Targeting parp11 small molecule inhibitors and their use in the preparation of anti-tumor drugs

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and discloses a small-molecule inhibitor targeting PARP11 and application thereof in preparation of an antitumor drug. Through systematic optimization of the skeleton structure of ITK7, a new type of PARP11 inhibitor with high efficiency, high selectivity and high safety is obtained. The compound can effectively activate the IFN-gamma / STAT1 signal pathway, inhibit AKT signal activation, improve the tumor immune microenvironment, and significantly inhibit tumor growth and metastasis, thereby providing a new chemical entity and treatment strategy for development of a new type of immunomodulatory antitumor drug.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to small molecule inhibitors targeting PARP11 and their application in the preparation of antitumor drugs. Background Technology

[0002] The poly(ADP-ribose) polymerase (PARP) family is a class of enzymes that use NAD+. + PARP inhibitors are enzymes that catalyze the ADP-ribosylation modification of proteins, participating in DNA repair, cell metabolism, immune regulation, and tumorigenesis. Existing PARP inhibitors (such as olaparib and niraparib) primarily target PARP1 / 2 and are used to treat DNA repair-deficient tumors. However, their applicability is limited, and they are prone to developing resistance. Recent studies have found that PARP11 (also known as ARTD11) plays a crucial immunosuppressive role in the tumor immune microenvironment, becoming a potential anti-tumor immune target.

[0003] Previous studies have shown that PARP11 interferes with the type I interferon signaling pathway through mono(ADP-ribose) modification, thereby weakening CD8. + T cell activation and cytotoxic function. Zhang et al. (Cancer Immunol Res, 2022) reported that in solid tumors, tumor-associated factors can induce PARP11 upregulation, thereby promoting IFNAR1 (interferon α / β receptor) degradation, leading to impaired interferon signaling and immune escape. Inhibiting PARP11 activity can restore IFNAR1 stability and enhance the anti-tumor activity of CAR-T cells.

[0004] Furthermore, Basavaraja et al. (Nature Cancer, 2024) found that PARP11 expression levels were significantly elevated in tumor-infiltrating regulatory T cells (TI-Tregs) and were associated with poor efficacy of immune checkpoint blockade (ICB). Knocking out PARP11 or treating with small molecule inhibitors reduced the suppressive activity of Tregs and enhanced the efficacy of immunotherapy. This study is the first to reveal the core role of PARP11 in tumor immunosuppression from an immune cell perspective.

[0005] In drug discovery, Kirby et al. (Cell Chemical Biology, 2018) first reported ITK7, a highly selective inhibitor of PARP11. This molecule exhibits over 200-fold selectivity for PARP11 and can induce PARP11 to detach from the nuclear membrane, suggesting that the localization of PARP11 is closely related to its catalytic activity. Although ITK7 has laid the foundation for PARP11 inhibition research, its binding affinity and pharmacokinetics still need further improvement.

[0006] In summary, selective small-molecule inhibitors targeting PARP11 hold promise for relieving tumor immunosuppression, enhancing immunotherapy responses, and improving clinical efficacy. Therefore, developing PARP11 inhibitors with novel structures, higher activity, and broader mechanisms of action to simultaneously improve the function of tumor cells and immune cells, overcome drug resistance, and expand clinical applications is of significant scientific importance and practical value. Summary of the Invention

[0007] The purpose of this invention is to address the technical deficiencies of existing PARP inhibitors, such as insufficient activity, limited selectivity, and strong drug resistance, by providing a small molecule inhibitor targeting PARP11 and its application in the preparation of antitumor drugs.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] Small molecule inhibitors targeting PARP11, having the structural formula of general formula I or general formula II, or pharmaceutically acceptable salts, solvates or stereoisomers of both;

[0010] General formula I is:

[0011] ;

[0012] General Formula II is:

[0013] ;

[0014] R 1 It is selected from H, C1-C6 alkyl or C1-C6 alkyl containing a substituent, aryl or aryl containing a substituent, wherein the substituent in the C1-C6 alkyl is halogen, cyano, C1-C4 alkoxy or phenyl, and the substituent on the aryl is halogen, C1-C4 alkyl, C1-C4 alkoxy;

[0015] R 2 It is selected from C1-C10 alkyl or C1-C10 alkyl containing substituents, C2-C10 alkenyl or C2-C10 alkenyl containing aryl substituents, C2-C10 alkynyl, aryl or 5-10 heteroaryl, wherein the substituents in the C1-C10 alkyl are cyano, amide, C3-C8 cycloalkyl, aryl, or 5-10 heteroaryl;

[0016] X represents C or does not exist. When X represents non-existence, -SR 2 The group is attached to the C-2 position of the quinazolinone core;

[0017] R 3 Selected from H, C1-C6 alkyl, and C6-C10 aryl;

[0018] R 4 Selected from H, C1-C6 alkyl, and benzyl groups;

[0019] R 5 It is selected from aryl, naphthyl, 5-10 member monocyclic or bicyclic heteroaryl, or aryl, naphthyl, 5-10 member monocyclic or bicyclic heteroaryl containing substituents, wherein the substituents are halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy or -CF3.

[0020] In the above technical solution, the structural formula of the PARP11-targeting small molecule inhibitor is as follows:

[0021] ;

[0022] Preferably, , .

[0023] Another aspect of the invention includes a pharmaceutical composition comprising the said PARP11-targeting small molecule inhibitor and a pharmaceutically acceptable carrier, diluent, or excipient.

