Heterocyclic compounds as inhibitors of DNA polymerase Theta

Abstract

The present invention relates to use of a heterocyclic compound or a pharmaceutically acceptable salt thereof that inhibits DNA polymerase Theta (Polθ) activity in treating or preventing cancer, and also relates to a composition containing the compound and a preparation method thereof.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a DNA polymerase Theta inhibitor or a pharmaceutically acceptable salt thereof, and relates to a method for preparing the compound, a pharmaceutical composition comprising the compound as an active ingredient, and the use of the compound in treating cancer. Background Art

[0002] DNA damage repair pathways include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double-strand break (DSB) repair. Double-strand DNA damage repair pathways primarily include homologous recombination (HR), nonhomologous end joining (NHEJ), and microhomology-mediated end joining (alt-EJ or MMEJ). Homologous recombination primarily relies on homologous chromosomes to guide the repair of damaged DNA, while nonhomologous end joining directly joins the two broken DNA strands to achieve repair. BRCA1 and BRCA2, key proteins involved in homologous recombination, are important tumor suppressors. Loss of these proteins significantly reduces the rate of homologous recombination and increases sensitivity to ionizing radiation, ultimately leading to an increased risk of breast and ovarian cancer. Furthermore, the proper functioning of the nonhomologous end joining (NHEJ) repair pathway is crucial; defects in related genes can lead to diseases such as ataxia telangiectasia and Fanconi anemia. MMEJ repair is driven by annealing of microhomology sequences flanking the DNA ends and was initially considered a backup pathway for gene repair. However, as research progressed, it was discovered that MMEJ is not a backup repair mechanism.

[0003] DNA polymerase θ (Polθ or POLQ) is a synthetically lethal target of homologous recombination (HR) defects and plays a crucial role in the DNA damage response (DDR) pathway for double-strand breaks (DSBs). When DNA end resection occurs, in the presence of BRCA2, BRCA2 not only recruits the recombinase RAD51 to the DSB to promote HR but also inhibits repair pathways such as alt-NHEJ. When homologous recombination-mediated repair is impaired (HR deficiency), as in the case of BRCA1 or BRCA2 mutations, Polθ becomes highly expressed and directs DSB repair toward alt-NHEJ, initiating the DNA repair process of microhomology-mediated end joining (MMEJ). In the context of HR deficiency, inhibition of Polθ leads to cell death through the accumulation of toxic RAD51 intermediates and inhibition of the alt-NHEJ repair pathway.

[0004] Pol θ is expressed in only a few tissue types but is highly expressed in many cancer cells. Pol θ can confer resistance to cancer therapies and promote the survival of abnormal cells lacking DNA damage repair pathways. The primary function of DNA polymerase θ (Pol θ) is to act as a reverse transcriptase. In healthy cells, Pol θ acts as an RNA-mediated DNA repair enzyme. In cancer cells, Pol θ is highly expressed and promotes cancer cell growth and drug resistance.

[0005] Researchers from Thomas Jefferson University and the University of Southern California published a research paper titled "Polθ reverse transcribes RNA and promotes RNA-templated DNA repair" in Science Advances, a subsidiary of the world's top academic journal Science. The paper revealed the activity of DNA polymerase θ (Polθ) on RNA and how it promotes DNA repair and cancer cell proliferation. This not only challenges the fundamental law of life science, the "central dogma," but also suggests that Polθ is a promising anti-cancer drug target.

[0006] CN114127062A, CN114667167A, WO2020160213, WO2022118210, and WO2021123785 disclose DNA polymerase Theta (Polθ) inhibitors. Summary of the Invention SUMMARY OF THE INVENTION

[0008] The present inventors have conducted extensive research and discovered that the compound represented by the following formula I has a strong Polθ inhibitory function, overcoming the shortcomings of Polθ inhibitors in the prior art. The heterocyclic compound provided by the present invention has a good inhibitory effect on Polθ.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] In one aspect, the present invention provides a compound represented by formula (I) or a tautomer or stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof:

[0011]

[0012] in,

[0013] Z is selected from -NH, -CH2;

[0014] X is selected from -C(O), -NH, -CH2, -CF2;

[0015] Y is selected from -C(O), S(O)2-;

[0016] R1 is selected from hydrogen, C 1-6 Alkyl, halogenated C 1-6 alkyl;

[0017] R2, R3, R4, R5, R6 are independently selected from hydrogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxyl;

[0018] R7, R8, R9, R 10 are independently selected from hydrogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogen, C 1-6 Alkoxy, hydroxy, cyano.

