A DNA methyltransferase 1 inhibitor and preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to a compound with inhibitory activity on DNA methyltransferase 1 (DNMT1) and a preparation method and application thereof. In the application, a DNMT1 small-molecule inhibitor with a 2-isoindole-3-carbazolyl propionic acid skeleton is found and prepared, and is expected to be applied to the treatment of diseases related to DNA hypermethylation, especially tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a compound with DNA methyltransferase 1 inhibitory activity, its preparation method, and its application. Background Technology

[0002] DNA methylation is an important epigenetic modification that plays a crucial role in maintaining chromosome structure, X chromosome inactivation, gene imprinting, and the development of many human genetic diseases, such as cancer, cardiovascular disease, and diabetes. Among these, gene silencing caused by hypermethylation of tumor suppressor gene promoters is a significant contributing factor to tumorigenesis.

[0003] DNA methylation primarily refers to the process by which DNA methyltransferases (DNMTs) catalyze the conversion of cytosine on CpG islands in DNA to 5-methylcytosine using S-adenosylmethionine as a methyl donor, thereby silencing the gene and causing it to lose function. The DNMT family mainly includes DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. Among them, DNMT1 is the most studied methyltransferase, and studies have shown a direct correlation between DNMT1 expression and the degree of gene methylation. When DNMT1 expression is excessive or its activity is too high, the CpG islands in the promoter region of tumor suppressor genes change from a hypomethylated state to a hypermethylated state, inactivating the tumor suppressor gene and activating oncogenes, leading to cancer. During cancer invasion, DNMT1 can influence tumor development by regulating cancer cell proliferation, matrix degradation, cancer cell metastasis, and evasion of the immune system through DNA methylation.

[0004] Currently, reported DNMT1 inhibitors are mainly divided into two categories: nucleoside and non-nucleoside inhibitors. Nucleoside inhibitors include decitabine, azacitidine, and guarditabine. Among them, azacitidine, decitabine, and clofarabine have been approved for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML). Non-nucleoside small molecule inhibitors include procainamide, RG108, SGI-1027, MC3343, and GSK3685032, but none of these compounds have yet entered clinical trials. Summary of the Invention

[0005] To address the above problems, this invention provides a DNA methyltransferase 1 inhibitor, with the structural formula shown in Formula I.

[0006]

[0007] Wherein, R1 to R4 are substituents on the isoindole ring, selected from independent hydrogen, halogen, hydroxyl, amino, nitro, cyano, substituted or unsubstituted alkyl containing 1-4 carbon atoms, substituted or unsubstituted alkoxy containing 1-4 carbon atoms, acyloxy containing 1-4 carbon atoms, substituted or unsubstituted alkylamino containing 1-4 carbon atoms, substituted or unsubstituted acylamino containing 1-4 carbon atoms, or combinations of the above groups; R1, R2, R3, and R4 can be the same or different groups; R5 to R12 are substituents on the carbazole ring, selected from independent hydrogen, halogen, hydroxyl, amino, nitro, Cyanoyl, substituted or unsubstituted alkyl groups containing 1-6 carbon atoms, substituted or unsubstituted alkoxy groups containing 1-6 carbon atoms, substituted or unsubstituted acyl groups containing 1-6 carbon atoms, substituted or unsubstituted acyl groups containing 1-6 carbon atoms, substituted or unsubstituted sulfonyl groups containing 1-6 carbon atoms, substituted or unsubstituted cycloalkyl groups containing 3-6 carbon atoms, substituted or unsubstituted aryl groups containing 6-12 carbon atoms, substituted or unsubstituted aromatic heteroyl groups containing 3-12 carbon atoms, or combinations of the above groups; R5 to R12 can be the same or different groups, or adjacent groups can form a ring.

[0008] Furthermore, the compound comprises:

[0009]

[0010]

[0011] The present invention also provides a method for preparing the above-mentioned compound, characterized by comprising the following reaction:

[0012]