[0024] In the above technical solutions, the dosage form of the drug composition is an injection, a lyophilized powder injection, or a nanoparticle formulation.

[0025] Another aspect of the present invention includes the use of the PARP11-targeting small molecule inhibitor in the preparation of antitumor drugs.

[0026] In the above technical solution, the anti-tumor drug is a drug for treating colorectal cancer, melanoma, or ovarian cancer.

[0027] Another aspect of the invention includes a combination drug comprising the aforementioned PARP11-targeting small molecule inhibitor and an immune checkpoint inhibitor.

[0028] In the above technical solution, the combined drug also includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0029] In the above technical solution, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0030] In the above technical solution, the dosage form of the combined drug is an injection, a lyophilized powder injection, or a nanoparticle formulation.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. The PARP11-targeting small molecule inhibitor of the present invention has a more significant anti-tumor effect, even with the same number of subcutaneous tumor cells (2×10⁻⁶). 6 Under MC38 cell conditions, the ITK7 group could no longer significantly inhibit tumor growth, while the compound C11, the representative compound of this invention, could still significantly delay tumor volume growth and improve survival rate, showing superior in vivo anti-tumor activity.

[0033] 2. The PARP11-targeting small molecule inhibitor of the present invention can overcome the drug resistance of traditional PARP inhibitors. Long-term use of traditional PARP1 / 2 inhibitors easily leads to upregulation of the AKT pathway, resulting in drug resistance. However, the PARP11-targeting small molecule inhibitor of the present invention can directly target the PARP11–AKT axis, effectively inhibit AKT activation, and restore sensitivity to treatment.

[0034] 3. The PARP11-targeting small molecule inhibitor of this invention has a broader mechanism of action. PARP1 / 2 inhibitors mainly function in tumors carrying BRCA mutations, while the PARP11-targeting inhibitor of this invention can simultaneously regulate the function of tumor cells and immune cells: enhancing the interferon signaling pathway in tumor cells and promoting CD8 in immune cells. + T cell activation and reduction of Treg inhibitory activity, thereby achieving a dual anti-tumor effect;

[0035] 4. The PARP11-targeting small molecule inhibitor of this invention has high safety. Experiments have confirmed that PARP11 knockout mice can develop and survive normally, with no obvious physiological abnormalities observed. Furthermore, the subcutaneous tumors formed in PARP11 knockout mice grow significantly slower compared to wild-type mice, indicating that PARP11 is a safe and feasible drug target. The small molecule drug developed based on this target in this invention has high safety and clinical application potential.

[0036] 5. The PARP11-targeting small molecule inhibitor of the present invention significantly inhibits tumor metastasis. In a melanoma lung metastasis model, ITK7 had limited inhibitory effect on metastatic nodules, while C11 significantly reduced the number of lung metastases, suggesting that this compound can not only inhibit the growth of tumors in situ, but also effectively block distant metastasis of tumors.

[0037] In summary, this invention, through systematic optimization of the ITK7 scaffold structure, yields a class of highly efficient, selective, and safe small-molecule inhibitors targeting PARP11. These compounds can effectively activate the IFN-γ / STAT1 signaling pathway, inhibit AKT signaling activation, improve the tumor immune microenvironment, and significantly inhibit tumor growth and metastasis, providing new chemical entities and therapeutic strategies for the development of novel immunomodulatory antitumor drugs. Attached Figure Description

[0038] Figure 1 It is the structural formula of C1-C20.

[0039] Figure 2 This is a flowchart of the virtual screening process for small molecule inhibitors targeting PARP11.

[0040] Figure 3This is the binding curve of C1-C20 to PARP11 protein.

[0041] Figure 4 It is the anti-tumor effect of C1-C20 and ITK7.

[0042] Figure 5 This refers to the tumor-inhibiting effect of C11 compared to olaparib.

[0043] Figure 6 This shows the tumor growth status in PARP11 knockout mice.

[0044] Figure 7 It is the effect of C11 and ITK7 in inhibiting lung metastasis of melanoma.

[0045] Figure 8 It refers to the cell-killing activity of CAR-T cells after C11 treatment of tumor cells.

[0046] Figure 9 It is the anti-tumor effect of C11 combined with anti-PD-L1 antibody. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0048] Example 1

[0049] A multi-level virtual screening process for small molecule inhibitors targeting PARP11 was implemented, virtually screening a commercial database containing over 10 million compounds. For example... Figure 2 As shown, the multi-level virtual screening process includes similarity search, multi-ligand pharmacophores, and molecular docking.

[0050] First, using the ITK7 molecule as a template structure, its MACCS molecular fingerprint was calculated. Based on the similarity between the fingerprint and commercial databases, molecules with a Tanimoto Coefficient greater than 0.85 were used for subsequent molecular docking. Second, known PARP11 inhibitors were collected, and pharmacophore models were constructed using these known inhibitors. The models were validated, and those with good screening performance were used to screen commercial compound databases. Finally, the three-dimensional structure of PARP11 was obtained through homology modeling. Then, the molecular docking tool (AutoDock Vina) was used to perform molecular docking studies on the molecules selected after similarity search and pharmacophore screening. Based on the docking results, molecules were selected for activity determination. The selected molecular structures are shown below. Figure 1 As shown. The preparation methods for each molecular structure are as follows.