[0019] Furthermore, the compound of the present invention is a compound of formula (I-1):

[0020]

[0021] Furthermore, the compound of the present invention is a compound of formula (I-2):

[0022]

[0023] Among them, X, Z, R2, R3, R4, R5, R6, R7, R8, R9, R 10 As defined above;

[0024] As a further preferred technical solution, wherein the R1 is selected from methyl, CD3, ethyl, isopropyl, preferably methyl, ethyl, more preferably methyl.

[0025] As a further preferred technical solution, the R2 is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and methoxy, preferably hydrogen; the R3 is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and methoxy, preferably methyl; the R4 is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and methoxy, preferably hydrogen; the R5 is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and methoxy, preferably hydrogen.

[0026] As a further preferred technical solution, R7, R8, R9, R 10 Independently selected from hydrogen, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyano.

[0027] The present invention also provides the following compound, wherein the compound is selected from:

[0028]

[0029]

[0030] The present invention also provides a pharmaceutical composition comprising the above-mentioned floral compound and its isomers, or pharmaceutically acceptable salts thereof, and one or more pharmaceutical excipients.

[0031] The present invention also provides the use of the above-mentioned compound and its isomers, or pharmaceutically acceptable salts thereof in the preparation of a drug for inhibiting a disease of Polθ overexpression, wherein the disease is cancer, and more preferably the cancer is a solid tumor, breast cancer, lung cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, or cervical cancer.

[0032] As a preferred technical solution, the cancer is characterized by reduced or absent BRCA gene expression, absent BRCA gene, or reduced BRCA protein function.

[0033] Without being bound by theory, it is expected that the Pol θ inhibitors of the present invention exhibit certain properties that make them particularly useful in the treatment of certain cancers. For example, in one embodiment, the Pol θ inhibitors of the present invention are moderately lethal in BRCA1 and BRCA2 deficient primary and secondary solid tumors, including breast, ovarian, prostate, and pancreatic.

[0034] In another embodiment, the Polθ inhibitors of the present invention have moderate lethality in a variety of primary and secondary solid tumors that are HRD by mechanisms other than BRCA deficiency, such as those with promoter hypermethylation. In these tumors where no DSB repair pathways may be completely downregulated, Polθi can be administered together with another DDR modulator such as a PARP inhibitor, DNA-PK inhibitor, ATR inhibitor, ATM inhibitor, wee1 inhibitor, or CHK1 inhibitor.

[0035] In another embodiment, Pol θ inhibitors of the invention are moderately lethal in primary and secondary breast, ovarian, prostate, and pancreatic tumors that retain BRCA1 deficiency but are resistant to PARPi treatment with or without exposure to PARPi drugs.

[0036] In another embodiment, the Polθ inhibitors of the present invention modestly increase ORR, including CRR, will delay the onset of PARPi resistance, will increase time to relapse and DFS, and will increase OS in HRD (BRCA1 / 2 deficiency and other HRD mechanisms) primary and secondary tumors (breast, ovary, prostate, and pancreas) when administered with PARPi treatment regimens.

[0037] The present inventors have discovered that these compounds are highly effective Polθ inhibitors with extremely strong Polθ inhibitory activity and can be used to prepare therapeutics for the prevention and / or treatment of indications associated with Polθ inhibition, including solid tumors, breast cancer, lung cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, and cervical cancer. The present invention is based on these discoveries. Detailed Description of the Invention

[0039] Various aspects and features of the present invention are further described below.

[0040] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail. The following are definitions of various terms used in the present invention, which apply to the terms used throughout the specification of this application, unless otherwise specified in the specific case.

[0041] The compounds according to the present invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

[0042] The compounds of the present invention possess asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art will appreciate how to prepare optically active forms, such as by racemate resolution or synthesis from optically active starting materials. Unless otherwise indicated with respect to specific stereochemistry or isomeric forms, the present invention encompasses all chiral, diastereoisomer, and racemic forms. Methods for preparing the compounds of the present invention and intermediates thereto are also intended to be included in the present invention. All tautomers of the compounds of the present invention are also intended to be included in the present invention.