[0013] Methyl 2-(1,3-dioxoisoindol-2-yl)acrylates (A) with different substitutions and carbazole (B) with different substitutions were dissolved in a solvent and condensed under alkaline conditions via Michael addition reaction to generate methyl 3-(9H-carbazole-9-yl)-2-(1,3-dioxoisoindol-2-yl)propionate (C) with different substitutions. Compound C was dissolved and subjected to ester hydrolysis under heating and stirring with a catalyst to obtain the target product. In methyl 2-(1,3-dioxoisoindol-2-yl)acrylates (A) with different substitutions, R1 to R4 were selected from independent hydrogen, halogen, hydroxyl, amino, nitro, cyano, substituted or unsubstituted alkyl groups containing 1-4 carbon atoms, substituted or unsubstituted alkoxy groups containing 1-4 carbon atoms, acyloxy groups containing 1-4 carbon atoms, and substituted or unsubstituted alkylamino groups containing 1-4 carbon atoms. The groups R1, R2, R3, and R4 can be the same or different groups; in carbazole (B) with different substituents, R5 to R12 are selected from independent hydrogen, halogen, hydroxyl, amino, nitro, cyano, substituted or unsubstituted alkyl with 1 to 6 carbon atoms, substituted or unsubstituted alkoxy with 1 to 6 carbon atoms, substituted or unsubstituted acyloxy with 1 to 6 carbon atoms, substituted or unsubstituted acyl with 1 to 6 carbon atoms, substituted or unsubstituted sulfonyl with 1 to 6 carbon atoms, substituted or unsubstituted cycloalkyl with 3 to 6 carbon atoms, substituted or unsubstituted aryl with 6 to 12 carbon atoms, substituted or unsubstituted aromatic heteroyl with 3 to 12 carbon atoms, or combinations of the above groups. R5 to R12 can be the same or different groups, or adjacent groups can form a ring.

[0014] The present invention also provides a method for preparing the above-mentioned compound, comprising the following reaction: methyl 2-(1,3-dioxoisoindol-2-yl)acrylate and carbazole are dissolved in a solvent and condensed under alkaline conditions via Michael addition reaction to generate methyl 3-(9H-carbazole-9-yl)-2-(1,3-dioxoisoindol-2-yl)propionate; methyl 3-(9H-carbazole-9-yl)-2-(1,3-dioxoisoindol-2-yl)propionate is dissolved and subjected to ester hydrolysis reaction under the action of a catalyst and heating and stirring to obtain the target product.

[0015] Furthermore, the solvent in the condensation reaction is acetone, tetrahydrofuran, ethyl acetate, acetonitrile, dioxane, N,N-dimethylformamide, methanol, ethanol, isopropanol, or tert-butanol, preferably acetone and acetonitrile; the base is selected from at least one of potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium hydrogen hydride, sodium ethoxide, sodium methoxide, potassium tert-butoxide, potassium diisopropylamino, pyridine, N,N-dimethylaminopyridine, triethylamine, and diisopropylamine.

[0016] Preferably, the above-mentioned alkali is selected from potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide.

[0017] Furthermore, in the ester hydrolysis reaction, the solvent for dissolving methyl 3-(9H-carbazole-9-yl)-2-(1,3-dioxoisoindol-2-yl)propionate is selected from at least one of tetrahydrofuran, acetone, ethyl acetate, and acetonitrile; the catalyst is selected from at least one of lithium iodide, lithium hydroxide, and lithium carbonate.

[0018] The present invention also provides the use of the above-described compounds and their stereoisomers, including R-configurations and S-configurations, or pharmaceutically acceptable salts, in the preparation of drugs that inhibit DNA methyltransferase 1.

[0019] The present invention also provides a pharmaceutical composition for inhibiting DNA methyltransferase 1, wherein the pharmaceutical composition comprises the above-mentioned compound as the active ingredient; the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0020] The present invention has the following beneficial effects:

[0021] In this invention, we discovered and prepared a class of small molecule inhibitors of DNMT1 with a 2-isoindolyl-3-carbazole propionic acid backbone, which are expected to be applied to the treatment of DNA hypermethylation-related diseases, especially in the treatment of tumors. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Example 3: CD and ECD spectra of the two isomers;

[0024] Figure 2 Example 7: CD and ECD spectra of the two isomers;

[0025] Figure 3 Example 8: CD and ECD spectra of the two isomers. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0032] Example 1. 3-(9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0033]

[0034] 1 H NMR (400MHz, DMSO-d6) δ8.06(d,J=6.9Hz,2H),7.74(d,J=3.0Hz,2H),7.34(dd,J=5.1,3.0Hz,2H),7.37~7.27 (m,4H),7.21~7.14(m,2H),4.81(dd,J=5.0,5.2Hz,1H),4.65(d,J=5.0Hz,1H),4.44(d,J=5.2Hz,1H).ESI-MS m / z 385.7(M+H +).

[0035] Example 2.3-(3,6-dichloro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0036]

[0037] 1 H NMR (400MHz, DMSO-d6) δ8.25 (s, 2H), 7.74~7.71 (m, 4H), 7.46 (d, J=8.8Hz, 2H ),7.39(d,J=8.7,2H),5.16(dd,J=3.3,3.7Hz,1H),4.71~4.66(m,2H).ESI-MS m / z453.1(M+H + ).