[0051] The compounds of the present invention can be prepared via the following general route:

[0052] Step 1, Synthesis of 2-thio-quinazolinone / pyrimidinone intermediate: Using anthranilic acid derivative (or the corresponding heterocyclic precursor) as a starting material, react with isothiocyanate or thiourea under acidic or basic conditions to close the ring, and obtain 2-mercapto-4(3H)-quinazolinone or 2-mercapto-4(3H)-pyrimidinone intermediate;

[0053] Step 2, S-alkylation reaction: Under alkaline conditions (such as K2CO3, TEA, NaH, etc.), the above thiolated intermediate is reacted with the corresponding haloalkane (R'-X, where X is Cl, Br, I) in a polar solvent (such as DMF, DMSO, ethanol, etc.) to introduce the key side chain -S-R', and the target compound is obtained after post-treatment purification. 1.1

[0055] Preparation of 2-((pyridin-3-yl)methylthio)-6-methyl-1H-pyrimidin-4-one (compound C12):

[0056] ;

[0057] 6-Methyl-2-thio-1H-pyrimidin-4-one (144.5 mg, 1.02 mmol) was dissolved in DMF (2 mL), and triethylamine (TEA, 123.5 mg, 1.22 mmol) was added. The mixture was stirred at room temperature for 30 minutes. Then, 3-(chloromethyl)pyridine hydrochloride (202 mg, 1.12 mmol) was added, and the reaction mixture was stirred at 25°C for 12 hours. After the reaction was complete, water (20 mL) was added to the mixture, and a precipitate formed. The precipitate was filtered, dried, and purified by preparative liquid chromatography (Prep-HPLC) to give a white solid product (119 mg, yield 39.5%).

[0058] 1 H NMR (400 MHz, DMSO-d6) δ 2.21 (s, 3H), 4.38 (s, 2H), 6.01 (br s,1H), 7.35 (dd, J=7.8, 4.8 Hz, 1H), 7.84 (dt, J=7.8, 1.8 Hz, 1H), 8.45 (dd, J=4.8, 1.5 Hz, 1H), 8.64 (d, J=1.9 Hz, 1H), 12.2-12.8 (m, 1H).

[0059] ESI-MS: m / z calcd for C11H11N3OS 233.1 found 234.1 [M + H] + . 1.2

[0061] Preparation of 4-((4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thiomethyl)benzonitrile (compound C16):

[0062] ;

[0063] Following the method for compound C12, 4-(bromomethyl)benzonitrile and 6-methyl-2-thio-1H-pyrimidin-4-one were reacted in DMF to prepare a white solid (414.6 mg, yield 45.7%).

[0064] 1 H NMR (400 MHz, DMSO-d6) δ 2.19 (s, 3H), 4.44 (s, 2H), 6.01 (br s,1H), 7.63 (d, J=8.1 Hz, 2H), 7.78 (d, J=8.1 Hz, 2H), 12.1-12.9 (m, 1H).

[0065] ESI-MS: m / z calcd for C13H11N3OS 257.1, found 258.1 [M + H] + . 1.3

[0067] Preparation of 2-((1H-benzimidazol-2-yl)methylthio)-6-phenyl-1H-pyrimidin-4-one (compound C20):

[0068] ;

[0069] 6-Phenylacetyl-2-thio-1H-pyrimidin-4-one (500 mg, 2.45 mmol) was dissolved in DMF (5 mL), and TEA (297.3 mg, 2.94 mmol) was added. The mixture was stirred at 25°C for 30 minutes. Subsequently, 2-(chloromethyl)-1H-benzimidazole (489.4 mg, 2.94 mmol) was added, and the reaction mixture was stirred at 25°C for 2 hours. After the reaction was completed, the mixture was quenched with water (20 mL), filtered, and the filter cake was purified by Prep-HPLC to give a pale yellow solid product (331.8 mg, yield 40.4%).

[0070] 1H NMR (400 MHz, DMSO-d6) δ 4.77 (s, 2H), 6.74 (s, 1H), 7.08-7.20 (m,2H), 7.24-7.71 (m, 5H), 7.97-8.15 (m, 2H), 11.3-13.9 (m, 1H).

[0071] ESI-MS: m / z calcd for C13H11N3OS 334.1, found 335.1 [M + H] + . 1.4

[0073] Preparation of 3-methyl-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one (compound C15):

[0074] ;

[0075] 3-Methyl-2-thio-2,3-dihydroquinazolin-4(1H)-one (450 mg, 2.34 mmol) was dissolved in DMF (5 mL), and TEA (947.5 mg, 9.36 mmol) was added. Then 3-bromopropyne (835.4 mg, 7.02 mmol) was added, and the mixture was stirred at 25°C for 2 hours. After water treatment, the mixture was purified by filtration and washing with water to give a pale yellow solid (475 mg, yield 87.2%).

[0076] 1 H NMR (400 MHz, DMSO-d6) δ 3.24 (t, J=2.6 Hz, 1H), 3.50 (s, 3H), 4.17 (d, J=2.6 Hz, 2H), 7.40-7.52 (m, 1H), 7.57 (d, J=8.1 Hz, 1H), 7.80 (m,1H), 8.09 (dd, J=7.9, 1.2 Hz, 1H).

