compound 20 (2- (5, 6-dichloro-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (56 mg,0.248mmol,1.0 eq) was dissolved in ethanol (1 mL), and compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (60 mg,0.248mmol,1.0 eq) and piperidine (21 mg,0.248mmol,1.0 eq) were added. The mixture was heated to reflux and stirred for 2 hours. After completion of the reaction, the yellow solid powder compound AB24214 ((E) -2- (3- (2-cyano-2- (5, 6-dichloro-1H-benzo [ d)) was obtained by filtration and drying ]Imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrole-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (37 mg, yield: 28.6%). MS (ESI) m/z 450[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO)δ8.16(s,1H),7.81(s,2H),6.95(s,1H),2.31(d,J＝15.5Hz,6H),2.10(d,J＝20.0Hz,6H)。

Example 20: synthesis of Compound (E) -2- (3- (2-cyano-2- (5, 6-dimethoxy-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24229)

Compound AB24229 (yield: 45.6%) was obtained in the same manner as in example 32, ¹ H NMR(400MHz,dmso)δ12.60(s,1H),7.96(s,1H),7.16(s,1H),6.91(s,2H),3.76(s,6H),2.29(d,J＝5.8Hz,6H),2.09(d,J＝18.5Hz,6H)。

example 21: synthesis of the Compound (E) -2- (3- (2-cyano-2- (6, 7-dihydro-1H- [1,4] dioxin [2',3':4,5] benzo [1,2-d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24287)

Compound AB24287 (yield: 31.3%) was obtained in the same manner as in example 32, ¹ H NMR(400MHz,dmso)δ8.06(s,1H),6.92(s,3H),4.22(s,4H),2.31(d,J＝12.6Hz,6H),2.10(d,J＝17.2Hz,6H)。

example 22: synthesis of Compound (E) -2- (3- (2-cyano-2- (7-methyl-1H-benzo [ d ] imidazol-2-yl) vinyl ] -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24242)

(1) Synthesis of Compound 22 (N- (2-amino-6-methylphenyl) -2-cyanoacetamide)

Compound 21 (2, 3-diaminotoluene) (2.0 g,16.37mmol,1.0 eq) was dissolved in methylene chloride (20 mL), and compound 6 (cyanoacetic acid) (2.1 g,24.56mmol,1.5 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.71 g,24.56mmol,1.5 eq) and 1-hydroxybenzotriazole (3.32 g,24.56mmol,1.5 eq) were added. The mixture was stirred at room temperature overnight. Then diluted with water and extracted with dichloromethane. After the organic layer was washed with brine, Dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=100/1 to 50/1) to obtain compound 22 (N- (2-amino-6-methylphenyl) -2-cyanoacetamide) (1.48 g, yield: 47.8%) as a black solid powder. TLC: DCM/MeOH (10:1); r is R _f (compound 21) =0.2; r is R _f (compound 22) =0.3.

(2) Synthesis of Compound 23 (2- (7-methyl-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 22 (N- (2-amino-6-methylphenyl) -2-cyanoacetamide) (1.178 mg,7.82mmol,1.0 eq) was dissolved in acetic acid (10 mL) and heated to 110℃and stirred overnight. Then diluted with water and extracted with dichloromethane. After washing the organic layer with brine, it was dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (dichloromethane/methanol=100/1 to 20/1) to obtain compound 23 (2- (7-methyl-1H-benzo [ d ]) as a black solid powder]Imidazol-2-yl) acetonitrile) (300 mg, yield: 22.4%). TLC: DCM/MeOH (20:1); r is R _f (compound 22) =0.7; r is R _f (compound 23) =0.75. ¹ H NMR(400MHz,CDCl ₃ )δ7.54(d,J＝7.9Hz,1H),7.40(t,J＝7.6Hz,1H),7.29(d,J＝7.1Hz,1H),4.56(s,2H),2.63(s,3H)。

(3) Synthesis of Compound AB24242 ((E) -2- (3- (2-cyano-2- (7-methyl-1H-benzo [ d ] imidazol-2-yl) vinyl ] -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

Compound 23 (2- (7-methyl-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (56 mg, 0.227 mmol,1.0 eq) was dissolved in ethanol (1 mL), and compound 4 (79 mg, 0.227 mmol,1.0 eq) and piperidine (28 mg, 0.227 mmol,1.0 eq) were added. The mixture was heated to reflux and stirred for 2 hours. After the reaction is completed, the yellow solid powder compound AB24242 is obtained after filtration(E) -2- (3- (2-cyano-2- (7-methyl-1H-benzo [ d ])]Imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (37 mg, yield: 28.6%). MS (ESI) m/z 396[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO)δ12.66(d,J＝10.8Hz,1H),8.11(s,1H),7.35(d,J＝45.7Hz,1H),7.02(d,J＝43.9Hz,3H),2.47(s,4H),2.32(d,J＝19.9Hz,6H),2.11(d,J＝20.7Hz,6H)。

