WRN helicase inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a WRN helicase inhibitor as well as a preparation method and application thereof. The WRN helicase inhibitor has a structure of a compound as shown in a formula I: # imgabs0. The compound disclosed by the invention has better inhibitory activity on WRN and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly relates to a WRN helicase inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Human RecQ family genes encode a class of nucleic acid-binding proteins with conserved structural features, consisting of five family members, all of which are important "guardians" for maintaining genomic stability. Research shows that the RecQ family gene WRN (Werner) is a "synthetic lethal" gene in MSI tumor cells. Knocking out the WRN gene will selectively induce the death of MSI tumor cells, indicating that the function of the WRN gene is crucial for the occurrence and development of MSI tumors. At the same time, knocking out WRN can also enhance the sensitivity of clinical MSI colorectal cancer to radiotherapy, chemotherapy, and immunotherapy, suggesting that the WRN protein has important biological functions in MSI tumors and has the potential to become a therapeutic target. However, up to now, only a very small number of small molecule inhibitors of WRN helicase have been reported. Currently, the discovery of small molecule inhibitors targeting the WRN protein mainly faces two difficulties: ① The WRN protein has large flexibility. Except for the ATP binding site, there is no clear small molecule binding pocket, making it difficult to design selective inhibitors specifically; ② The reported screening methods based on helicase activity have poor effects, hindering the screening of new molecules. Therefore, there is an urgent need to develop novel anti-cancer lead compounds with high helicase inhibitory activity and strong selectivity targeting the WRN protein, conduct systematic research on the anti-cancer mechanism of action, and explore the feasibility of using the WRN protein as a "synthetic lethal" target for MSI tumors. Summary of the Invention

[0003] The present invention aims to at least solve the above technical problems existing in the prior art. For this reason, the object of the present invention is to provide a WRN helicase inhibitor, a preparation method thereof, and an application thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In the first aspect of the present invention, there is provided a compound of formula I, or a stereoisomer thereof, a deuterated compound thereof, a pharmaceutically acceptable salt thereof:

[0006]

[0007] L is selected from a carbon-carbon triple bond, an unsubstituted or halogen-substituted carbon-carbon double bond;

[0008] Each R1 is independently selected from deuterium, hydrogen, halogen, azido, -C1-C8 alkyl, -C1-C8 alkoxy, -NR 11 R 12 、-C(O)OR 13 、-C(O)NR 14 R15 ; wherein, the alkyl and alkoxy groups are optionally unsubstituted or substituted by one or more R a substituents;

[0009] R 11 and R 12 are each independently selected from deuterium, hydrogen, -C1-C8 alkyl, -C0-C6 alkylene-NR 111 R 112 ;

[0010] R 13 and R 14 and R 15 and R 111 and R 112 are each independently selected from deuterium, hydrogen, -C1-C8 alkyl;

[0011] Each R a is independently selected from deuterium, hydroxy, halogen, -C1-C8 alkyl;

[0012] Each R2 is independently selected from deuterium, hydrogen, halogen, hydroxy, carboxy, diethyl phosphate group, -C1-C8 alkyl, -C1-C8 alkoxy, -NR 11 R 12 , 3-10 membered heterocycloalkyl, -L1-C0-C6 alkylene-Ar;

[0013] -L1- is selected from -NH-, -NHC(O)-, -C(O)NH-, -N=C-, -O-;

[0014] Ar is selected from -C1-C8 alkyl, -C1-C8 alkoxy, -C2-C8 alkenyl, -C2-C8 alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aryl, 5-20 membered heteroaryl; wherein, the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally unsubstituted or substituted by one or more R b substituents;

[0015] Each R b is selected from deuterium, halogen, hydroxy, amino, -C1-C8 alkyl, -S(O)2R a , -NR 11 R 12 , -C2-C8 alkenyl, -C2-C8 alkynyl; wherein, the alkyl is optionally unsubstituted or substituted by one or more R c substituents;

[0016] Each R c is selected from hydroxy, cyano, -C(O)OR a , 3-10 membered heterocycloalkyl;

[0017] n is a natural number selected from 0 to 4; m is a natural number selected from 0 to 5.

[0018] In some embodiments of the present invention, the compound of formula I has the structures of formula Ia, formula Ib, and formula Ic:

[0019] Wherein,

[0020] X is a halogen.

[0021] In some embodiments of the present invention, the heteroatoms of the heterocycloalkyl and heteroaryl include at least one of N, O, and S.

[0022] In some embodiments of the present invention, Ar is selected from cyclopentyl, cyclohexyl, phenyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, 1,3-dioxolanyl, piperazinyl, imidazolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, and 5-imidazolyl, etc.), oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, oxadiazolyl, triazolyl, tetrazolyl, carbazolyl, indolyl, quinolinyl, isoquinolinyl, benzothienyl; these groups are optionally unsubstituted or substituted by one or more R b substituted.

[0023] In some embodiments of the present invention, Ar is selected from

[0024] These groups are optionally unsubstituted or substituted by one or more R b substituted; such as can be

[0025] In some embodiments of the present invention, R2 is selected from

[0026] In some embodiments of the present invention, -NR 11 R 12 is selected from amino, -N(C1-C8 alkyl)2, -NH-C0-C6 alkylene-NR 111 R 112 ; such as -NH2, -N(CH3)2, -NH(CH2)3N(CH2CH3)2.