[0077] ESI-MS: m / z calcd for C13H11N3OS 230.1, found 231.1 [M + H] + . 1.5

[0079] Preparation of 5-benzyl-2-(benzylthio)-6-methyl-1H-pyrimidin-4-one (compound C14):

[0080] ;

[0081] 5-Benzyl-6-methyl-2-thio-1H-pyrimidin-4-one (500 mg, 2.15 mmol) was dissolved in DMF (5 mL), and TEAS (261.4 mg, 2.58 mmol) and benzyl bromide (441.8 mg, 2.58 mmol) were added. The reaction mixture was stirred at 25°C for 2 hours. The solution was then purified by Prep-HPLC to give a white solid (159 mg, yield 22.8%).

[0082] 1 H NMR (400 MHz, DMSO-d6) δ 2.22 (s, 3H), 3.74 (s, 2H), 4.37 (s, 2H), 7.13-7.34 (m, 8H), 7.39-7.44 (m, 2H), 12.4-12.8 (br s, 1H).

[0083] ESI-MS: m / z calcd for C19H18N2OS 322.1, found 323.1 [M + H] + . 1.6

[0085] Preparation of 4,6-dimethyl-2-oxo-5-[(pyrimidin-2-ylthio)methyl]-1,2-dihydropyridine-3-nitrile (compound C2):

[0086] ;

[0087] Ethyl acetoacetate (10 g, 58.08 mmol), 2-cyanoacetamide (14.65 g, 174.24 mmol), and TEA (17.63 g) were mixed in ethanol (100 mL) and stirred under reflux at 80°C for 4 hours. After cooling to 0°C, the mixture was filtered, and the filter cake was the target product (1.5 g, yield 10.6%).

[0088] 1 H NMR (400 MHz, DMSO-d6) δ 1.28 (t, J=7.1 Hz, 3H), 2.34 (s, 3H), 2.38 (s, 3H), 4.24-4.32 (m, 2H), 12.72 (br s, 1H).

[0089] ESI-MS: m / z calcd for C14H14N4OS 286.1, found 287.1 [M + H] + . 1.7

[0091] Preparation of compound C1 (2-(3-methylbenzylthio)pyrimidin-4(3H)-one):

[0092] ;

[0093] Following the method used for compound C12, 2-thio-1H-pyrimidin-4-one was reacted with 1-(bromomethyl)-3-methylbenzene. A white solid (349.1 mg, yield 38.4%) was obtained.

[0094] 1 H NMR (400 MHz, DMSO-d6) δ 2.28 (s, 3H), 4.36 (s, 2H), 6.13 (d, J=6.4 Hz, 1H), 7.04-7.11 (m, 1H), 7.14-7.26 (m, 3H), 7.91 (d, J=5.6 Hz, 1H),12.3-13.0 (br s, 1H).

[0095] ESI-MS: m / z calcd for C12H12N2OS 232.1, found 233.1 [M + H] + . 1.8

[0097] Preparation of compound C3 (6-methyl-2-((1-methyl-1H-benzimidazol-2-yl)methylthio)-1H-pyrimidin-4-one):

[0098] ;

[0099] Following the synthetic method for compound C20, 6-methyl-2-thio-1H-pyrimidin-4-one and 2-(chloromethyl)-1-methyl-1H-benzimidazole were reacted. White solid (127.5 mg, yield 29.5%);

[0100] 1 H NMR (400 MHz, DMSO-d6) δ 2.20 (s, 3H), 3.86 (s, 3H), 4.72 (s, 2H), 6.03 (s, 1H), 7.14-7.28 (m, 2H), 7.49-7.60 (m, 2H).

[0101] ESI-MS: m / z calcd for C14H14N4OS 286.1, found 287.1 [M + H] + . 1.9

[0103] Preparation of compound C4(2-((1H-benzimidazol-2-yl)methylthio)-6-methyl-1H-pyrimidin-4-one):

[0104] ;

[0105] Following the synthetic method for compound C20, 6-methyl-2-thio-1H-pyrimidin-4-one was reacted with 2-(chloromethyl)-1H-benzimidazole. White solid (127.5 mg, yield 29.5%).

[0106] 1 H NMR (400 MHz, DMSO-d6) δ 2.20 (s, 3H), 4.63 (s, 2H), 6.05 (s, 1H), 7.09-7.21 (m, 2H), 7.45-7.57 (m, 2H), 11.4-13.3 (br s, 1H).

[0107] ESI-MS: m / z calcd for C13H12N4OS 272.1, found 273.1 [M + H] + . 1.10

[0109] Preparation of compound C5(2-((1H-benzimidazol-2-yl)methylthio)quinazolin-4(3H)-one):

[0110] ;

[0111] Following the synthetic method for compound C20, 2-thioquinazolinone was reacted with 2-(chloromethyl)-1H-benzimidazole;

[0112] 1 H NMR (400 MHz, DMSO-d6) δ 4.75 (s, 2H), 7.12-7.19 (m, 2H), 7.40-7.47 (m, 1H), 7.47-7.54 (m, 2H), 7.60 (d, J=7.9 Hz, 1H), 7.73-7.81 (m, 1H),8.01-8.08 (m, 1H), 12.1-12.9 (br s, 1H).

[0113] MS (ESI) m / z: 309.1 [M+H] + .