[0038] Example 3.3-(3,6-dibromo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0039]

[0040] 1 H NMR (400MHz, DMSO-d6) δ8.40 (s, 2H), 7.77~7.68 (m, 4H), 7.51 (d, J = 7.0Hz, 2H) ,7.40(d,J=6.8Hz,2H),5.13~5.07(m,2H),4.67(dd,J=5.8,4.6Hz,1H).ESI-MS m / z540.4(M+H + ).

[0041] Example 4.3-(3,6-Diiodo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0042]

[0043] 1 H NMR (400MHz, DMSO-d6) δ8.53 (s, 2H), 7.76~7.69 (m, 4H), 7.63 (d, J=8.6Hz, 2H) ,7.28(d,J=8.6Hz,2H),5.17~5.05(m,2H),4.67(dd,J=5.6,4.5Hz,1H).ESI-MS m / z636.7(M+H + ).

[0044] Example 5.3-(2,7-dibromo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0045]

[0046] 1 H NMR (400MHz, DMSO-d6) δ8.05 (d, J = 8.3Hz, 2H), 7.77~7.71 (m, 4H), 7.60 (s, 2H), 7.28~7. 24(m,2H),5.18(dd,J=4.0,3.3Hz,1H),5.04(d,3.8Hz,1H),4.63(d,3.4Hz,1H).ESI-MS m / z 540.3(M+H + ).

[0047] Example 6.3-(3,6-dimethyl-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0048]

[0049] 1 H NMR (400MHz, DMSO-d6) δ7.80 (s, 2H), 7.74 ~ 7.68 (m, 4H), 7.28 (d, J = 8.3Hz, 2H), 7.13 ( d,J=8.4Hz,2H),5.15~5.10(m,2H),4.73(dd,J=5.6,5.5Hz,1H),2.39(s,6H).ESI-MS m / z 413.1(M+H + ).

[0050] Example 7.3-(3,6-di-tert-butyl-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0051]

[0052] 1 H NMR (400MHz, DMSO-d6) δ7.98 (s, 2H), 7.68 ~ 7.62 (m, 4H), 7.34 (d, J = 8.9Hz, 2H), 7.27 ( d,J=8.9Hz,2H),5.41(dd,J=6.5,5.2Hz,1H),5.13~5.08(m,2H),1.36(s,18H).ESI-MS m / z 497.5(M+H + ).

[0053] Example 8.3-(1,3,6-trichloro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0054]

[0055] 1 H NMR (400MHz, DMSO-d6) δ8.34(s,1H),8.23(s,1H),7.77~7.72(m,4H),7.62(s,1H),7.37(d, J=7.7Hz,1H),7.28(d,J=7.5Hz,1H),5.56(dd,J=5.5,4.5Hz,1H),5.14~5.10(m,2H).ESI-MS m / z 487.3(M+H + ).

[0056] Example 9.3-(3-bromo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0057]

[0058] 1 H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.96(d,J=7.7Hz,1H),7.54(dd,J=8.7,7.8Hz,2H),7.32~7.17(m,4 H),7.06(d,J=8.5Hz,1H),6.95(d,J=7.2Hz,2H),4.66(dd,J=6.5,5.5Hz,1H),4.54~4.37(m,2H).ESI-MS m / z 463.4(M+H + ).

[0059] Example 10. 3-(3-iodo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0060]

[0061] 1H NMR (400MHz, DMSO-d6) δ8.47(s,1H),8.11(d,J=7.8Hz,1H),7.73~7.69(m,4H),7.61(d,J=9.5Hz,1H),7.41(d, J=9.2Hz,1H),7.38~7.28(m,2H),7.12(d,J=7.4Hz,1H),5.16~5.12(m,2H),4.71(dd,J=6.8,5.5Hz,1H).ESI-MS m / z 511.0(M+H + ).

[0062] Example 11.2-(1,3-dioxoisoindoline-2-yl)-3-(4-(epoxyethylene-2-ylmethoxy)-9H-carbazole-9-yl)propionic acid

[0063]

[0064] 1 H NMR(400MHz, DMSO-d6)δ8.10(d,J=7.5Hz,1H),7.77~7.67(m,4H),7.47(d,J=8.3H z,2H),7.30~7.20(m,2H),7.11(dd,J=7.6,7.1Hz,1H),6.67(d,J=7.3Hz,1H),5.51 (dd,J=7.6,3.8Hz,1H),5.02(d,J=7.6Hz,1H),4.52(d,J=3.8Hz,1H),4.07~3.99( m,2H),3.30(d,J=3.1Hz,1H),2.92(d,J=2.8Hz,1H),2.82(d,J=2.4Hz,1H).ESI-MS m / z457.1(M+H + ).