[0043] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent, as long as the valence of the particular atom is normal and the resulting compound after the substitution is stable.

[0044] The terms "alkoxy" and "alkylamino" are customary expressions and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or amino group, respectively, wherein the alkyl group is as described herein.

[0045] As used herein, the terms "halogen", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine and bromine, with fluorine and chlorine being particularly preferred.

[0046] As used herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include its subgroups. For example, when referring to "C1-C6 alkyl", it may also include sub-ranges of groups represented by C1-C4 alkyl, C1-C3 alkyl, C2-C6 alkyl, C2-C4 alkyl, etc., as well as specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[0047] The terms "heterocycle," "heterocyclic," or "heterocyclyl" are used interchangeably and refer to substituted and unsubstituted 3 to 7 membered monocyclic groups, 7 to 11 membered bicyclic groups, and 10 to 15 membered tricyclic groups having at least one heteroatom (O, S, or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2, or 3 heteroatoms selected from O, S, and N. Each ring of such heteroatom-containing groups may contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be saturated, partially saturated, or fully unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom. As used herein, the terms "heterocycle," "heterocycloalkyl," "heterocyclo," "heterocyclic," and "heterocyclyl" include "heteroaryl" groups, as defined below.

[0048] In addition to the heteroaryl groups described below, exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane, and tetrahydro-1,1-dioxythienyl, etc. Exemplary bicyclic heterocyclic groups include quinuclidinyl.

[0049] The term "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 9- or 10-membered bicyclic groups, and 11- to 14-membered tricyclic groups having at least one heteroatom (O, S, or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2, or 3 heteroatoms selected from O, S, and N. Each ring of the heteroaryl containing heteroatoms can contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. A bicyclic or tricyclic heteroaryl must include at least one fully aromatic ring, but the other one or more fused rings may be aromatic or non-aromatic. The heteroaryl group may be attached to any available nitrogen or carbon atom of any ring. If the other ring is cycloalkyl or heterocycle, it is additionally optionally substituted with =0 (oxo), where valence permits.

[0050] Unless otherwise indicated, when referring to a specifically named aryl (e.g., phenyl), heterocyclyl (e.g., pyrrolidinyl, piperidinyl, and morpholinyl), or heteroaryl (e.g., tetrazolyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, and furanyl) group, the reference is intended to include rings optionally having 0 to 3, preferably 0 to 2, substituents selected from the substituents listed above for arylheterocyclyl and / or heteroaryl groups.

[0051] As used herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate during chiral resolution. Although the salt of this intermediate cannot be directly administered to a subject, the salt can play a role in obtaining the final product of the present invention.

[0052] As described herein, the term "disease" refers to a physical condition of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegenerative diseases described in the present invention.

[0053] Cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy, and hormone therapy.

[0054] "Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other sites in the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cell" refers to a cell that is undergoing an early, intermediate, or advanced stage of multistep neoplastic progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumors, head and neck cancer, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, advanced solid tumors, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myeloma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma, lymphoma, pancreatic ductal adenocarcinoma.

[0055] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0056] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0057] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known method of administration.

[0058] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0059] The solvents used in this application can be obtained commercially. The abbreviations used in this application are as follows:

[0060] Table 1: Meaning of abbreviations

[0061]

[0062] Beneficial technical effects

[0063] The inventors have discovered that the compounds of the present invention exhibit excellent Polθ inhibitory activity, with IC50 values ​​lower than those of the positive control drug ART558. The present invention provides a class of novel, highly active Polθ inhibitor compounds with promising application prospects in preventing and / or treating indications associated with Polθ inhibition, such as decreased or absent MTAP expression, MTAP gene deletion, and decreased MTAP protein function. DETAILED DESCRIPTION

[0064] The following embodiments are intended to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the present invention in any way.

[0065] For all of the following examples, standard procedures and methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectroscopy (MS).

[0066] The structures of the compounds of the present invention were confirmed by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are expressed in parts per million (ppm). NMR measurements were performed using a Bruker Avance-400 NMR spectrometer. The solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0067] Liquid phase mass spectrometry LC-MS measurement The liquid phase part used ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part used Xevo G2-S Qtof mass spectrometer.