[0114] ESI-MS: m / z calcd for C16H12N4OS 308.1, found 309.1 [M + H] + . 1.11

[0116] Preparation of compound C6 (2-(sec-butylthio)quinazolin-4(3H)-one):

[0117] ;

[0118] Following the synthetic method of compound C15, 2-thioquinazolinone was reacted with 2-bromobutane in DMF in the presence of potassium carbonate (K2CO3);

[0119] 1 H NMR (400 MHz, DMSO-d6) δ 0.99 (t, J=7.4 Hz, 3H), 1.39 (d, J=6.9Hz, 3H), 1.71 (m, 2H), 3.95 (m, 1H), 7.38-7.44 (m, 1H), 7.51 (d, J=8.0 Hz,1H), 7.71-7.77 (m, 1H), 8.02 (dd, J=7.9, 1.1 Hz, 1H), 12.49 (br s, 1H).

[0120] ESI-MS: m / z calcd for C12H14N2OS 234.1, found 235.1 [M + H] + . 1.12

[0122] Preparation of compound C7 (2-(cyanomethylthio)quinazolin-4(3H)-one):

[0123] ;

[0124] Following the synthetic method for compound C15, 3-methyl-2-thioquinazolinone was reacted with 2-chloroacetonitrile. The product was a gray solid (220 mg, yield 81.6%).

[0125] 1H NMR (400 MHz, DMSO-d6) δ 4.22 (s, 2H), 7.43 (t, J=7.6 Hz, 1H), 7.56 (d, J=8.1 Hz, 1H), 7.78 (t, J=7.8 Hz, 1H), 8.08 (d, J=7.9 Hz, 1H), 12.7(br s, 1H).

[0126] ESI-MS: m / z calcd for C11H9N3OS 231.0, found 232.0 [M + H] + . 1.13

[0128] Preparation of compound C8 (3-(4-methylphenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one):

[0129] ;

[0130] Step 1: Methyl 2-aminobenzoate and p-Tolylisothiocyanate were reacted by heating in DMSO to prepare 3-(4-methylphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one intermediate;

[0131] Step 2: Following the general method of Example 4, the above intermediate and 3-bromopropyne were subjected to an S-alkylation reaction in DMF in the presence of TEA to obtain the target compound;

[0132] 1 H NMR (400 MHz, DMSO-d6) δ 2.41 (s, 3H), 3.17 (t, J=2.63 Hz, 1H), 3.99 (d, J=2.63 Hz, 2H), 7.30-7.40 (m, 4H), 7.47-7.53 (m, 1H), 7.63 (d, J=7.88 Hz, 1H), 7.81-7.88 (m, 1H), 8.05-8.12 (m, 1H).

[0133] ESI-MS: m / z calcd for C18H14N2OS 306.1, found 307.1 [M + H] + . 1.14

[0135] Preparation of compound C9 (2-(chloromethyl)-8-methyl-7-(prop-1-yn-1-yl)quinazolin-4(3H)-one):

[0136] ;

[0137] Step 1: 2-Amino-N-phenylbenzamide and chloroacetyl chloride were reacted with acetic acid by heating to prepare 2-(chloromethyl)-3-phenylquinazoline-4(3H)-one intermediate;

[0138] Step 2: The above intermediate was reacted with 4,6-dimethylpyrimidine-2-thiol in DMA in the presence of NaH to obtain the target compound;

[0139] 1 H NMR (400 MHz, DMSO-d6) δ 2.25 (s, 6H), 4.23 (s, 2H), 6.89 (s, 1H), 7.40-7.51 (m, 5H), 7.53-7.60 (m, 1H), 7.70 (d, J=7.88 Hz, 1H), 7.84-7.91 (m,1H), 8.12 (dd, J=7.94, 1.06 Hz, 1H).

[0140] ESI-MS: m / z calcd for C21H20N4OS 374.1, found 375.1 [M + H] + . 1.15

[0142] Preparation of compound C10 (2-((E)-cinnamylthio)quinazolin-4(3H)-one):

[0143] ;

[0144] Referring to compound C12, 2-thioquinazolinone and (E)-cinnamyl bromide were reacted in DMF;

[0145] 1 H NMR (400 MHz, DMSO-d6) δ 4.09 (d, J=7.3 Hz, 2H), 6.33-6.49 (m,1H), 6.75 (d, J=15.8 Hz, 1H), 7.19-7.46 (m, 5H), 7.61 (d, J=8.0 Hz, 1H),7.73-7.83 (m, 1H), 8.03 (dd, J=7.9, 1.2 Hz, 1H), 12.59 (br s, 1H).

[0146] ESI-MS: m / z calcd for C17H14N2OS 294.1, found 295.1 [M + H] + . 1.16

[0148] Preparation of compound C11(2-((pyridin-4-yl)methylthio)quinazolin-4(3H)-one):

[0149] ;

[0150] Referring to compound C12, 2-thioquinazolinone and 4-(chloromethyl)pyridine hydrochloride were reacted in a DMF / TEA system;

[0151] 1 H NMR (400 MHz, DMSO-d6) δ 4.48 (s, 2H), 7.39-7.45 (m, 1H), 7.46-7.53 (m, 2H), 7.58 (d, J=7.9 Hz, 1H), 7.73-7.80 (m, 1H), 8.02 (dd, J=7.9, 1.3Hz, 1H), 8.47-8.53 (m, 2H), 12.56-12.75 (br s, 1H).