[0027] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0028]

[0029]

[0030]

[0031]

[0032] The second aspect of the present invention provides a method for preparing the compound of formula I as described above, comprising the following steps:

[0033] Reacting a compound of formula II with a compound of formula III to obtain a compound of formula I;

[0034] wherein X is halogen or methyl; the definitions of R1, R2, m, and n are as described above.

[0035] In some embodiments of the present invention, a compound of formula IIa is subjected to a coupling reaction with a compound of formula IIIa to obtain a compound of formula Ic;

[0036] wherein X1 is halogen.

[0037] In some embodiments of the present invention, a compound of formula IIb is subjected to a condensation reaction with a compound of formula IIIb to obtain a compound of formula Ia;

[0038]

[0039] In some embodiments of the present invention, a compound of formula Ic is subjected to an addition and substitution reaction to obtain a compound of formula Ib.

[0040] In some embodiments of the present invention, a compound of formula Ic-1 is subjected to a condensation reaction to obtain a compound of formula Ic-2;

[0041] wherein R2 is selected from hydroxyl, amino, carboxyl; k is a natural number between 0 and 6; the definition of L1 is as described above.

[0042] In some embodiments of the present invention, a compound of formula Ic-3 is subjected to a click reaction to obtain a compound of formula Ic-5;

[0043]

[0044] The third aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I as described above, or its stereoisomers, its deuterated compounds, its pharmaceutically acceptable salts.

[0045] In some embodiments of the present invention, the pharmaceutical composition further comprises other therapeutic agents.

[0046] In some embodiments of the present invention, the other therapeutic agent is a chemotherapeutic drug, an immunotherapeutic drug, or a targeted drug.

[0047] In some embodiments of the present invention, the chemotherapeutic drugs are selected from: anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytarabine arabinoside, cytarabine liposome injection, dacarbazine, actinomycin, daunorubicin hydrochloride, daunorubicin citrate, liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5-fluorouracil, flutamide, telbivudine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, calcium folinate, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, gemtuzumab, paclitaxel, pentostatin, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine; preferably 5-fluorouracil (5-FU) and irinotecan.

[0048] In some embodiments of the present invention, the immunotherapeutic drugs are selected from: PD-1 inhibitors (including small molecules and antibody drugs), PD-L1 inhibitors (including small molecules and antibody drugs), CTLA-4 inhibitors, PD-1 / CTLA-4 bispecific antibodies, PD-1 / TIGIT bispecific antibodies, PD-L1 / 4-1BB bispecific antibodies, PD1 / VEGF-A bispecific antibodies, PD-1 / LAG-3 bispecific antibodies;

[0049] In some embodiments of the present invention, the PD-1 inhibitors are selected from: nivolumab, pembrolizumab, camrelizumab, toripalimab, sintilimab, tislelizumab, penpulimab, sepacilimab, surufatinib, Dostarlimab, and PDR001;

[0050] In some embodiments of the present invention, the PD-L1 inhibitors are selected from: durvalumab, atezolizumab, envafolimab, and sugemalimab;

[0051] In some embodiments of the present invention, the CTLA-4 inhibitors are selected from ipilimumab;

[0052] In some embodiments of the present invention, the PD-1 / CTLA-4 bispecific antibodies are selected from cadonilimab.

[0053] In some embodiments of the present invention, the targeted drugs include, but are not limited to, VEGFR inhibitors (lenvatinib), NTRK inhibitors (entrectinib), BRAFV600 inhibitors (dabrafenib), MEK inhibitors (trametinib), EGFR monoclonal antibodies (cetuximab), PARP inhibitors (olaparib, niraparib, fluzoparib, pamiparib, etc.), and ATR inhibitors.

[0054] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, and vehicle.

[0055] The pharmaceutical composition of the present invention is suitable for various administration routes and can thus be formulated into any pharmaceutically acceptable dosage form. For example, the above-mentioned pharmaceutical composition can be administered to patients or subjects in need of such treatment by oral, parenteral, rectal, or pulmonary administration, etc. For oral administration, the pharmaceutical composition can be formulated into oral preparations, such as conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be formulated into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added. For parenteral administration, the above-mentioned pharmaceutical composition can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating into injections, conventional methods in the existing pharmaceutical field can be used for production. When formulating injections, no additives can be added, or suitable additives can be added according to the properties of the drug. For rectal administration, the pharmaceutical composition can be formulated into suppositories, etc. For pulmonary administration, the pharmaceutical composition can be formulated into inhalation preparations, aerosols, powder aerosols, or sprays, etc.

[0056] In a fourth aspect of the present invention, there is provided the use of the compound of formula I, or its stereoisomers, its deuterated compounds, its pharmaceutically acceptable salts, or the pharmaceutical composition in the preparation of anti-tumor drugs.

[0057] In some embodiments of the present invention, the drug is a WRN helicase inhibitor.