[0065] Example 12.3-(3,6-dinitro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0066]

[0067] 1H NMR (400MHz, DMSO-d6) δ9.43 (s, 2H), 8.31 (d, J = 7.46Hz, 2H), 7.98 ~ 7.87 (m, 4H), 7.33 (d, J = 7.4 6Hz, 2H), 4.95 (dd, J=5.91, 5.27Hz, 1H), 4.79 (d, J=5.37Hz, 1H), 4.51 (d, J=5.91Hz, 1H).ESI-MS m / z 475.1(M+H + ).

[0068] Example 13. 3-(3,6-diacetyl-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0069]

[0070] 1 H NMR (400MHz, DMSO-d6) δ8.96 (s, 2H), 7.99 (d, J = 8.6Hz, 2H), 7.70 ~ 7.65 (m, 4H), 7.55 ( d,J=8.7Hz,2H),5.32~5.14(m,2H),4.75(dd,J=5.2,3.4Hz,1H),2.65(s,6H).ESI-MS m / z 469.2(M+H + ).

[0071] Example 14.2-(1,3-dioxoisoindoline-2-yl)-3-(1,2,3,4-tetrahydro-11H-benzo[a]carbazole-11-yl)propionic acid

[0072]

[0073] 1 H NMR (400MHz, DMSO-d6) δ8.19~8.11(m,1H),7.87(dd,J=5.0,3.1Hz,2H),7.78~7.59(m,4H),7.37~7.29(m,2H),6.90(d,J=7.8Hz,1H),4.70 (dd,J=3.4,3.4Hz,1H),4.59(d,3.4Hz,1H),4.53(d,3.4Hz,1H),3.01~2.87(m,2H),2.78(dd,J=6.9,6.2Hz,2H),1.83~1.68(m,4H).ESI-MS m / z 439.2(M+H + ).

[0074] Example 15. 3-(11H-benzo[a]carbazole-11-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0075]

[0076] 1 H NMR (400MHz, DMSO-d6) δ8.31 (d, J=8.4Hz, 1H), 8.11~8.09 (m, 3H), 7.87 (dd, J=7.6, 5.0Hz, 2H), 7.84~7.89 (m, 4H), 7.54 (d, J=7. 6Hz,1H),7.39(d,J=7.5Hz,1H),7.33(d,J=7.6,1H),7.17(d,J=5.0,1H),4.96(dd,J=5.2,3.4Hz,1H),4.58~4.44(m,2H).ESI-MS m / z 435.1(M+H + ).

[0077] Example 16. 3-(3,6-diacetoxy-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0078]

[0079] 1 H NMR (400MHz, DMSO-d6) δ7.84 (s, 2H), 7.74~7.70 (m, 4H), 7.45 (d, J = 8.9Hz, 2H), 7.30 ( d,J=8.6Hz,2H),5.25~5.11(m,2H),4.80(dd,J=5.4,3.5Hz,1H),2.27(s,6H).ESI-MS m / z 501.1(M+H + ).

[0080] Example 17. 3-(3,6-diacetoxy-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0081]

[0082] 1H NMR (400MHz, DMSO-d6) δ10.11(s,2H),7.85,(s,2H),7.78~7.71(m,4H),7.56(d,J=9.0,2H ),7.38(d,J=9.0,2H),5.13~4.96(m,2H),4.77(dd,J=6.1,5.2Hz,1H),2.04(s,6H).ESI-MS m / z 499.1(M+H + ).

[0083] Example 18.2-(1,3-dioxoisoindoline-2-yl)-3-(7,8,9,10-tetrahydro-5H-benzo[b]carbazole-5-yl)propionic acid

[0084]

[0085] 1 H NMR(400MHz, DMSO-d6)δ8.19(s,1H),7.87(dd,J=5.0,3.1Hz,2H),7.74~7.67(m,4H),7.60(d,J=3.1Hz,1H),7.37(d,J=3.1Hz,1H),7 .26(s,1H),4.75(dd,J=3.4,3.3Hz,1H),4.61(d,J=3.5Hz,1H),4.52(d,J=3.3Hz,1H),2.78~2.65(m,4H),1.79~1.70(m,4H).ESI-MS m / z 439.1(M+H + ).