[0152] ESI-MS: m / z calcd for C14H11N3OS 269.1, found 270.1 [M + H] + . 1.17

[0154] Preparation of compound C13 (3-benzyl-5,6-dimethylthiopheno[2,3-d]pyrimidine-4(3H)-one):

[0155] ;

[0156] Step 1: Ethyl 2-amino-4,5-dimethylthiophene-3-carboxylate was reacted with 2-phenylacetyl chloride in a DIEA / THF system to prepare an amide intermediate;

[0157] Step 2: Dissolve the intermediate in ethanol, add sodium ethoxide (EtONa), and heat under reflux to cyclize, thus obtaining the target compound;

[0158] 1H NMR (400 MHz, DMSO-d6) δ 2.33 (d, J=15.26 Hz, 6H), 3.90 (s, 2H), 7.17-7.40 (m, 5H).

[0159] ESI-MS: m / z calcd for C15H14N2OS 270.1, found 271.1 [M + H] + . 1.18

[0161] Preparation of compound C17(2-(((4,6-dimethylpyrimidin-2-yl)thio)methyl)benzo[4,5]imidazo[1,2-a]pyrimidin-4(10H)-one):

[0162] ;

[0163] Step 1: Ethyl 4-chloroacetoacetate was reacted with 4,6-dimethyl-2-mercaptopyrimidine under alkaline conditions (such as TEA / DMF or EtONa / EtOH) to prepare the key intermediate β-keto ester.

[0164] Step 2: The intermediate obtained in Step 1 is reacted with 2-aminobenzimidazole in a solvent (such as DMF, glacial acetic acid, or ethanol) and heated under reflux (usually 80-120°C). After dehydration condensation cyclization, the solid is filtered after cooling, washed, and dried to obtain the target product C17.

[0165] 1 H NMR (400 MHz, DMSO-d6) δ 2.37 (s, 6H), 4.33 (s, 2H), 6.14 (s, 1H), 6.99 (s, 1H), 7.25-7.36 (m, 1H), 7.38-7.53 (m, 2H), 8.40 (d, J=8.13 Hz, 1H),13.04 (br s, 1H).

[0166] ESI-MS: m / z calcd for C17H15N5OS 337.1, found 338.0 [M + H] + . 1.19

[0168] Preparation of compound C18 (N-(2,6-dimethylphenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide):

[0169] ;

[0170] Following the method for compound C20, C18 was prepared by reacting 2-mercaptoquinazolin-4(3H)-one and 2-chloro-N-(2,6-dimethylphenyl)acetamide in DMF.

[0171] 1 H NMR (400 MHz, DMSO-d6) δ 2.11 (s, 6H), 4.18 (s, 2H), 7.00-7.06 (m,3H), 7.39-7.45 (m, 1H), 7.50-7.57 (m, 1H), 7.73-7.79 (m, 1H), 8.01-8.06 (m,1H), 9.63 (br s, 1H), 12.69 (br s, 1H).

[0172] ESI-MS: m / z calcd for C18H17N3O2S 339.1, found 340.1 [M + H] + . 1.20

[0174] Preparation of compound C19 (2-((1H-benzimidazol-2-yl)methylthio)-5,6-dimethyl-3-phenylthiopheno[2,3-d]pyrimidin-4(3H)-one):

[0175] ;

[0176] Following the method for compound C20, the compound was prepared by reacting 5,6-dimethyl-3-phenyl-2-thio-2,3-dihydrothiopheno[2,3-d]pyrimidin-4(1H)-one and 2-(chloromethyl)-1H-benzimidazole in DMF.

[0177] 1 H NMR (400 MHz, DMSO-d6) δ 2.34 (s, 3H), 2.38 (s, 3H), 4.58 (s, 2H), 7.08-7.17 (m, 2H), 7.42-7.49 (m, 4H), 7.53-7.61 (m, 3H), 11.59-12.82 (m, 1H).

[0178] ESI-MS: m / z calcd for C22H18N4OS2 418.1, found 419.2 [M + H] + .

[0179] Example 2

[0180] In this embodiment, the binding affinity of each compound to the PARP11 protein was detected by SPR:

[0181] like Figure 3 As shown, all four compounds exhibit concentration-dependent binding reactions with the PARP11 protein, but the affinities of the different compounds vary significantly. The equilibrium dissociation constant (K0) of ITK7... The value is 1.55 × 10 -5 M, while the K of the structurally optimized compound C11 1.21×10 -5 M indicates that the binding affinity of C11 to PARP11 is significantly enhanced compared to ITK7. Furthermore, among all the tested compounds, only C5 has a binding affinity of 2.768 × 10⁻⁶. -5 M and C6 are 2.838 × 10 -6 M and C7 are 5.415 × 10 -5 M and C10 are 1.815 × 10 -5 M and C11 are 1.21 × 10 -5 M and C16 are 1.49 × 10 -4 The SPR curve of M exhibits a clear saturation trend, enabling reliable steady-state fitting, thus yielding a credible K. Value. While other compounds showed some dose-dependency, reliable K values ​​could not be obtained because the binding curves did not reach the saturation range or the steady-state data deviated from the ideal 1:1 binding model. Therefore, this study did not report it quantitatively.

[0182] These results demonstrate that by optimizing the ITK7 scaffold structure, some derivatives obtained in this invention (especially C11) can bind more efficiently to PARP11, thus providing a structural basis for their enhanced biological activity. This result validates the rationale for computer-aided drug design and lays the foundation for subsequent efficacy validation.