[0058] In some embodiments of the present invention, the drug inhibits the activity of WRN helicase.

[0059] In some embodiments of the present invention, the tumors include at least one of colon cancer, colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, skin cancer, and microsatellite instability-high (MSI-H) cancers.

[0060] The positive and progressive effects of the present invention are as follows: The compounds of the present invention have good inhibitory activity against WRN. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The inhibitory effect of compound B18 of the present invention on various colon cancer cells. Specific embodiments

[0062] The content of the present invention will be further described in detail below through specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0063] Definition of terms used in the present invention: Unless otherwise specified, the initial definitions provided for groups or terms herein apply to such groups or terms throughout the specification; for terms not specifically defined herein, meanings that can be given to them by those skilled in the art should be given according to the disclosure content and context.

[0064] "Plurality" means two or more quantities. Thus, when described in the present invention as being substituted by a plurality of groups, it means being substituted by two or more groups. The specific number of substituents is affected by the number of substitutable sites and steric hindrance of the group being substituted, and usually means being substituted by two, three, four, five or six groups, and more preferably being substituted by two or three groups.

[0065] The term "alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. The alkyl group can be straight-chain or branched-chain. Representative branched-chain alkyl groups have one, two or three branches. The alkyl group can be optionally substituted by one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl.

[0066] The term "alkylene" refers to a divalent saturated aliphatic hydrocarbon group having a specified number of member atoms. C a ~C b alkylene refers to an alkylene group having a to b carbon atoms. The alkylene group includes branched-chain and straight-chain hydrocarbon groups. The term "dimethylbutylene" can be exemplified by any of the following structures:

[0067] The -C0-C6 alkylene of the present invention can be C0 alkylene, C1 alkylene (e.g., -CH2-), C2 alkylene (e.g., -CH2CH2-), C3 alkylene, C4 alkylene, C5 alkylene or C6 alkylene; C0 alkylene means that the group does not exist here and is connected in the form of a chemical bond. A-C0 alkylene-B means A-B, that is, group A and group B are directly connected by a chemical bond.

[0068] The term "C1-C6 alkoxy" refers to a chemical formula -O-C1-C8 alkyl, including but not limited to, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, sec-pentyloxy, neopentyloxy, hexyloxy, etc. In one embodiment, the alkoxy may be substituted with one or more substituents, such as 1 to 3 halogens or C1-C6 alkyl.

[0069] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having at least 1 vinyl unsaturation site (>C=C<). For example, C a -C b alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0070] The term "alkynyl" refers to a straight-chain monovalent hydrocarbon group or a branched-chain monovalent hydrocarbon group containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, C2-C6 alkynyl is intended to include ethynyl, propynyl, etc.

[0071] The term "cycloalkyl" refers to a saturated or partially saturated cyclic group having multiple carbon atoms, no ring heteroatoms, and having a single ring or multiple rings (including fused). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and polycycloalkyl rings, such as bicyclopropyl, bicyclohexyl, bicyclopentyl, bicyclooctyl, etc.

[0072] The term "heterocycloalkyl" refers to a saturated ring or a non-aromatic partially saturated ring having a single ring or multiple rings (fused, bridged, spiro-fused) containing at least one heteroatom; where the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. Generally represents a monovalent saturated or partially unsaturated monocyclic or polycyclic ring system having multiple ring atoms, which contains 1, 2 or 3 ring heteroatoms selected from N, O and S, and the remaining ring atoms are carbon. 3-membered rings, such as oxiranyl, aziridinyl; 4-membered rings, such as azetidinyl, oxetanyl, thietanyl; 5-membered rings, such as tetrahydrofuranyl, 1,3-dioxolanyl, tetrahydrothienyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, triazolidinyl, isoxazolidinyl, oxazolidinyl, oxadiazolidinyl, thiazolidinyl, isothiazolidinyl, thiadiazolidinyl; 6-membered rings, such as piperidinyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazinyl, triazinyl, hexahydrotriazinyl, tetrahydropyranyl, dioxanyl, tetrahydrothiopyranyl, dithianyl, morpholinyl, 1,2-oxazepanyl, oxathiazepanyl, thiomorpholinyl; or 7-membered rings, such as oxepanyl, azepanyl, 1,4-diazepanyl and 1,4-oxazepanyl.

[0073] The term "heteroaryl" refers to an aromatic 5- to 10-membered monocyclic or bicyclic heterocycle containing 1 to 4 heteroatoms selected from N, O, and S. That is, heteroaryl refers to a 5- or 6-membered aromatic heterocycle containing 1 to 4 heteroatoms selected from N, O, and S, or a bicyclic ring where the heteroaryl ring is fused to a benzene ring or another heteroaryl ring. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of the heteroaryl can be substituted by substituents. Examples of monocyclic heteroaryls include, but are not limited to, thiazolyl, oxazolyl, phenylthio, furyl, pyrrolyl, imidazolyl, isoxazolyl, isothiazolyl, pyrazolyl, triazolyl, triazinyl, thiadiazolyl, tetrazolyl, dioxazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and similar groups. Examples of bicyclic heteroaryls include, but are not limited to, indolyl, azaindolyl, dihydroindolyl, benzothienyl, benzofuryl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, purinyl, furanopyridyl, and similar groups.