Example 23: synthesis of Compound (E) -2- (3- (2- (7-chloro-1H-benzo [ d ] imidazol-2-yl) -2-cyanovinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24218)

Compound AB24218 (yield: 61%) was obtained in the same manner as in example 35, ¹ H NMR(400MHz,DMSO-d6)δ13.16(s,1H),8.27-8.12(m,1H),7.57-7.43(m,1H),7.19-7.17(m,2H),6.95(s,1H),2.45(s,3H),2.32(s,3H),2.11(s,3H),2.06(s,3H)。

example 24: synthesis of Compound (E) -2- (3- (2-cyano-2- (7-methoxy-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24241)

Compound AB24241 (yield: 55.6%) was obtained in the same manner as in example 35, ¹ H NMR(400MHz,DMSO-d6)δ13.00(s,1H),8.35–7.79(m,1H),7.07(d,J＝23.3Hz,2H),6.89(s,1H),6.69(d,J＝11.6Hz,1H),3.86(s,3H),2.28(s,3H),2.23(s,3H),2.06(s,3H),2.02(s,3H)。

example 25: synthesis of Compound (E) -2- (3- (2-cyano-2- (6-isopropoxy-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24297)

(1) Synthesis of Compound 25 (5-isopropoxy-2-nitroaniline)

Compound 24 (5-fluoro-2-nitroaniline) (3.0 g,19.23mmol,1.0 eq) was dissolved in isopropanol (i-PrOH) (30 mL) and sodium hydroxide (1.1 g,27.5mmol,1.5 eq) was added. The mixture was stirred at 90℃for 4 hours. After cooling, the mixture was poured into ice water. The residue was purified using Flash Column Chromatography (FCC) (petroleum ether: ethyl acetate=5:1) and gave compound 25 (5-isopropoxy-2-nitroaniline) (2.3 g, 63% yield) as a yellow solid. MS (ESI) m/z 197[ M+H ]] ⁺ 。TLC：PE/EA(5/1)；R _f (compound 24) =0.5; r is R _f (compound 25) =0.2.

(2) Synthesis of Compound 26 (4-isopropoxy-benzene-1, 2-diamine)

Compound 25 (5-isopropoxy-2-nitroaniline) (1.0 g,5.1mmol,1.0 eq) was dissolved in methanol (10 mL) and palladium on carbon (200 mg,0.2 wt%) was added. The mixture was stirred at room temperature for 16 hours under hydrogen. The solid was filtered off and the filtrate was concentrated to give a brown solid, compound 26 (4-isopropoxyphenyl-1, 2-diamine) (800 mg, 94.4% yield). MS (ESI) m/z 167[ M+H ]] ⁺ 。TLC：DCM/MeOH(20:1)；R _f (compound 25) =0.7; r is R _f (compound 26) =0.2.

(3) Synthesis of Compound 28 (2- (6-isopropoxy-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 26 (4-isopropoxybenzene-1, 2-diamine) (500 mg,3.01mmol,1.0 eq) and compound 27 (ethyl cyanoacetate) (850 mg,7.53mmol,2.5 eq) were added to N-methylpyrrolidone (NMP) (2 ml) and stirred at 180℃for 4 hours. After cooling, the mixture was diluted with water and extracted with the mixed solution (dichloromethane: methanol=10:1). The organic phase was dried and filtered and concentrated. The residue was purified using preparative high performance liquid chromatograph and gave compound 28 (2- (6-isopropoxy-1H-benzo [ d ]) as a yellow solid ]Imidazol-2-yl) acetonitrile) (310 mg, yield 47.9%). MS (ESI) m/z 216.0[ M+H ]] ⁺ 。

(4) Synthesis of Compound AB24297 ((E) -2- (3- (2-cyano-2- (6-isopropoxy-1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