[0183] Example 3

[0184] This embodiment verifies the antitumor activity of each compound:

[0185] To evaluate the antitumor activity of the designed compounds, the inhibitory effects of 20 ITK7 analogs (C1-C20) on the proliferation of colorectal cancer cell line MC38 were first detected in vitro using the CCK-8 assay.

[0186] like Figure 4As shown in Figure A, most compounds exhibited varying degrees of enhanced inhibitory effects on MC38 cells, with C5 and C11 showing the most significant cytotoxic activity, reducing cell viability to below 30%. In contrast, the parent compound ITK7 showed a weaker inhibitory effect, suggesting that structural optimization effectively improved antitumor activity.

[0187] To further verify the in vivo efficacy, a subcutaneous tumor-forming model was established in MC38 mice (2 million doses per mouse). After tumor formation, mice were treated with DMSO, ITK7, C1, C2, and C7 (150 μg / mouse, every two days); and DMSO, ITK7, C5, C10, and C11 (150 μg / mouse, every three days). Figure 4 As shown in Figure B, the ITK7 group had limited inhibitory effect on tumor growth, while the tumor volume growth of the C11 group was significantly slowed down. Specifically, the tumor volume of the C11 group was significantly inhibited in both animal experiments. Body weight changes showed that the body weight of the mice in the C11 group remained stable, and no obvious toxic reactions were observed.

[0188] The results in summary indicate that C11 exhibits superior antitumor activity compared to ITK7 both in vitro and in vivo, demonstrating stronger tumor-suppressing potential and better tolerability, providing a basis for subsequent pharmacodynamic mechanism research and clinical application.

[0189] Example 4

[0190] This embodiment tests the antitumor efficacy of compound C11:

[0191] To further verify the antitumor efficacy of compound C11, the representative compound of this invention, BALB / c mice were subcutaneously inoculated with 2 × 10⁶ mol / L mice. 6 4T1 breast cancer cells were randomly divided into three groups after tumor formation for 7 days. The groups were given intraperitoneal injections of DMSO, Olaparib (50 mg / kg), and C11 (10 mg / kg) every three days.

[0192] like Figure 5 As shown in Figure A, C11 treatment significantly inhibited tumor growth, and the tumor volume on day 21 of administration was significantly lower than that in the Olaparib group and the control group, with statistically significant differences. P 0.001). Weight monitoring results ( Figure 5 (B) showed that the body weight of mice in each group remained stable, with no obvious toxic side effects. Endpoint measurement results ( Figure 5 Further, it was shown that the tumor weight in the C11 group mice was significantly lower than that in the Olaparib group and the DMSO group (C11). P 0.01).

[0193] To analyze the effects of drugs on the immune microenvironment, flow cytometry was used to detect CD8+ in tumor tissue. + The activation status of T cells was shown. Results showed that CD69 in the C11-treated group... + CD8 + The proportion of T cells increased significantly ( Figure 5 The presence of C11 in the middle D group indicates that C11 can enhance the activation of T cells within the tumor. Simultaneously, Tim3 in the C11 group... + PD1 + CD8 + The proportion of T cells decreased significantly. Figure 5 The presence of E in the middle suggests that it can alleviate T cell exhaustion and improve the immunosuppressive microenvironment.

[0194] In summary, these results demonstrate that C11 exhibits superior antitumor activity compared to olaparib in the 4T1 breast cancer model, effectively inhibiting tumor growth while simultaneously activating the antitumor immune response, showcasing a dual-mechanism advantage. This further validates the unique potential of targeting PARP11 compared to traditional PARP1 / 2 inhibitors in antitumor immunotherapy.

[0195] Example 5

[0196] Examples of the study verify the physiological safety of the PARP11 target and its anti-tumor effects in vivo.

[0197] To verify the physiological safety of the PARP11 target and its antitumor effect in vivo, PARP11 gene knockout mice (Parp11) were selected. - / - A comparative experiment was conducted between mice and wild-type (WT) mice. Both groups of mice were injected with MC38 colorectal cancer cells under the same conditions, and tumor growth and immune cell composition were monitored.

[0198] like Figure 6 As shown in A and B, Parp11 - / - Tumor growth in mice was significantly slowed, and tumor volume and endpoint tumor weight ( Figure 6 The values ​​of C in the medium and low concentrations were significantly lower than those in the WT control group (P). The result was 0.01, suggesting that PARP11 deficiency can inhibit tumor development.

[0199] Flow cytometry analysis results showed ( Figure 6 (D, E, F), Parp11 - / - CD8 in mouse tumor tissue + The proportion of T cells increased significantly, while CD4... + The relative decrease in the proportion of T cells leads to CD8 + / CD4 + The significantly increased ratio indicates that PARP11 deficiency can promote effector T cell infiltration and enhance anti-tumor immune responses.

[0200] In addition, Parp11 - / - The mice maintained stable weight throughout the experiment, with no abnormal behavior or tissue damage observed, indicating that PARP11 deficiency does not cause significant physiological toxicity and that the target has good safety.

[0201] In conclusion, PARP11 gene deletion not only does not affect the normal development and physiological state of mice, but can also enhance CD8... + T-cell activity inhibits tumor growth. This result genetically validates the feasibility and safety of PARP11 as a drug target, providing a solid basis for the clinical translation of the small molecule inhibitor in this invention.