[0068] The starting materials used in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0069] Example 1: (S)-1-[3-cyano-6-methyl-4-(trifluoromethyl)pyridin-2-yl]-N-methyl-5-oxo-N-(m-tolyl)pyrrolidine-2-carboxamide

[0070]

[0071] (S)-1-(3-cyano-6-methyl-4-(trifluoromethyl)pyridin-2-yl)-5-oxopyrrolidine-2-carboxylic acid (100.0 mg, 0.32 mmol, 1.0 eq), N-methyl-m-toluidine (42.56 mg, 0.35 mmol, 1.1 eq), tetramethylchlorouronium hexafluorophosphate (107.49 mg, 0.38 mmol, 1.2 eq), and N-methylimidazole (55.05 mg, 0.67 mmol, 2.1 eq) were added to acetonitrile (3.0 mL) and reacted at room temperature for 12 hours. After completion of the reaction, the reaction solution was directly concentrated. Purification by column chromatography (petroleum ether / ethyl acetate system, 40% polarity) gave (S)-1-[3-cyano-6-methyl-4-(trifluoromethyl)pyridin-2-yl]-N-methyl-5-oxo-N-(m-tolyl)pyrrolidine-2-carboxamide (30.0 mg) as a solid. LCMS (TOF MS ES+) m / z [M+H]+: 417.155. 1 H NMR(400MHz, DMSO-d6)δ7.83(s,1H),7.43(t,J=7.9Hz,1H),7.30–7.22(m,3H),4.93(t,J= 7.1Hz,1H),3.14(s,3H),2.70(s,3H),2.67–2.52(m,2H),2.37(s,3H),2.12–2.03(m,2H).

[0072] The compounds in Table 2 were obtained by referring to the method of Example 1:

[0073] Table 2: Structure and characterization of compounds 2-12

[0074]

[0075]

[0076] Experimental Example 1: Testing the binding ability of the compound of the present invention to Polθ protein

[0077] Experimental purpose: To detect the binding ability of the compound to Polθ protein using the CETSA experimental method

[0078] Background and Principle: The CETSA assay is a molecular test that measures the affinity of a drug for its target protein. The principle is that drug binding to the target protein stabilizes its structure. Cells or tissue samples are treated with a candidate drug. If the candidate drug is a Polθ inhibitor, it will bind to Polθ, stabilizing the Polθ protein. After heating the sample, the PRMT5 protein in the sample will be more easily detected by specific antibodies, making Polθ more readily detectable by Western blot. Conversely, heating will destabilize the Polθ protein, resulting in a lower amount of protein detected. This allows the assessment of the drug's binding ability to the target protein and is used to screen for Polθ inhibitors.

[0079] Specific experimental process:

[0080] Polθ-polymerase domain protein expression: The Polθ-polymerase domain sequence (residues 1819-2590) was cloned into the pET24N vector containing an N-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site. The constructed vector was transformed into Escherichia coli Rosetta (DE3) and incubated at an OD of 0.6. 1 mM IPTG was added and cultured at 12°C for 16 hours. The cells were harvested, disrupted by sonication, and the supernatant was centrifuged. The protein was purified using Ni-NTA and dialyzed for determination of protein concentration and purity.

[0081] CETSA sample preparation: Incubate samples with the candidate drug, control drug, and control reagent for 30 minutes, heating each sample at approximately 10 pre-set temperature points. Return to room temperature, centrifuge the sample at 20,000 g, and collect the supernatant. Denature the protein sample by heating it at 100°C for 10 minutes using sample buffer. After returning to room temperature, perform western blot analysis on the sample. The protein loading volume is controlled at 20 μg. After determining the mutation temperature, set a concentration gradient of the compound, typically 9 points, incubate the sample, and perform western blot analysis as above. Protein electrophoresis: Set the voltage of the stacking gel to 60V and the voltage of the separation gel to 120V. After electrophoresis, begin electrotransfer. Electroporation was performed at 250 mA for 2 hours, followed by 5% BSA blocking for 1 hour. The cells were then incubated with the primary specific antibody overnight at 4°C on a shaker. The cells were washed four times with TBST for 2.5 minutes each. The cells were then incubated with the secondary specific antibody for 1 hour at room temperature on a shaker. The cells were washed four times with TBST for 2.5 minutes each. TYK2 protein expression was detected using ECL development in different groups and at various temperatures. Western blots were processed using Image J and GraphPad software, and the EC50 values ​​were calculated.