Compound 28 (2- (6-isopropoxy-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (70 mg,0.32mmol,1.0 eq) was dissolved in ethanol (1 mL), and compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (79 mg,0.32mmol,1.0 eq) and piperidine (55 mg,0.64mmol,2.0 eq) were added. The mixture was heated under reflux and stirred for 1 hour. The mixture was cooled to room temperature and filtered. The solid was collected and dried to give a yellow solid, AB24297 (32 mg, 22.8% yield). MS (ESI) m/z 440.0[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d6)δ12.69(s,1H),8.05(s,1H),7.42(s,1H),7.01(s,1H),6.94(s,1H),6.79(d,J＝8Hz,1H),4.60-4.58(m,1H),2.31(d,J＝8Hz,6H),6.79(d,J＝20Hz,6H),1.25(d,J＝4Hz,6H)。

Example 26: synthesis of Compound (E) -2- (3- (2-cyano-2- (1-methyl-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24231)

(1) Synthesis of Compound 30 (2- (1-methyl-1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Benzyl triethylammonium chloride (TEBAC) (18 mg,0.08mmol,0.08 eq) was dissolved in acetonitrile (3 mL), and potassium carbonate (138 mg,1mmol,1 eq) and compound 29 (2-cyanomethyl benzimidazole) (157 mg,1mmol,1 eq) dissolved in 1mL of dichloromethane were added. The mixture was stirred at room temperature for half an hour, and dimethyl sulfate (139 mg,1.1mmol,1.1 eq) was added. The mixture was stirred at room temperature for 16 hours. After concentration, the residue was quenched with water and then extracted with dichloromethane. The organic layer was dried, filtered and concentrated. The residue was purified by preparative thin layer chromatography (dichloromethane/methanol=20/1) to give compound 30 (2- (1-methyl-1H-benzo) as a yellow oil [d]Imidazol-2-yl) acetonitrile) (76 mg, yield: 44.4%). MS (ESI) m/z 172[ M+H ]] ⁺ . TLC: dichloromethane/methanol (20/1); r is R _f (compound 29) =0.5; r is R _f (compound 30) =0.2; ¹ H NMR(400MHz,CDCl ₃ )δ7.76(d,J＝8Hz,1H),7.36-7.31(m,3H),4.08(s,2H),3.86(s,3H)。

(2) Synthesis of Compound AB24231 ((E) -2- (3- (2-cyano-2- (1-methyl-1H-benzo [ d ] imidazol-2-yl ] vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

After dissolving compound 30 (2- (1-methyl-1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (75 mg, 0.433 mmol,1.0 eq) was added compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (105 mg,0.436mmol,1.0 eq) and piperidine (37 mg,0.436mmol,1.0 eq) and the mixture was heated to reflux and stirred for 1 hour. After the reaction was completed, the mixture was cooled to room temperature and filtered. The solid was collected and dried to give compound AB24231 ((E) -2- (3- (2-cyano-2- (1-methyl-1H-benzo [ d)) as a yellow solid]Imidazol-2-yl]Vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (102 mg, yield: 67.7%). MS (ESI) m/z 396[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d6)δ7.89(s,1H),7.62-7.45(m,2H),7.27-7.18(m,2H),6.94(s,1H),3.90(s,3H),2.25(d,J＝8Hz,6H),2.10(s,3H),2.05(s,3H)。

Example 27: synthesis of Compound (E) -2- (3- (2-cyano-2- (1- (methoxymethyl) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (AB 24263)

(1) Synthesis of Compound 31 (2- (1- (methoxymethyl) -1H-benzo [ d ] imidazol-2-yl) acetonitrile)

Compound 29 (2-cyanomethyl benzimidazole)) (1.0 g,6.36mmol,1.0 eq) was dissolved in 16ml tetrahydrofuran and NaH (267 mg,6.68mmol,1.05eq, 60% in mineral oil) was added at 0deg.C. The mixture was stirred at 0℃for 15 minutes. Chloromethyl methyl ether (665 mg,8.27mmol,1.3 eq) was then added. The mixture was stirred at room temperature for 2 hours. The reaction was quenched with sodium bicarbonate and then extracted with ethyl acetate. The organic layer was dried, filtered and concentrated. The residue was purified by preparative high pressure liquid chromatography to give compound 31 (2- (1- (methoxymethyl) -1H-benzo [ d ]) as a yellow oil]Imidazol-2-yl) acetonitrile) (230 mg, yield: 17.9%). MS (ESI) m/z 202[ M+H ]] ⁺ . TLC: petroleum ether/ethyl acetate (2/1); r is R _f (compound 29) =0.5; r is R _f (compound 31) =0.3. ¹ H NMR(400MHz,CDCl ₃ )δ7.64(d,J＝9.2Hz,2H),7.29-7.20(m,2H),5.56(s,2H),4.50(s,2H),3.18(s,3H)。

(2) Synthesis of Compound AB24263 ((E) -2- (3- (2-cyano-2- (1- (methoxymethyl) -1H-benzo [ d ] imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile)