[0086] Example 19.2-(1,3-dioxoisoindoline-2-yl)-3-(1,2,3,4-tetrahydro-7H-benzo[c]carbazole-7-yl)propionic acid

[0087]

[0088] 1H NMR (400MHz, DMSO-d6) δ8.15 (d, J=3.1Hz, 1H), 7.78~7.73 (m, 4H), 7.70 (dd, J=5.1, 3.1Hz,1H),7.55(dd,J=5.4,3.1Hz,1H),7.33(d,J=3.1Hz,2H),7.27(d,J=3.1Hz,1H ),4.75(dd,J=3.5,3.4Hz,1H),4.61(d,J=3.5Hz,1H),4.52(d,J=3.3Hz,1H),2.90(d ,J=3.8Hz,2H),2.86(d,J=3.8Hz,2H),2.81~2.72(m,2H),1.74~1.70(m,2H).ESI-MS m / z386.1(M+H + ).

[0089] Example 20.2-(1,3-dioxoisoindoline-2-yl)-3-(1-formyl-9H-carbazole-9-yl)propionic acid

[0090]

[0091] 1 H NMR (400MHz, DMSO-d6) δ8.00 (d, J=7.7Hz, 1H), 7.68~7.61 (m, 4H), 7.39 (dd, J=8.2, 7.4Hz, 2H), 7.32 (dd, J=7.6, 7.4Hz, 1H), 7.32 (d, J=7.4Hz, 1H), 7.15 (d, J=7.4Hz, 2H), 5.49 (dd, J=5.8, 4.6Hz, 1H), 5.18~5.15 (m, 2H).ESI-MS m / z 413.1(M+H + ).

[0092] Example 21.3-(2,7-di-tert-butyl-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0093]

[0094] 1H NMR (400MHz, DMSO-d6) δ7.86 (d, J=8.2Hz, 2H), 7.72~7.61 (m, 4H), 7.33 (s, 2H), 7.19 (d, J=8.2Hz, 2 H),5.40(dd,J=5.5,4.6Hz,1H),5.21(d,J=5.6Hz,1H),5.14(d,J=4.6Hz,1H),1.33(s,18H).ESI-MS m / z 497.2(M+H + ).

[0095] Example 22. 3-(3-bromo-6-methoxy-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0096]

[0097] 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=5.1Hz,1H),7.73(s,1H),7.56~7.53(m,4H),7.35(s,1H),7.20(d,J=5.1Hz,1H) ,6.88(d,J=8.3Hz,1H),6.69(d,J=8.3Hz,1H),5.18~5.01(m,2H),4.63(dd,J=4.6,3.5Hz,1H),3.72(s,3H).ESI-MS m / z 493.0(M+H + ).

[0098] Example 23. 3-(7H-benzo[c]carbazole-7-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0099]

[0100] 1 H NMR (400MHz, DMSO-d6) δ8.73(d,J=8.3Hz,1H),8.56(d,J=7.6Hz,2H),8.03(dd,J=8.3,6.0Hz,2H),7.76~7.64(m,4H), 7.48(d,J=7.6Hz,1H)7.31(d,J=6.0Hz,1H),7.29~7.10(m,3H),5.46~4.94(m,2H),4.86(dd,J=5.9,4.3Hz,1H).ESI-MS m / z 435.1(M+H + ).

[0101] Example 24.2-(1,3-dioxoisoindoline-2-yl)-3-(3,6-dipropionyl-9H-carbazole-9-yl)propionic acid

[0102]

[0103] 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=1.8Hz,2H),8.44(s,2H),7.90~7.81(m,4H),7.81(d,J=1.5Hz,2H),4.75(dd,J= 3.4,3.3Hz,1H),4.58(d,J=3.4Hz,1H),4.46(d,J=3.3Hz,1H),2.95(q,J=7.6Hz,4H),1.20(t,J=7.6Hz,6H).ESI-MS m / z 497.2(M+H + ).

[0104] Example 25. 3-(2,7-diethoxy-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0105]

[0106] 1 H NMR (400MHz, DMSO-d6) δ7.90~7.83(m,4H),7.81(d,J=7.8Hz

[0107] ,2H),7.74(s,2H),7.18(d,J=7.8Hz,2H),4.75(dd,J=5.1,3.4Hz,1H),4.62(d,J=3. 5Hz,1H),4.51(d,J=5.1Hz,1H),4.03(q,J=6.7Hz,4H),1.43(t,J=6.7Hz,6H).ESI-MS m / z 473.1(M+H + ).

[0108] Example 26. 3-(2-cyclopropyl-7-methyl-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0109]

[0110] 1H NMR (400MHz, DMSO-d6) δ7.94(d,J=4.9Hz,1H),7.87~7.74(m,4H),7.37(d,J=2.1,Hz,1H),7.27(s,2H),7.20(d,J=4.9Hz,1H),7.17(d,J=2.1 Hz,1H),4.75(dd,J=3.4,3.3Hz,1H),4.64(d,J=3.5Hz,1H),4.54(d,J=3.3Hz,1H),2.46(s,3H),1.04–0.92(m,2H),0.85–0.73(m,2H).ESI-MS m / z 439.1(M+H + ).