[0202] Example 6

[0203] This embodiment verifies the inhibitory effect of C11 on tumor metastasis:

[0204] To evaluate the inhibitory effect of C11 on tumor metastasis, a B16-F10 melanoma lung metastasis model was established. Mice were injected with B16-F10 cells via the tail vein and then treated with DMSO, ITK7 (150 μg / mouse), or C11 (150 μg / mouse) every three days. Mice were sacrificed on day 21, and lung tissue was collected for statistical analysis of metastatic nodules and immune cell analysis.

[0205] like Figure 7 As shown in Figures A and B, numerous black nodules were visible on the lung surface of mice in the DMSO group, while the number of nodules was significantly reduced in the ITK7 and C11 groups. Among them, the C11 group had the fewest metastatic lesions and a significantly smaller metastatic area (P < 0.05). 0.05). For example... Figure 7 As shown in C, D, and E, the flow cytometry analysis results show that CD11b in the lung tissue of group C11 is... + Gr1 + The proportion of myeloid-derived suppressor cells (MDSCs) decreased significantly (P 0.01), while CD8 + The proportion of T cells increased (P 0.05), Treg (Foxp3) + CD4 + The cell ratio did not change significantly.

[0206] The overall results indicate that C11 reduces the infiltration of immunosuppressive cells and enhances CD8. +T cell activity was significantly inhibited, thus suppressing lung metastasis of melanoma. Compared to ITK7, C11 exhibited higher biological activity in both immunomodulation and anti-metastatic effects.

[0207] Example 7

[0208] This example evaluates the impact of PARP11 inhibitors on sensitivity to immunotherapy:

[0209] To evaluate the effect of PARP11 inhibitors on the sensitivity to immunotherapy, an MC38-HER2-Luc subcutaneous tumor model was constructed, and hHER2-CAR-T cells were prepared.

[0210] In in vitro experiments, MC38-HER2-Luc cells were pretreated with DMSO (control), Olaparib (1µM), ITK7 (1µM), and C11 (1µM) for 24 h, respectively. CAR-T cells with comparable infection efficiency were then co-cultured for 24 h at an E:T ratio of 10:1. The CAR-T cells were derived from BALB / c mouse spleen-derived T cells activated by CD3 / CD28 co-stimulation and infected with hHER2-CAR virus using retrolectin-coated plates. The culture system contained IL-2 (5 ng / mL).

[0211] like Figure 8 As shown in Figure A, the transduction efficiency of CAR-T cells in each group is similar. The results of the kill experiment show that ( Figure 8 Both ITK7 and C11 pretreatment significantly improved the killing rate of CAR-T cells against tumor cells. Among them, the C11 group had the lowest cell survival rate and its killing activity was significantly better than that of the Olaparib group and the control group (P < 0.05). 0.01).

[0212] These results indicate that C11 enhances the sensitivity of tumor cells to CAR-T cells by inhibiting PARP11, suggesting that PARP11 inhibitors can serve as effective synergistic agents for immunotherapy and expand the application scope of existing PARP inhibitors.

[0213] Example 8

[0214] This embodiment verifies the synergistic effect of C11 and immune checkpoint blockade therapy:

[0215] To verify the synergistic effect of C11 and immune checkpoint blockade therapy, an MC38 xenograft model was established in C57BL / 6J mice. The tumor volume was increased to approximately 100 mm². 3Mice were randomly divided into PBS, anti-PD-L1, C11, and C11+anti-PD-L14 groups. C11 nanoparticles were formed using the amphiphilic phospholipid DSPE-PEG and administered via tail vein injection (150 μg / mouse) every three days. Anti-PD-L1 antibody was administered intravenously on days 8, 11, 14, and 17 (100 µg / mouse). Figure 9 The results showed that C11 monotherapy significantly inhibited tumor growth, while the combination of C11 and anti-PD-L1 showed the most significant tumor-suppressing effect, with a significant delay in tumor volume growth and a final volume reduction of approximately 70% compared to the control group. The body weight of mice in all groups remained stable, and no obvious toxic side effects were observed.

[0216] These results indicate that C11 can have a significant synergistic effect with anti-PD-L1 antibodies, enhancing the immune response and inhibiting tumor growth, thereby further improving the efficacy of immunotherapy while maintaining safety. These findings provide a new approach for the combined application of PARP11 inhibitors and immune checkpoint inhibitors.

[0217] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of PARP11-targeting small molecule inhibitors in the preparation of anti-colorectal cancer drugs, characterized in that, The structural formula of the small molecule inhibitor targeting PARP11 is as follows: 。 2. The application of PARP11-targeting small molecule inhibitors in the preparation of anti-melanoma drugs, characterized in that, The structural formula of the small molecule inhibitor targeting PARP11 is as follows: 。 3. The application of PARP11-targeting small molecule inhibitors in the preparation of anti-breast cancer drugs, characterized in that, The structural formula of the small molecule inhibitor targeting PARP11 is as follows: 。 4. A combination drug, characterized in that, This includes small molecule inhibitors targeting PARP11 and immune checkpoint inhibitors. The structural formula of the small molecule inhibitor targeting PARP11 is as follows: ； The immune checkpoint inhibitor is an anti-PD-L1 antibody.

5. The combination drug as described in claim 4, characterized in that, It also includes pharmaceutically acceptable excipients.

6. The combination drug as described in claim 4, characterized in that, The dosage form of the combined drugs is an injection, a lyophilized powder for injection, or a nanoparticle formulation.

Images