Purino[1,2-a]carbazolin derivatives, their preparation methods and applications

Abstract

This application discloses a purino[1,2-a]carbazolin derivative, its preparation method, and its application. Specifically, it discloses a compound having the structure shown in Formula I, or its stereoisomers, solvates, metabolites, pharmaceutically acceptable salts, cocrystals, or prodrugs: ; wherein R1 and R2 are each independently selected from hydrogen, hydroxyl, carboxyl, substituted or unsubstituted C1-C6 alkyl, -CONH-R3, and -COO-R4; wherein R3 is a substituted or unsubstituted C1-C5 alkyl, and the substituent can be any of cyano, halogen, alkylC1-C5 alkoxy, C1-C5 alkylsulfonyl, di(C1-C5 alkyl)amino, nitrogen-containing heterocyclic, aryl, and C1-C5 heteroaryl; R4 is selected from any of substituted or unsubstituted aryl C1-C5 alkyl and nitrogen-containing saturated heterocyclic C1-C5 alkyl. This compound can be used as a TDP1 inhibitor for antitumor therapy.

Description

Technical Field

[0001] This application relates to the field of compound synthesis technology, and in particular to a purino[1,2-a]carbazolin derivative and its preparation method and application. Background Technology

[0002] Tyrosine-DNA phosphodiesterase 1 (TDP1) is a key DNA repair enzyme whose core function is to specifically hydrolyze the phosphotyrosine bonds at the ends of DNA, thus playing a crucial role in repairing topoisomerase I (TOP1)-induced DNA damage. Specifically, when TOP1 inhibitors capture and stabilize the TOP1-DNA covalent complex (TOP1cc), they prevent DNA reconnection, ultimately causing persistent DNA damage and triggering apoptosis. TDP1, on the other hand, specifically hydrolyzes the 3'-phosphotyrosine bonds in TOP1cc, generating DNA fragments with 5'-hydroxyl and 3'-phosphate ends. Subsequently, poly(ADP-ribose) polymerase 1 (PARP-1) recruits and activates the X-ray repair cross-complementation gene 1 (XRCC-1) repair pathway, which relies on polynucleotide kinase phosphatase (PNKP), DNA polymerases (such as POLβ), and DNA ligase III (LIG3) to complete the entire repair process of TOP1cc-induced DNA damage. TDP1-mediated efficient DNA repair reduces the cytotoxic effects of TOP1 inhibitors, ultimately leading to drug resistance in tumor cells. Conversely, inhibiting TDP1 activity can block this repair pathway, enhance the damaging effects of TOP1 inhibitors on tumor cells, and reverse their drug resistance—this is the core mechanism by which TDP1 inhibitors can act as TOP1 inhibitor sensitizers.

[0003] To date, the number of reported TDP1 inhibitors remains very limited, and most belong to different structural types: Steroidal inhibitor 1, discovered through novel high-throughput screening, is a competitive TDP1 inhibitor with a half-maximal inhibitory concentration (IC50). 50 The concentration was 7.7 μM, and its mechanism of action was to mimic the oligonucleotide-peptide substrate of this enzyme; puromycin (inhibitor 2), which belongs to the class of ribosome inhibitors, had weak inhibitory activity against TDP1, with an IC50 value of 7.7 μM. 50 The value was 18 mM; the coumarin inhibitor 3 isolated from Daphne plants had potent inhibitory activity, IC50 value 18 mM. 50 The value was 1.1 μM; the usnic acid-derived Schiff base inhibitor 4, validated by affinity experiments and molecular simulations, not only showed no cytotoxicity but also significantly enhanced the antitumor efficacy of topotecan (TPT) against Lewis lung cancer in both in vitro and in vivo experiments, with an IC50 value of 1.1 μM.50 The value is 26 nM; substrate mimicking inhibitors of the natural tyrosine-DNA phosphodiester bond substrate of TDP1 (such as the ferulic acid derivative ATA and the multi-target inhibitor suramin), of which ATA has an IC50 value of 26 nM. 50 The value is 22 nM, and its inhibitory activity is highly dependent on the substitution positions of the two hydroxyl groups in the molecule. The IC50 value of suramin for TDP1 is... 50 The inhibitor has a concentration of 5 μM and is active against a variety of DNA-interacting proteases. Benzophenanthrene-7, a selective TDP1 inhibitor developed by introducing an α-alkoxypyridine structure into the B ring, is a benzophenanthrene-7 derivative inhibitor with an IC50 value of 5 μM. 50 The concentration was 8.2 μM, which, when used in combination with TPT, produced a synergistic anti-tumor effect on human breast cancer MCF-7 cells; the indole isoquinoline derivative inhibitor 8 is the first reported dual-target inhibitor of TOP1 / TDP1, and its IC50 against TDP1 is 8.2 μM. 50 The effective concentration was 1.52 μM. However, these reported TDP1 inhibitors generally have certain limitations in their development, such as poor absorption, insufficient bioavailability, high cytotoxicity, and off-target effects. These problems largely stem from inherent defects in their chemical backbones. Therefore, developing novel TDP1 inhibitors with entirely new chemical backbones has significant research value and application prospects.