[0111] Example 27. 3-(2-allyl-6-bromo-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0112]

[0113] 1 H NMR (400MHz, DMSO-d6) δ7.94 (d, J=8.2Hz, 1H), 7.87~7.74 (m, 4H), 7.69 (s, 1H), 7.49 (dd,J=8.2,1.8Hz,1H),7.39(d,J=8.2Hz,1H),7.34(s,1H),7.17(dd,J=7.9,2.0Hz,1 H),5.94~5.92(m,1H),5.09(d,J=9.6Hz,1H),4.96(d,J=9.6Hz,1H),4.75(dd,J=3.4 ,3.3Hz,1H),4.63(d,J=3.5Hz,1H),4.49(d,J=3.3Hz,1H),3.35~3.23(m,2H).ESI-MS m / z 503.1(M+H + ).

[0114] Example 28. 3-(1,6-dibromo-3-(methylcarbamoyl)-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0115]

[0116] 1H NMR (400MHz, DMSO-d6) δ8.53(s,1H),7.99(s,1H),7.93(d,J=2.2Hz,1H),7.87~7.74(m,4H),7.70( s,1H),7.49(d,J=2.0Hz,1H),4.70(dd,J=3.4,3.2Hz,1H),4.59~4.48(m,2H),2.93(s,3H).ESI-MS m / z 598.1(M+H + ).

[0117] Example 29. 3-(3-Butyl-1,5,7-trichloro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid

[0118]

[0119] 1 H NMR (400MHz, DMSO-d6) δ7.87~7.74(m,4H),7.61(s,1H),7.41(s,1H),7.07(s,1H),6.95(s,1H),4.70(dd,J=3.4,3.3Hz, 1H), 4.61~4.59(m,2H),2.62(t,J=8.3,2.7Hz,2H),1.58~1.50(m,2H),1.33~1.28(m,2H),0.95(t,J=7.2Hz,3H).ESI-MS m / z543.1(M+H + ).

[0120] Example 30. 2-(5-acetamido-1,3-dioxoisoindoline-2-yl)-3-(3,6-di-tert-butyl-9H-carbazole-9-yl)propionic acid

[0121]

[0122] 1 H NMR (400MHz, DMSO-d6) δ9.47(s,1H),8.05(d,J=2.2Hz,1H),7.97(d,J=2.1Hz,1H),7.44(d,J=2.6Hz,2H),7.33(d,J=2.6Hz,2 H),7.24(s,2H),4.75(dd,J=3.4,3.3Hz,1H),4.58(d,J=3.3Hz,1H),4.46(d,J=3.3Hz,1H),2.0(s,3H),1.35(s,18H).ESI-MS m / z554.2(M+H + ).

[0123] Example 31.3-(3,6-di-tert-butyl-9H-carbazole-9-yl)-2-(5-methoxy-1,3-dioxoisoindoline-2-yl)propionic acid

[0124]

[0125] 1 H NMR (400MHz, DMSO-d6) δ7.97(d,J=2.2Hz,1H),7.91(s,1H),7.79(d,J=2.5Hz,1H),7.40(d,J=2.6Hz,2H),7.29(d,J=2.6Hz,2 H),7.24(s,2H),4.75(dd,J=3.4,3.3Hz,1H),4.58(d,J=3.3Hz,1H),4.46(d,J=3.3Hz,1H),2.8(s,3H),1.35(s,18H).ESI-MS m / z527.2(M+H + ).

[0126] Example 32.2-(5-acetoxy-4-bromo-1,3-dioxoisoindoline-2-yl)-3-(3,6-di-tert-butyl-9H-carbazole-9-yl)propionic acid

[0127]

[0128] 1 H NMR (400MHz, DMSO-d6) δ7.93(d,J=2.5Hz,1H),7.79(d,J=2.5Hz,1H),7.44(d,J=2.6Hz,2H),7.36(d,J=2.6Hz,2H),7. 25(s,2H),4.75(dd,J=3.4,3.3Hz,1H),4.58(d,J=3.3Hz,1H),4.46(d,J=3.3Hz,1H),2.0(s,3H),1.35(s,18H).ESI-MS m / z633.2(M+H + ).