[0074] When a wavy line is present on the valence bond of a group such as in the wavy line indicates the point of attachment of the group to the rest of the molecule

[0075] "Halogen" as used in the present invention refers to fluorine, chlorine, bromine, or iodine.

[0076] In "-S(O)2R" etc. as used in the present invention, the oxygen atom is double-bonded to the sulfur atom, and the R group is single-bonded to the sulfur atom.

[0077] The "deuterated compound" of the present invention refers to a molecule or group in which one or more hydrogen atoms are replaced by deuterium atoms, where the proportion of deuterium atoms is greater than the natural abundance of deuterium.

[0078] "Stereoisomers" include enantiomers and diastereoisomers, and also include cis-trans isomers, tautomers, etc.

[0079] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or the salt formed is generally chemically or physically compatible with the other components constituting a pharmaceutical dosage form and is physiologically compatible with the receptor.

[0080] "Salt" includes any and all salts. "Pharmaceutically acceptable salts" refer to salts that, within the scope of reasonable medical judgment, are suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic response, etc. and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutical salts in detail in the following literature: J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts include those derived from inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by reacting an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods employed in the art (such as ion exchange). Other pharmaceutical salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Pharmaceutical salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C1-C4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Additional pharmaceutical salts include (where appropriate) non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide ions, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0081] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers are 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0082] In each example, 1H NMR was recorded on an Avance III Avance TM 500 MHz nuclear magnetic resonance spectrometer, and the chemical shift was expressed as δ (ppm).

[0083] In the following experimental descriptions, the following abbreviations may be used:

[0084]

[0085] The present invention prepared a WRN helicase inhibitor, which can be divided into three series of compounds, namely A, B, and C.

[0086] In the present invention, the synthetic routes of the A series compounds are summarized into two routes. The synthesis of the target products A1 - A19 is carried out according to Reaction Route 1. Starting from commercially purchased brominated benzothiazole and alkynylbenzene, through the Sonogashira coupling reaction, Compound 1 is obtained. Addition and substitution reactions are carried out on Compound 1 to obtain Compound 2. The target product A21 is obtained through Synthesis Route 2 under the conditions of 50% KOH and DMSO as the solvent, through the condensation reaction of methyl and aldehyde groups.

[0087]

[0088] Synthesis Route 1: (a) CuI, TEA, PdCl2(PPh3)2, DMF, Ar, 80 °C, 2 h; (b) K2CO3, DMF, 90 °C, 6 - 12 h

[0089]

[0090] Synthesis Route 2: (c) 50% KOH, DMSO, 80 °C, 2 h

[0091] Example 1: Preparation steps of Compounds A1 - A8

[0092] Taking the 4-(benzo[d]thiazol-2-ylethynyl)aniline compound A1 as an example

[0093]

[0094] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylaniline (228 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and cuprous iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe, and heat the reaction to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and perform extraction and liquid separation. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain the yellow solid A1.

[0095] Replacing 4-ethynylaniline in the preparation method of A1 with 4-ethynylbenzaldehyde can obtain compound A2, and replacing 2-bromobenzothiazole in the preparation method of A1 with 2-bromo-6-methoxybenzothiazole can obtain compound A10. The preparation methods of the remaining compounds are the same.

[0096] Example 2: Preparation steps of 3-(benzo[d]thiazol-2-ylethynyl)phenyl ethyl phosphonate compound A9

[0097]

[0098] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 3-ethynylphenol (224 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and cuprous iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe, and heat the reaction to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and perform extraction and liquid separation. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain intermediate 1.

[0099] Dissolve the intermediate in DMF (4 mL), add carbon tetrachloride (0.5 mL, 5.1 mg) at -10 °C, then add DIPEA (13 mg, 0.1 mmol) and DMAP (13 mg, 0.1 mmol), and finally dropwise add diethyl phosphite (318 mg, 2.3 mmol). The reaction needs to be carried out below 0 °C. For the post-treatment process, add K2HPO4 (3 mL), stir at room temperature until a solid precipitates, and filter to obtain a white solid. Continue to extract the mother liquor with EA (50 mL), wash the organic phase with water, rotary evaporate the organic phase, and combine to obtain white solid A9.

[0100] Example 3: Preparation steps of compounds A16 - 20

[0101] Using (Z)-N 1 -(4-(2-(benzo[d]thiazol-2-yl)-1-fluorovinyl)phenyl)-N 3 ,N 3 -diethylpropane-1,3-diamine compound A16 as an example

[0102]

[0103] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-fluorophenylacetylene (228 mg, 1.9 mmol), tris(dibenzylideneacetone)dipalladium(0) (68 mg, 0.1 mmol) and copper(I) iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) using a syringe and heat to 80 °C for reaction. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and perform extraction and liquid separation. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain intermediate 1.

[0104] Dissolve intermediate 1 and K2CO3 (100 mg) in DMF (4 mL), add 3-diethylaminopropylamine (299 mL, 1.9 mmol) using a syringe, and heat to 120 °C for reaction. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and perform extraction and liquid separation. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain A16.