[0004] Based on this, the present invention proposes a new class of compounds based on the purinol[1,2-a]carbazolin skeleton and evaluates their TDP1 inhibitory activity, providing a novel potential therapeutic strategy for cancer treatment. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, embodiments of this application provide a purino[1,2-a]carbazolin derivative, its preparation method, and its application.

[0006] A first aspect of this application provides a compound having the structure shown in Formula I, or a stereoisomer, solvate, metabolite, pharmaceutically acceptable salt, eutectic, or prodrug thereof:

[0007] ;

[0008] In the formula, R1 and R2 are each independently selected from hydrogen, hydroxyl, carboxyl, substituted or unsubstituted C1-C6 alkyl, -CONH-R3, -COO-R4;

[0009] Wherein, in -CONH-R3, R3 is a substituted or unsubstituted C1-C5 alkyl group, and the substituent may be any one of cyano, halogen, alkyl C1-C5 alkoxy, C1-C5 alkyl sulfonyl, di(C1-C5 alkyl)amino, nitrogen-containing heterocyclic group, aryl, and C1-C5 heteroaryl.

[0010] In the -COO-R4, R4 is selected from any one of substituted or unsubstituted aryl C1-C5 alkyl groups and nitrogen-containing saturated heterocyclic C1-C5 alkyl groups.

[0011] In some embodiments, the nitrogen-containing heterocyclic group in -CONH-R3 includes any one of pyrrolidinyl, oxopyrrolidinyl, morpholinyl, C1–C4 alkylpiperazinyl, C1–C4 alkyl-substituted imidazolyl, and C1–C4 alkoxyphenyl.

[0012] In some embodiments, in -COO-R4, R4 is a substituted or unsubstituted aryl C1-C5 alkyl group, wherein the substituent group may be any one of morpholino, piperazine, pyrrolidinyl, piperidinyl, pyrimidinyl, or morpholino.

[0013] In some embodiments, in -COO-R4, R4 is a nitrogen-containing saturated heterocyclic C1-C5 alkyl group, wherein the nitrogen atom of the nitrogen-containing saturated heterocyclic group is optionally replaced by an aminopyrimidinyl group or a C1-C4 alkylpyrimidinyl group.

[0014] In some embodiments, R1 and R2 are each independently selected from hydrogen or any of the following groups;

[0015] , , , , , , , , , , , , ;

[0016] Where n or m is independently selected from any positive integer from 1 to 6.

[0017] In some embodiments, the compound comprises any one of formulas 4a-16a and 4b-16b.

[0018] The second aspect of this application provides a method for preparing a compound having the structure shown in Formula I as in the first aspect, comprising: mixing the compound shown in Formula 3 with an amine chain, triethylamine and N,N-dimethylformamide, subjecting it to an amide condensation reaction, and then purifying it to obtain the compound;

[0019] ;

[0020] In the formula, H or -COOH are selected independently.

[0021] A third aspect of this application provides a pharmaceutical composition whose active ingredient comprises a compound having the structure shown in Formula I, or a stereoisomer thereof, solvate, metabolite, pharmaceutically acceptable salt, cocrystal, or prodrug.

[0022] In some embodiments, the pharmaceutical composition further comprises topotecan.

[0023] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0024] The fourth aspect of this application provides the use of a compound having the structure shown in Formula I as in the first aspect, or a stereoisomer, solvate, metabolite, pharmaceutically acceptable salt, cocrystal, or prodrug thereof, in the preparation of a tyrosine DNA phosphodiesterase 1 (TDP1) inhibitor.

[0025] The fifth aspect of this application provides the use of compounds having the structure shown in Formula I as in the first aspect, their stereoisomers, solvates, metabolites, pharmaceutically acceptable salts, cocrystals or prodrugs, or pharmaceutical compositions as described in any of the third aspects, in the preparation of antitumor drugs.

[0026] In some embodiments, the tumor includes, but is not limited to,

[0027] This application includes at least the following beneficial effects:

[0028] This application is the first to discover that purino[1,2-a]carbazole derivative compounds exhibit good inhibitory activity against TDP1, with compound 16b showing the best inhibitory effect on TDP1, and its IC50 value being [missing information]. 50 The value was 1.52 ± 0.34 μM. Furthermore, this application, through the combined use of compound 16b and topotecan (TPT), found that they exhibited a synergistic effect in HeLa cells, enhancing the antitumor effect. Specifically, this combination significantly enhanced TPT-induced DNA damage, induced apoptosis in a dose-dependent manner, caused S-phase cell cycle arrest, and inhibited cell migration, invasion, and colony formation. In in vivo experiments, the combination of TPT and compound 16b also showed a significant inhibitory effect on mouse cervical cancer xenografts. The purino[1,2-a]carbazole derivative compound of this application, as a promising TDP1 inhibitor, can be used for antitumor therapy. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0030] Figure 1 The images show the TDP1 inhibitory activity detection and molecular docking analysis of compound 3b of this application. In the images, A is the fluorescence intensity-time curve of the fluorescent DNA strand in the presence of different concentrations of 3b; B is the effect of different concentrations of 3b on the rate of fluorescence intensity change; and C is the dose-response curve of the TDP1 inhibition rate of 3b.