[0129] Example 33.3-(3,6-dibromo-9H-carbazole-9-yl)-2-(5-methoxy-1,3-dioxoisoindoline-2-yl)propionic acid

[0130]

[0131] 1H NMR (400MHz, DMSO-d6) δ7.97(d,J=2.2Hz,1H),7.91(s,1H),7.79(d,J=2.5Hz,1H),7.45(d,J=2.6Hz,2H),7.33(d,J=2 .6Hz,2H),7.27(s,2H),4.75(dd,J=3.4,3.3Hz,1H),4.58(d,J=3.3Hz,1H),4.46(d,J=3.3Hz,1H),2.8(s,3H).ESI-MS m / z 633.2(M+H + ).

[0132] Example 34.

[0133] Preparation of Example 2 (Based on Example 2, the compounds shown in Examples 1, 3-33 can be obtained according to the following scheme)

[0134]

[0135] Synthesis of Methyl 3-(3,6-dichloro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionate (C2): In a 25 mL round-bottom flask, 118 mg of 3,6-dichloro-9H-carbazole (0.5 mmol, 1.15 eq) and 112 mg of KOH (3.5 eq) were added. The mixture was stirred at 60 °C for 1 h, followed by the addition of Al (100 mg, 0.7 mmol, 1 eq), and the reaction was allowed to proceed overnight. A TLC test with a PE:EA ratio of 4:1 showed the formation of new spots, with a polarity slightly greater than 1A21. After stopping the reaction, the insoluble matter was filtered off. The solution was evaporated to dryness in acetonitrile, extracted with ethyl acetate, and then subjected to column chromatography (PE:EA = 8:1) to give 140 mg of a yellow-green solid. Yield: 69%. 1 H NMR (400MHz, CDCl3) δ8.22(s,2H),7.75~7.71(m,4H),7.47(d,J=8.8Hz,2H),7.39( d,J=8.7,2H),5.16(dd,J=3.3,3.7Hz,1H),4.71~4.66(m,2H),3.82(s,3H).ESI-MS m / z 467.4(M+H + )

[0136] Synthesis of 3-(3,6-dichloro-9H-carbazole-9-yl)-2-(1,3-dioxoisoindoline-2-yl)propionic acid (Example 2): 100 mg of C2 and 3 eq of LiI were added to a 25 mL flask, dissolved in ethyl acetate, and the mixture was heated and stirred overnight at 80 °C. The reaction was carried out for 5 days. The precipitate was filtered and washed repeatedly with ethyl acetate to give 45 mg of a yellow-green solid. Yield: 45%. 1H NMR (400MHz, DMSO-d6) δ8.25(s,2H),7.74~7.71(m,4H),7.46(d,J=8.8Hz,2H),7.39(d,J=8.7,2H),5.16(dd,J=3.3,3.7Hz,1H), 4.71~4.66(m,2H).

[0137] ESI-MS m / z 453.1 (M+H) + )

[0138] Example 35. Decomposition and Structure Determination of Examples 3, 7, and 8

[0139] Preparation method: Chiral isomers were separated using HPLC and a chiral column, and the corresponding fractions were collected. The solvent was removed by rotary evaporation to obtain the pure optical isomer.

[0140] Column specifications: 0.46cm ID × 15cm L; Mobile phase: MeOH / TFA = 100 / 0.1 (V / V); Flow rate: 1.0 mL / min. The first eluting compound is numbered 1, and the second eluting compound is numbered 2.

[0141] Configuration determination: The configuration was determined by superimposing the predicted ECD spectra of the R-configuration and S-configuration compounds with the measured CD spectra of compounds-1 and-2.

[0142] The results are shown in Table 1:

[0143] Table 1. Results of isomer resolution and configuration determination for some compounds.

[0144]

[0145]

[0146]

[0147] Example 37. Binding ability of the compound with DNMT1 (SPR experimental results):

[0148] Experimental Procedure: The binding affinity of DNMT1 protein to a final concentration of 50.7 μg / mL was measured using 10 mM sodium acetate buffer at pH 5.5, 5.0, 4.5, and 4.0. Based on the results, the optimal coupling pH was selected, and DNMT1 protein was coupled to the CM5 chip at this concentration. The affinity of DNMT1 protein to a 10 μM small molecule inhibitor was then measured, with the DNMT1 inhibitor RG108 used as a positive control. The affinity measurement data were analyzed. Compounds binding to DNMT1 protein were selected and started at 5 μM, diluted 2-fold, resulting in 8 concentrations (including 0 concentration). The corresponding solutions were flowed into the Fc1 and Fc2 channels of the chip, using a binding time of 35 s and a dissociation time of 45 s. The chip was then regenerated using HBS-EP+ at a flow rate of 30 L / min for 30 s. After the binding assay, data were analyzed using Biacore T200 evaluation software (blank concentration: 0 nM). Finally, based on the affinity pattern, a fitting analysis was performed using either kinetics or affinity.