[0105] Replacing 3 - diethylaminopropylamine with N - methylpiperazine in the preparation method of A16 can obtain compound A17, and the preparation methods of the remaining compounds are the same.

[0106] Example 4: Preparation steps of compound A21 of 2 - (phenylethynyl)benzo[d]thiazole

[0107]

[0108] Dissolve 2 - methylbenzothiazole (500 mg, 2.3 mmol) and 3,5 - dichlorobenzaldehyde (332 mg, 1.9 mmol) in EtOH (4 mL), heat to 80 °C and reflux. Neutralize with dilute hydrochloric acid to pH = 7, dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain A21.

[0109] The synthetic routes of B - series compounds in the present invention are summarized into 3 routes. The synthesis of target products B1 - B13 and B20 - B26 is carried out according to Route 1. The bromobenzothiazole raw material undergoes a coupling reaction with the phenylacetylene raw material to construct the core part of the whole structure, and the amino group in the phenylacetylene raw material undergoes a condensation reaction with the raw material with a carboxyl group. The synthesis of target products B14 - B19 is carried out according to Route 2. The bromobenzothiazole raw material undergoes a coupling reaction with the phenylacetylene raw material to construct the core part of the whole structure, and the carboxyl group in the phenylacetylene raw material undergoes a condensation reaction with the raw material with an amino group. For compounds B27 and B28 connected by an imine bond, they are condensed from aldehyde and amine intermediates according to Route 3.

[0110]

[0111] Route 1:

[0112] (a) CuI, Et3N, PdCl2(PPh3)2, DMF, 80 °C, 2 h; (b) R2COOH, HATU, DIPEA, DCM, r.t, 3 h (c) CF3COOH, DCM, r.t, 6 - 12 h

[0113]

[0114] Route 2:

[0115] (a) CuI, Et3N, PdCl2(PPh3)2, DMF, 80 °C, 2 h; (b) R2NH2, HATU, DIPEA, DCM, r.t, 3 h (c) CF3COOH, DCM, r.t, 6 - 12 h

[0116]

[0117] Synthetic Route Three: (a) CuI, Et3N, PdCl2(PPh3)2, DMF, 80 °C, 2 h; (b) EtOH, r.t, 3 h

[0118] Example 4: Preparation Steps of Compounds B1 - B5 and B20 - B26

[0119] Taking N-(4-(benzo[d]thiazol-2-ylethynyl)phenyl)-3-phenylpropanamide Compound B1 as an example

[0120]

[0121] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylaniline (228 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and cuprous iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe and heat to 80 °C for reaction. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0122] Dissolve Intermediate 1 (50 mg, 200 μmol), phenylpropionic acid (25 mg, 167 μmol) and HATU reagent (76 mg, 200 μmol) in DCM (2 mL), add TEA (0.1 mL) dropwise, and continue stirring at room temperature for reaction. Dilute the reaction solution with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain white solid B1.

[0123] Compound B2 can be prepared by replacing benzoic acid with cyclohexanecarboxylic acid in the preparation method of B1, and the preparation methods of the other compounds are the same.

[0124] Example 5: Preparation Steps of Compound B6 - B13

[0125] Taking the compound (2S,4R)-N-(4-(benzo[d]thiazol-2-ylethynyl)phenyl)-4-hydroxypyrrolidine-2-carboxamide, Compound B7, as an example

[0126]

[0127] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylaniline (228 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and copper(I) iodide (19 mg, 0.2 mmol) in DMF (4 mL), and evacuate and refill with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe, and heat the reaction to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0128] Dissolve Intermediate 1 (50 mg, 200 μmol), hydroxyproline raw material 2 (39 mg, 167 μmol) and HATU reagent (76 mg, 200 μmol) in DCM (2 mL), add dropwise TEA (0.1 mL), and continue stirring the reaction at room temperature. Dilute the reaction solution with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain white solid B7.

[0129]

[0130] Among them, hydroxyproline raw material 2 is obtained by dissolving hydroxyproline (160 μmol) in DCM (2 mL), adding DIPEA (300 μL, 0.02 mmol) with a syringe, dropping N-Boc-glycine dissolved in DCM (2 mL) at 0 °C, and reacting at room temperature. Dilute the reaction solution with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / EA (v / v) = 4 / 1).

[0131] Compound B8 can be prepared by replacing 2-bromobenzothiazole with 2-bromo-6-fluorobenzothiazole in the preparation method of B7, and the preparation methods of the remaining compounds are the same.

[0132] Example 6: Preparation steps of compounds B14 - B17

[0133] Taking 4-(benzo[d]thiazol-2-ylethynyl)-N-(2-hydroxycyclopentyl)benzamide compound B14 as an example

[0134]

[0135] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylbenzoic acid (277 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and copper iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe and heat the reaction to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain intermediate 1.

[0136] Dissolve intermediate 1 (56 mg, 200 μmol), 2-aminocyclopentanol (16 μL, 167 μmol) and HATU reagent (76 mg, 200 μmol) in DCM (2 mL), add TEA (0.1 mL) dropwise, and continue stirring the reaction at room temperature. Dilute the reaction solution with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain white solid B14.