Compound 31 (2- (1- (methoxymethyl) -1H-benzo [ d)]Imidazol-2-yl) acetonitrile) (50 mg, 0.219 mmol,1.0 eq) was dissolved in 1.5ml ethanol, and compound 4 (2- (3-formyl-2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile) (60 mg, 0.219 mmol,1.0 eq) and piperidine (21 mg, 0.219 mmol,1.0 eq) were added. The mixture was stirred under reflux for 1 hour. After completion of the reaction, the mixture was cooled to room temperature, filtered and the solid was collected and dried to give AB24263 ((E) -2- (3- (2-cyano-2- (1- (methoxymethyl) -1H-benzo [ d)) as a yellow solid ]Imidazol-2-yl) vinyl) -2, 5-dimethyl-1H-pyrrol-1-yl) -4, 5-dimethylfuran-3-carbonitrile (22 mg, yield: 20.9%). MS (ESI) m/z 426[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,DMSO-d6)δ8.05(s,1H),7.67-7.58(m,2H),7.27-7.20(m,2H),6.98(s,1H),5.66(s,2H),3.38(s,3H),2.29(s,3H),2.24(s,3H),2.14(s,3H),2.08(s,3H)。

Pharmacological Activity test

Preparation of recombinant RNA helicase DHX33

The separation and purification of the protein is described in Wang X, ge W, and Zhang Y.Recombinat DHX33 Protein Possesses Dual DNA/RNA-helix Activity.biochemistry.2019;58 (4):250-8. The RNA helicase gene (mouse DHX33 gene) was cloned between BamH I/Not I cleavage sites of the pET32M-3C vector. The plasmid was then transformed into E.coli strain BL-21pLysS (DE 3), 0.5mM isopropyl 1-thio- β -D-galactopyranoside (IPTG) was added and recombinant protein expression was induced at 16℃for 16 hours. Cells were pelleted and resuspended in cell lysis buffer [50mM Tris-HCl (pH 7.2), 150mM NaCl,1%Triton X-100 and 50mM imidazole with protease inhibitor added ]. The cells were then sonicated and centrifuged at 13000rpm for 25 minutes. The supernatant was incubated with Tris buffer equilibrated nickel-nitrilotriacetic acid beads and then washed extensively. The purified protein was then eluted with 300mM imidazole in Tris buffer, and then dialyzed against Tris buffer without imidazole at 4℃overnight.

DHX33 helicase activity assay

The helicase active reaction components were added to a 96-well opaque color plate. The method is summarized as follows: neutravidin (Neutravidin) was coated on 96-well plates overnight at 4℃at a final concentration of 10. Mu.g/mL (100. Mu.L/well). The neutravidin coated plates were then blocked with 100. Mu.L of 0.1% (w/v) BSA (in conventional PBS) for 2 hours at 22 ℃. After washing, DNA duplex [2.5ng was added, which was annealed from two oligo DNA single strands (one strand was 5'-GCTGACCCTGCTCCCAATCGTAATCTATAG-3' labeled with biotin; the other strand was 5'-CGATTGGGAGCAGGGTCAGC-3' labeled with DIG), in 1M PBS (pH 7.0) containing 1M NaCl]And incubated at 22℃for 4 hours. After addition of 90. Mu.L of the reaction mixture, the helicase reaction was started [ purified full-length DHX33 protein 0.25. Mu.g, dissolved in 25mM 4-MOPS (pH 7.0), 5mM ATP,2mM DTT,3mM MnCl ] ₂ And 100. Mu.g/mL BSA]. The reaction was carried out at 37℃for 60 minutes. After washing, each well was treated with blocking solution [10% (w/v) BSA in 0.1M maleic acid and 0.15M NaCl (pH 7.5)]The incubation time was allowed to run for 30 minutes,then incubated with 20. Mu.L of antibody solution (anti-DIG-AP, roche in blocking buffer) for 30 min. mu.L of detection buffer [0.1M Tris-HCl and 0.1M NaCl (pH 9.5) ]After washing, 1. Mu.L of chemiluminescent substrate (CSPD-0.25 mM) was then added to each well and the plate incubated at 17℃for 5 minutes. Plates were then patted dry and incubated at 37℃for 30 minutes. The DIG-AP marker control remaining in each well was counted for 10 minutes by a luminescent multiwell plate reader (Enspire, perkinelmer).