[0082] EC50 is the concentration for 50% of maximal effect (EC50), which refers to the drug concentration that can induce effective response in 50% of individuals. ART558 was used as a positive reference compound, and its preparation method was based on Example 24 of patent document CN114667167A.

[0083] The ART558 structure is as follows:

[0084]

[0085] The test results are shown in Table 3 below. The EC50 values ​​of each compound are classified according to the following description:

[0086] “+” indicates EC50 value greater than 1 μM;

[0087] “++” indicates that the EC50 value is less than 1 μM and greater than 100 nM;

[0088] “+++” indicates that the EC50 value is less than 100 nM and greater than 10 nM;

[0089] “++++” indicates that the EC50 value is less than 10 nM.

[0090] Table 3: EC50 results

[0091] Compound number EC50 1 ++ 2 +++ 3 +++ 4 +++ 5 +++ 6 ++ 7 ++++ 8 +++ 9 +++ 10 +++ 11 +++ 12 +++ Positive drug ART558 ++

[0092] The experimental results show that the compound of the present invention has a good ability to bind to the Polθ-polymerase domain protein, which is equivalent to or stronger than the in vitro binding ability of the positive control compound. Therefore, the compound of the present invention can be used as an effective Polθ protein regulator.

[0093] Experimental Example 2: Determination of the effect of the compounds of the present invention on the proliferation of DLD1 BRCA2- / - cells

[0094] Experimental purpose: The purpose of this test case is to test the effect of the compound on DLD1 BRCA2 - / - Cell proliferation effect.

[0095] Background principle: DNA polymerase theta (Polθ) is a synthetic lethal target with homologous recombination deficiency and plays an important role in the DNA damage response pathway of double-strand breaks (DSBs). When homologous recombination-mediated repair is impaired (HR deficiency), such as BRCA1 or BRCA2 mutations, Polθ is highly expressed and guides DSB repair towards alt-EJ, turning on the DNA repair process of microhomology-mediated end joining (MMEJ). In the case of HR deficiency, inhibition of Polθ leads to cell death through the accumulation of toxic RAD51 intermediates and inhibition of the alt-EJ repair pathway. Therefore, by testing the effects of compounds on DLD1 BRCA2 - / -The inhibition rate of cell proliferation can be used to screen Polθ protein inhibitors.

[0096] Specific experimental process:

[0097] DLD1 BRCA2 knockout cells were constructed and single clones were screened. - / - WT cells were seeded into 96-well plates, with 90 μL per well, at a concentration of 1,000 cells / well. The cells were incubated overnight at 37°C. The next day, 10 μL of compound at various concentrations was added, with a maximum concentration of 100 μM. A 10-fold dilution gradient was created (final DMSO concentration was 1%), and the cells were incubated at 37°C for 6 days. On the 6th day, the old culture medium was aspirated and 110 μL of culture medium (culture medium to CCK8 ratio of 100:10) was added. The cells were incubated at 37°C for 1-4 hours. The absorbance was measured at 450 nM, and the IC50 value was calculated using GraphPad software. Compounds were screened by comparing the results with those of active drugs.

[0098] IC50 (half maximal inhibitory concentration) is the half-maximal inhibitory concentration of the antagonist being measured. It indicates the half-maximum inhibitory concentration of a drug or substance (inhibitor) in inhibiting a biological process (or substances involved in that process, such as enzymes, cell receptors, or microorganisms). ART558 was used as a positive reference compound.

[0099] The test results are shown in Table 4 below, where the IC50 values ​​of the compounds are classified according to the following description:

[0100] “+” indicates IC50 value greater than 1 μM;

[0101] “++” indicates IC50 value less than 1 μM and greater than 100 nM;

[0102] "+++" indicates that the IC50 value is less than 100 nM and greater than 10 nM.

[0103] The test results are shown in Table 4 below:

[0104] Table 4: IC50 experimental data

[0105]

[0106]

[0107] The results showed that the compounds of the present invention can inhibit DLD1 BRCA2 - / - The IC50 value for cell proliferation reached the nanomolar range, which was comparable to or stronger than the positive control drug ART558. This strong inhibitory effect has important therapeutic significance for the treatment of conditions or diseases associated with Polθ inhibition.