[0137] Compound B15 can be prepared by replacing 2-aminocyclopentanol with 3-aminomethylpyridine in the preparation method of B14, and the preparation methods of the remaining compounds are the same.

[0138] Example 7: Preparation steps of compounds B18 and B19

[0139] Taking the compound B18 of N-butyl-2-((4-((pyrazin-2-ylmethyl)carbamoyl)phenyl)ethynyl)benzo[d]thiazole-6-carboxamide as an example

[0140]

[0141] Dissolve 2-bromobenzothiazole-6-carboxylic acid (590 mg, 2.3 mmol), 4-ethynylbenzoic acid (277 mg, 1.9 mmol), tris(dibenzylideneacetone)dipalladium(0) (68 mg, 0.1 mmol) and copper(I) iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) using a syringe, and heat the reaction to 80 °C. Dilute the reaction mixture with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0142] Dissolve Intermediate 1 (64 mg, 200 μmol), 2-(aminomethyl)pyrazine (20 μL, 200 μmol) and HATU reagent (76 mg, 200 μmol) in DCM (2 mL), add dropwise TEA (0.1 mL), and continue stirring the reaction at room temperature. Dilute the reaction mixture with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain Intermediate 2.

[0143] Dissolve Intermediate 2 (41 mg, 100 μmol), n-butylamine (10 μL, 100 μmol) and HATU reagent (38 mg, 100 μmol) in DCM (2 mL), add dropwise TEA (0.1 mL), and continue stirring the reaction at room temperature. Dilute the reaction mixture with DCM (30 mL) and saturated NH4Cl solution (30 mL), extract and separate the layers, wash the organic phase with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 50 / 1) to obtain the yellow solid B18.

[0144] Replacing n-butylamine with n-hexylamine in the preparation method of B18 can obtain B19, and the preparation methods of the other compounds are the same.

[0145] Example 8: Preparation steps of (E)-N-(4-(benzo[d]thiazol-2-ylethynyl)phenyl)-1-(3,5-

[0146] dichlorophenyl)methanimine Compound B27

[0147]

[0148] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylaniline (228 mg, 1.9 mmol), palladium dichloride triphenylphosphine (68 mg, 0.1 mmol) and cuprous iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe, and heat the reaction to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic phase with water (50 mL), then with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0149] Dissolve Intermediate 1 (24 mg, 96 μmol) and 3,5-dichlorobenzaldehyde (20 mg, 115 μmol) in EtOH (1 mL), and continue stirring the reaction at room temperature. After the reaction is complete, yellow particles precipitate. Remove the solvent under reduced pressure, and recrystallize the solid from DCM / n-hexane to obtain golden yellow solid B27.

[0150] Example 9: Preparation steps of (E)-N-(4-(benzo[d]thiazol-2-ylethynyl)phenyl)-1-(9-ethyl-9H-carbazol-3-yl)methanimine B28

[0151]

[0152] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 4-ethynylaniline (228 mg, 1.9 mmol), dichlorobis(triphenylphosphine)palladium(II) (68 mg, 0.1 mmol) and copper(I) iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) with a syringe, and heat the reaction mixture to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract and separate the layers. Wash the organic layer with water (50 mL), then wash with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0153] Dissolve Intermediate 1 (24 mg, 96 μmol) and the aldehyde raw material (25 mg, 115 μmol) in EtOH (1 mL), and continue stirring the reaction at room temperature. After the reaction is completed, yellow particles precipitate. Remove the solvent under reduced pressure, and recrystallize the solid from DCM / n-hexane to obtain yellow solid B28.

[0154] In the present invention, the bromobenzothiazole raw material of the C series compounds undergoes a one-step Sonogashira coupling reaction with 3-hydroxyphenylacetylene to construct the parent nucleus to obtain Intermediate 1. React with NaH to remove the H atom in the phenolic hydroxyl group of Intermediate 1, and introduce an alkynyl side chain to obtain Compound 2. At the same time, react the haloalkyl side chain with sodium azide to obtain the diazoalkyl side chain 3. Finally, introduce the side chain into the benzene ring through a click reaction to obtain the target product 4.

[0155]

[0156] (a) CuI, Et3N, PCl2(PPh3)2, DMF, 80 °C, 2 h; (b) 3-Bromopropyne, NaH, DMF, 0 °C; (c) copper(II)

[0157] sulfate pentahydrate; sodium L-ascorbate In water, tert-butyl alcohol, 80 °C, 3 h

[0158] Example 10: Preparation steps of Compound C1

[0159]

[0160] Dissolve 2-bromobenzothiazole (500 mg, 2.3 mmol), 3-ethynylbenzyl alcohol (224 mg, 1.9 mmol), bis(triphenylphosphine)palladium(II) dichloride (68 mg, 0.1 mmol) and copper(I) iodide (19 mg, 0.2 mmol) in DMF (4 mL), and purge with Ar. Add TEA (0.8 mL, 5.7 mmol) using a syringe, and heat the reaction mixture to 80 °C. Dilute the reaction solution with EA (50 mL) and saturated NH4Cl solution (50 mL), and extract with a separatory funnel. Wash the organic phase with water (50 mL), then with saturated NaCl solution (50 mL), dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate the concentrate by silica gel column chromatography (eluent: PE / DCM (v / v) = 1 / 1) to obtain Intermediate 1.