[0149] Experimental Results: The results are shown in Table 2. The optimal coupling pH for DNMT1 protein and the CM5 chip is 4.5. KD assays were performed on all examples. 50 Value testing. All embodiments showed a significantly improved affinity for DNMT1 compared to RG108.

[0150] Table 2. Experimental results of affinity determination between small molecules and DNMT1

[0151]

[0152]

[0153] Conclusion: The interaction force between the embodiments involved in this invention and DNMT1 is significantly higher than that between RG108 and DNMT1, mostly by nearly two orders of magnitude.

[0154] Example 38. Test results of the inhibitory activity of the compound against the DNMT1 enzyme.

[0155] Experimental Procedure: Prepare the enzyme solution: Prepare the enzyme solution in 1x assay buffer. Prepare the substrate solution: Prepare the substrate solution in 1x assay buffer. Dilute the compound to a final DMSO-D6 concentration of 1%. Prepare the [3H]-SAM solution. Transfer 10 μL of the enzyme solution to the assay plate, and transfer 10 μL of 1x assay buffer to the assay plate for the minimum control group. Incubate at room temperature for 15 minutes.

[0156] Add 10 μL of substrate solution to each well. Add 10 μL of [3H]-SAM solution to each well to start the reaction. Incubate at 37 °C for 180 min. Add 10 μL of cold SAM solution to each well to stop the reaction.

[0157] Pre-incubate the filter plate with 0.5% PEI for 15 minutes and then evacuate it. Transfer 40 μL of the reaction mixture to the filter plate and wash the filter plate three times under vacuum with ddH2O. Read the counts on a MicroBeta.

[0158] Inhibition rate = (maximum value - measured value) / (maximum value - minimum value) * 100%.

[0159] The inhibition rate of DNMT1 enzyme in Examples 1-11 was tested at 250 μM, and several examples with better activity were selected and dissected for DNMT1 inhibition rate testing at 125 μM.

[0160] The experimental results are shown in Table 3:

[0161] Table 3. Results of the inhibitory activity assay of small molecule inhibitors against DNMT1

[0162]

[0163]

[0164] Experimental conclusion: The compound of this invention has a significant inhibitory effect on DNMT1 protease.

[0165] Example 39: Inhibition of Cancer Cell Proliferation Activity

[0166] Experimental procedure: A2780 cell line (ovarian cancer cells) and HeLa cell line (cervical cancer cells) were selected to test the inhibitory activity of cell proliferation.

[0167] Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, add 100 μL to each well, and seed the cells to a density of 5000-8000 cells per well (fill the edge wells with sterile PBS). 24 h after cell seeding, add 100 μL of a 400 μM inhibitor (D++ formulation) to each well, with three replicates for each example. Incubate at 37°C with 5% CO2 for 48 h, observing under an inverted microscope. Add 150 μL of MTT solution (0.5 mg / ml, i.e., 0.5% MTT diluted 10 times) to each well and continue culturing for 4 h. Terminate the culture and carefully aspirate the culture medium from the wells. Add 150 μL of dimethyl sulfoxide to each well to fully dissolve any crystals. Measure the absorbance of each well at OD 570 nm using an ELISA reader.

[0168] The results are shown in Table 4. Inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%

[0169] As: Reading of experimental wells (containing cell culture medium, MTT, and drugs)

[0170] Ac: Reading from control wells (cell-containing culture medium, MTT, no drug).

[0171] Ab: Reading of blank wells (medium medium without cells and drugs, DMSO)

[0172] Table 4. Effects of small molecule inhibitors on tumor cell proliferation

[0173]

[0174]

[0175] This invention relates to compounds that can effectively inhibit the proliferation of cancer cells and can be used to treat diseases related to high DNMT1 expression, especially tumors such as cervical cancer and ovarian cancer.

[0176] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound that inhibits DNA methyltransferase 1 (DNMT1), said compound being: ， 。 2. The use of the compound of claim 1 and its stereoisomers, including the R-configuration and the S-configuration, or a pharmaceutically acceptable salt in the preparation of a medicament for an inhibitor of DNA methyltransferase 1.

3. A pharmaceutical composition of a DNA methyltransferase 1 inhibitor, characterized in that, The pharmaceutical composition uses the compound of claim 1 as the active ingredient; the pharmaceutical composition further includes pharmaceutically acceptable excipients.

4. A pharmaceutical composition of a DNA methyltransferase 1 inhibitor, characterized in that, The pharmaceutically acceptable excipient is a pharmaceutically acceptable carrier.

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