Concentration of cell half inhibition (EC ₅₀ ) Measurement

DHX33 overexpressing cancer cell line U251-MG cells at 1 x 10 ⁴ 100 ul/well was plated onto 96-well plates. Waiting for cell attachment to be complete, compounds were added to the cell culture medium at concentrations of 5nM, 10nM, 25nM, 50nM, 100nM, 250nM, 500nM, 1000nM, 2000nM and mixed well using a multichannel lance. After waiting for the compound and cell incubation time to reach 48 hours, the compound was added to the medium of 96-well plates according to standard procedure using CCK-8 reagent (hao-halibut biosciences, inc.) and incubated for 2 hours, the experiment was repeated three times using a microplate reader (od=450 nm), and inhibition curves of the compound at different concentrations were plotted to calculate the half-inhibition concentration (EC ₅₀ )。

Cell culture and sources

U251-MG cells were purchased from the cell bank of the department of Chinese sciences. Culturing in MEM medium containing 10% Fetal Bovine Serum (FBS), 2mM L-glutamine and streptomycin and penicillin, and setting growth condition at 37deg.C and 5% CO ₂ In a cell incubator with humidity. Cells were passaged every 3 days and discarded after 10 passages.

Quantitative real-time PCR

Primers were designed by IDT (http:// sg. Idtdna. Com/site) on line "Realtime PCR Tool", available from BGI (Shenzhen) Inc. Total RNA was extracted by High Pure RNA isolation kit (Roche) and then transcribed into cDNA using PrimeScript mixing kit (Takara). Real-time PCR was performed using ABI One step plus cycler and managed using corresponding software. For analysis of mRNA levels SYBR green Supermix (Bio-Rad) was used and passed after normalization to GAPDH values ^ΔΔ Transcript quantification was calculated by CT values. The melting curve was used to confirm amplification of a single product. The primer sequences are shown in Yuan B, wang X, fan C, you J, liu Y, weber JD, zhong H, and Zhang Y.DHX33 Transcriptionally Controls Genes Involved in the Cell cycle.molecular and cellular biology.2016;36 (23):2903-17.

Modulation of known DHX33 downstream genes by Compounds

U251 cells were treated with AB24202 (0.5 μm, 1 μm respectively) or AB245203 (0.5 μm concentration) for 16 hours, control groups were treated with equal volumes of DMSO. After extraction of total RNA, reverse transcription into cDNA is performed. Then, real-time quantitative PCR was performed to analyze the transcript level of the target gene in each sample, and GAPDH was used as an internal reference.

Test results and analysis

FIG. 1 shows analysis of the results of SDS-PAGE separation of recombinant DHX33 protein prepared as described above, stained with Coomassie brilliant blue. The arrow in the figure indicates the recombinant DHX33 protein of interest (containing a thioredoxin tag) with a molecular weight of the band of interest of 90kDa.

FIG. 2 shows the regulation of known DHX33 downstream genes by compounds of the present invention, wherein selected CCNE2, MCM3, AURKB, E2F1, MCM2 genes are all downstream genes identified in published paper as being DHX33 regulated (Yuan B, wang X, fan C, you J, liu Y, weber JD, zhong H, and Zhang Y.DHX33 Transcriptionally Controls Genes Involved in the Cell cycle.molecular and cellular biology.2016;36 (23): 2903-17) that are significantly down-regulated in the event of a DHX33 genetic knockout or suppression. As can be seen from fig. 2, the compounds of the invention have a significant inhibitory effect on the transcription of these downstream genes (P is less than 0.05, n=3, data were taken from three independent experiments).

In vitro recombinant DHX33 protein helicase activity assays were performed using a range of concentrations of compounds (concentration ranges set to 1nM, 5nM, 10nM, 20nM, 50nM, 100nM, 250nM, 500 nM). The half inhibitory concentrations of the compounds of the present invention on the helicase activity of DHX33 protein are shown in table 1. As can be seen from table 1, the compounds of the present invention have a significant inhibitory effect on the helicase activity of DHX33 protein.

Table 1: helicase activity inhibition assay of compounds on DHX33 protein

Compounds of formula (I)	IC ₅₀ (nM)
		Control compounds	450
AB24202	50
		AB24205	190
AB24243	100
		AB24283	180

Table 2: semi-inhibitory concentration analysis of compounds for U251-MG

As can be seen from Table 2, the compounds of the present invention have a remarkable inhibitory effect on the cells of the cancer cell line U251-MG over-expressing DHX 33.

Claims

1. A compound or a pharmaceutically acceptable salt thereof selected from the following compounds or pharmaceutically acceptable salts thereof:

2. a pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

3. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder mediated by DHX 33.

4. The use according to claim 3, wherein the disease is cancer.