[0161] Dissolve Intermediate 1 (300 mg, 1.2 mmol) and 60% NaH (70 mg, 1.8 mmol) in DMF (5 mL), and place the reaction mixture in an ice bath for 20 min. Add bromoacetylene (168 mg, 1.4 mmol) dropwise, and raise the reaction temperature to room temperature and continue stirring. Quench the reaction by adding ice water dropwise, extract with EA, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1) to obtain white solid C1.

[0162] Example 11: Preparation steps of Compounds C2 - C17

[0163] Taking methyl 2-(4-((3-(benzo[d]thiazol-2-ylethynyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetate Compound C2 as an example

[0164]

[0165] Add 260 μL of 100 mM aqueous copper sulfate pentahydrate solution and 780 μL of 100 mM aqueous sodium ascorbate solution to a 500 μL solution of t-butanol / water (2:1) of C1 (40 mg, 138.2 μmol), stir at room temperature for 10 min, and the system turns green. Add methyl 2-azidoacetate (276.5 μmol), and heat the reaction mixture to 80 °C to continue the reaction. Quench the reaction by adding water, extract with DCM, wash twice with saturated brine, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 100 / 1) to obtain pale yellow solid C2.

[0166]

[0167] Among them, methyl 2-azidoacetate, namely compound 1, is obtained by the substitution reaction of the bromoalkyl side chain with sodium azide. Under the condition of using DMF as the solvent, it is heated to 60 °C and reacted in a pressure-resistant tube for 12 h. During the reaction process, the solvent should be added to the container first, and finally sodium azide should be added to avoid the situation of solvent-free stirring. After the reaction system is cooled, the pH needs to be adjusted to >9 before extraction to avoid the generation of highly toxic hydrazoic acid. Wash away the excess sodium azide and treat the waste liquid with sodium hypochlorite.

[0168] Replacing methyl 2-azidoacetate in the preparation method of compound C2 with methyl 2-azido-2-methylpropionate can obtain C3, and the preparation methods of the remaining compounds are the same.

[0169] A total of 66 target products are prepared, and the specific characterization data are shown in Table 1 below:

[0170] Table 1

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Example 2

[0179] In this example, the inhibitory effects of all the above compounds on tumor cells and the inhibition of the proliferation of various colon cancer cells by compound B18 were tested. The specific process is as follows:

[0180] 1. Detection of the inhibitory effect on tumor cells by the MTT method

[0181] Select the WRN helicase inhibitors (i.e., compounds A1 - A21, B1 - B28, C1 - C17) prepared in the above examples, and test their inhibitory effects on the growth of tumor cells (RKO). The in vitro cytotoxicity assay was carried out by the thiazolyl blue (MTT) method. Cells in the logarithmic growth phase were added with different concentrations of compounds A1 - A21, B1 - B28, and C1 - C17. After acting for 24 h, MTT was added, and its absorbance was measured. Calculate the compound concentration when the cell growth is inhibited by 50% respectively, and express it with the IC 50 value. The results are shown in the following table.

[0182] Table 2 Cytotoxicity of WRN helicase inhibitors (IC 50 value / μmol / L)

[0183]

[0184]

[0185] a Indicates that the compound has no inhibitory activity at the tested concentration or the IC 50

[0186] As can be seen from Table 2, the benzothiazole derivatives of the present invention have a certain inhibitory effect on the proliferation of colorectal cancer cells, and it is expected to select WRN helicase inhibitors therefrom.

[0187] 2. Detect the inhibitory effect of compound B18 on various colon cancer cells.

[0188] Select the WRN helicase inhibitor compound B18 prepared in the above embodiment, and test its inhibitory effect on the growth of various colon cancer cells (HCT116, DLD-1, RKO). The thiazolyl blue (MTT) method is used for in vitro cytotoxicity determination. After treating the cells in the logarithmic growth phase, they are stained with crystal violet solution, and the fluorescence signal is identified and recorded using a cell high-content detection system. Calculate the compound concentration when the cell growth is inhibited by 50%, and express it as IC 50 value, and the results are shown in the following table.

[0189] Table 3 Detection of the cytotoxicity of compound B18 in colon cells by the PI method

[0190]

[0191] According to the results in Table 3, compound B18 shows good cytotoxicity in each cell.

[0192] The experimental results are as Figure 1 shown. The colony formation of HCT116, DLD-1, and RKO is significantly reduced. When the compound concentration is 0.13 μM for RKO and DLD-1 cells, the number of colonies is reduced to half. This indicates that compound B18 shows good cytotoxicity in each cell and significantly inhibits the clone formation of cancer cells in a dose-dependent manner.