[0108] Experimental Example 3: Testing of the Effects of the Compounds of the Present Invention on the Function of Polθ Protein Polymerase

[0109] Experimental Purpose: The purpose of this test case is to test the effect of the compound on the function of Polθ protein polymerase.

[0110] Principle: Polθ consists of a C-terminal family A DNA polymerase and an N-terminal superfamily 2 (SF2) DNA helicase, separated by a long, poorly conserved central domain of unknown function. ART558 binds to an allosteric site within the Polθ polymerase catalytic domain and inhibits MMEJ in a dose-dependent manner. Therefore, compounds can be used to screen for Polθ inhibitors by detecting their inhibitory effects on Polθ protein polymerase function.

[0111] Specific experimental process:

[0112] Polθ-polymerase domain protein expression: The Polθ-polymerase domain sequence (residues 1819-2590) was cloned into the pET24N vector containing an N-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site. The constructed vector was transformed into Escherichia coli Rosetta (DE3) and incubated at an OD of 0.6. 1 mM IPTG was added and cultured at 12°C for 16 hours. The cells were harvested, disrupted by sonication, and the supernatant was centrifuged. The protein was purified using Ni-NTA and dialyzed for determination of protein concentration and purity.

[0113] Purified Polθ-polymerase domain protein was added to assay buffer (20 mM Tris, pH 7.80, 50 mM KCl, 10 mM MgCl, 1 mM DTT, 0.01% BSA, 0.01% Tween 20) at a protein concentration of 4 nM. Test compounds (an 11-point dilution series) were then added. The enzyme and test compound inhibitor mixture were incubated at room temperature for 15 minutes. dNTPs and primers (primer: 5-GCG GCT GTC ATA AG-3; template: 5-GCT ACATTG ACAATG GCATCA AAT CTC AGA TTG CGT CTT ATG ACA GCC GCG-3; primer: template ratio = 1:1.1) were added to all assay wells and incubated at room temperature for 60 minutes in the dark. The reaction was terminated by adding 16 μL of TE stop solution containing 0.5% PicoGreen dye to each well and incubated in the dark for 90 minutes. The fluorescence was read at 485 / 520 nm using a microplate reader, and the IC50 was calculated using GraphPad software. The compounds were screened by comparing with positive drugs.

[0114] The test results are shown in Table 5 below, where the IC50 values ​​of the compounds are classified according to the following description:

[0115] “+” indicates IC50 value greater than 1 μM;

[0116] “++” indicates IC50 value less than 1 μM and greater than 100 nM;

[0117] “+++” indicates that the IC50 value is less than 100 nM and greater than 10 nM;

[0118] The test results are shown in Table 5 below:

[0119] Table 5: IC50 experimental data

[0120] Compound number IC50 1 ++ 2 +++ 3 ++ 4 +++ 5 ++ 6 ++ 7 ++ 9 +++ 11 +++ 12 +++ ART558 +++

[0121] The results showed that the compounds of the present invention have very strong inhibitory effects on the polymerase function of the Polθ-polymerase domain protein, with IC50 values ​​reaching the nanomolar range, which is comparable to or lower than the positive control drug ART558. This strong inhibitory effect has important therapeutic implications for the treatment of conditions or diseases associated with Polθ inhibition.

[0122] The above examples are merely representative. It can be seen from the above examples that the compounds of the present invention are ideal and highly effective Pol θ inhibitors and can be expected to be used for treating or preventing conditions or diseases associated with Pol θ inhibition.

[0123] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and these changes should be covered by the scope of protection of the present invention.

Claims

1. A compound as shown below or a tautomer or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition comprising the compound according to claim 1, and one or more pharmaceutical excipients.

3. Use of the compound according to claim 1 or the composition according to claim 2 in the preparation of a medicament for inhibiting a disease caused by overexpression of Pol θ.

4. The use according to claim 3, characterized in that The disease is cancer.

5. The use according to claim 4, characterized in that The cancer is solid tumor, breast cancer, lung cancer, bladder cancer, esophageal cancer, prostate cancer, pancreatic cancer, and cervical cancer.

6. Use of the compound of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating homologous recombination (HR)-deficient cancer in a patient.

7. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating cancer in a patient, characterized in that The cancer has reduced or absent BRCA gene expression, absent BRCA genes, or reduced BRCA protein function.

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