[0193] 3. Detect the inhibitory activity against WRN helicase using a newly constructed method based on ELISA technology

[0194] In the ELISA method, the nucleic acid sequence coated on the well plate substrate can carry out a solid-phase reaction to collect the solution in the reaction system. For the collected reaction mixture, the ATPase activity of the helicase during the unwinding process can be synchronously detected. By detecting the ATP consumption of the recovered reaction solution using an ATPase kit, the helicase activity and ATPase activity of the WRN unwinding replication fork are synchronously detected. Based on this, the WRN helicase inhibitor prepared in the above example (the specific compounds selected are shown in the following table) was selected to determine its inhibitory effect on the WRN helicase at a single concentration point of 20 μM.

[0195] Table 4 Inhibitory activity of compounds on WRN helicase detected by ELISA method

[0196]

[0197]

[0198] As can be seen from Table 4, the benzothiazole derivatives of the present invention inhibit the WRN helicase activity, and it is expected to select WRN helicase inhibitors therefrom.

[0199] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A compound of formula I, or its stereoisomer, its deuterated compound, its pharmaceutically acceptable salt: L is selected from a carbon-carbon triple bond, an unsubstituted or halogen-substituted carbon-carbon double bond; Each R1 is independently selected from deuterium, hydrogen, halogen, azide, -C1-C8 alkyl, -C1-C8 alkoxy, -NR 11 R 12 , -C(O)OR 13 , -C(O)NR 14 R 15 ; wherein, The alkyl and alkoxy groups are optionally unsubstituted or substituted by one or more R a substituents; R 11 、R 12 are each independently selected from deuterium, hydrogen, -C1-C8 alkyl, -C0-C6 alkylene-NR 111 R 112 ; R 13 、R 14 、R 15 、R 111 、R 112 are each independently selected from deuterium, hydrogen, -C1-C8 alkyl; Each R a is independently selected from deuterium, a hydroxyl group, a halogen, and a -C1-C8 alkyl group; Each R2 is independently selected from deuterium, hydrogen, halogen, hydroxyl, carboxyl, diethyl phosphate group, -C1-C8 alkyl, -C1-C8 alkoxy, -NR 11 R 12 , 3- to 10-membered heterocycloalkyl, -L1-C0-C6 alkylene-Ar; -L1- is selected from -NH-, -NHC(O)-, -C(O)NH-, -N=C-, -O-; Ar is selected from -C1-C8 alkyl, -C1-C8 alkoxy, -C2-C8 alkenyl, -C2-C8 alkynyl, 3-10 membered cycloalkyl, 3-10 membered hetero cycloalkyl, 6-10 membered aryl, 5-20 membered heteroaryl; wherein, the alkyl, alkoxy, cycloalkyl, hetero cycloalkyl, aryl, heteroaryl are optionally unsubstituted, or substituted by one or more R b substituted; Each R b is selected from deuterium, halogen, hydroxy, amino, -C1-C8 alkyl, -S(O)2R a , -NR 11 R 12 , -C2-C8 alkenyl, -C2-C8 alkynyl; wherein said alkyl is optionally unsubstituted or substituted by one or more R c substituents; Each R c is selected from hydroxy, cyano, -C(O)OR a , and 3- to 10-membered heterocycloalkyl; n is a natural number between 0 and 4; m is a natural number between 0 and 5.

2. The compound of formula I according to claim 1, or its stereoisomers, its deuterated compounds, its pharmaceutically acceptable salts, characterized in that: The compound of formula I has the structures of formula Ia, formula Ib, and formula Ic: Among them, X is a halogen.

3. The compound of formula I according to claim 1, or its stereoisomers, its deuterated compounds, its pharmaceutically acceptable salts, characterized in that: Ar is selected from cyclopentyl, cyclohexyl, phenyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, 1,3-dioxolanyl, piperazinyl, imidazolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, oxadiazolyl, triazolyl, tetrazolyl, carbazolyl, indolyl, quinolinyl, isoquinolinyl, benzothienyl; these groups are optionally unsubstituted or substituted by one or more R b substituents.

4. The compound of formula I according to claim 1, or its stereoisomer, its deuterated compound, its pharmaceutically acceptable salt, characterized in that: The compound of formula I is selected from the following compounds:

5. A method for preparing the compound of formula I according to any one of claims 1 to 4, characterized in that, Comprising the following steps: Reacting a compound of formula II with a compound of formula III to obtain a compound of formula I; Wherein, X is halogen or methyl; R1, R2, m, and n are defined as described in any one of claims 1 to 4.

6. The method for preparing the compound of formula I according to claim 5, characterized in that, Carrying out a coupling reaction between a compound of formula Ia and a compound of formula IIIa to obtain a compound of formula Ic; Wherein X1 is a halogen.

7. The preparation method of the compound of formula I according to claim 5, characterized in that, Carrying out a condensation reaction between a compound of formula IIb and a compound of formula IIIb to obtain a compound of formula Ia; 8. A pharmaceutical composition comprising the compound of formula I according to any one of claims 1 to 4, or its stereoisomer, its deuterated compound, its pharmaceutically acceptable salt.

9. Use of the compound of formula I according to any one of claims 1 to 4, or its stereoisomer, its deuterated compound, its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating tumors.

10. The application according to claim 9, characterized in that: The tumors include at least one of colon cancer, colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, skin cancer, and high microsatellite instability cancer.

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