[0031] Figure 2 The above diagram shows the TDP1 inhibitory activity assay of compound 16b of this application. In the diagram, A is the fluorescence intensity-time curve of the fluorescent DNA strand in the presence of different concentrations of 16b; B is the effect of different concentrations of 16b on the rate of fluorescence intensity change; and C is the dose-response curve of the TDP1 inhibition rate of 16b.

[0032] Figure 3 The diagram shows the synergistic effect of compound 16b and TPT, where A is a bar graph of the enzyme-DNA complex immunoassay; B shows the effect of 16b, TPT alone and in combination on HeLa cell viability; C is the combination index graph; and D is the dose reduction index graph.

[0033] Figure 4 This paper presents the effects of compound 16b combined with TPT on the colony formation, migration, and invasion capabilities of HeLa cells. A shows representative crystal violet staining images (top) and microscopic images (bottom) of HeLa cell colonies after different treatments; B is a quantitative bar chart of the number of HeLa cell colonies in the colony formation experiment; C is a representative image of the wound closure process of HeLa cells in the scratch healing experiment; D shows the quantitative analysis results of the wound healing rate; E is a representative staining image of HeLa cells after transmembrane invasion in the Transwell invasion experiment; F is a quantitative bar chart of the relative number of invading HeLa cells. Statistical significance is indicated as follows: ns indicates no statistical significance; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.

[0034] Figure 5This is an in vivo anti-tumor experiment of compound 16b in combination with topotecan in this application. Among them, A shows the body weight changes of mice in each group during the drug administration period; B shows the tumor volume changes of mice in each group during the drug administration period. The experimental data are expressed as mean ± standard deviation. ns indicates no statistical difference, * indicates 0.01 < P < 0.05, and ** indicates 0.001 < P < 0.01; C shows the physical pictures of xenograft tumors of mice in each group after the drug administration; D shows the masses of the dissected xenograft tumors of mice in each group after the drug administration. The experimental data are expressed as mean ± standard deviation. ns indicates no statistical difference, *** indicates 0.0001 < P < 0.001, and **** indicates P < 0.0001; E shows the HE staining histopathological feature diagrams of cervical cancer xenograft tumors in different drug administration groups. Scale bars: 200 μm, 50 μm. Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of this application more clear and understandable, the following further details this application in combination with the attached drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0036] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic heterocyclic group, in which one or more heteroatomic ring members (ring-forming atoms) in at least one ring are each independently selected from oxygen (O), sulfur (S), and nitrogen (N).

[0037] The term "stereoisomer" refers to an isomer generated by the different arrangements of atoms in space in a molecule, including cis-trans isomers, enantiomers, and conformational isomers.

[0038] The term "solvate" refers to a substance formed by a stoichiometric or non-stoichiometric solvent in which a compound of the present invention or its salt is combined with intermolecular non-covalent forces. When the solvent is water, it is a hydrate.

[0039] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers, excipients, diluents, binders, fillers and other auxiliary materials, as well as anti-tumor drugs.

[0040] The term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" described in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially in humans.

[0041] The term "eutectic" refers to a crystal formed by the bonding of an active pharmaceutical ingredient (API) and a eutectic formation compound (CCF) through hydrogen bonding or other non-covalent bonds. Both API and CCF are solids in their pure states at room temperature, and a fixed stoichiometric ratio exists between the components. A eutectic is a multi-component substance, encompassing both binary eutectics formed between two neutral solids and multi-component eutectics formed between a neutral solid and a salt or solvate.

[0042] The term "pharmaceutically acceptable carrier" refers to a carrier that can be used to prepare a pharmaceutical composition. These carriers are generally safe, non-toxic, and not biologically or otherwise undesirable, and include carriers that are pharmaceutically acceptable to animals and humans.

[0043] All chemical reagents and starting materials used in this application were purchased from commercial suppliers and were of analytical grade; unless otherwise specified, all reagents were used directly without further purification. Nuclear magnetic resonance (NMR) spectra were determined on a spectrometer manufactured by Brookbyspin Ltd. at operating frequencies of 400 MHz, 500 MHz, and 600 MHz, with tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) data were obtained on a Waters SYNAPT G2-XS quadrupole-time-of-flight mass spectrometer (Milford, Massachusetts, USA) and a Shimadzu liquid chromatography-ion trap-time-of-flight mass spectrometer (Waltham, Massachusetts, USA, Thermo Fisher Scientific), respectively; high-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu LC-20AB liquid chromatograph. The mobile phase consisted of an aqueous phase (containing 0.1% formic acid) and an acetonitrile phase. Gradient elution was used, and the volume percentage of the acetonitrile phase gradually increased from 60% to 100% during the analysis. The flow rate was kept constant at 1.0 mL / min. The chromatographic peak area was quantitatively analyzed using the area normalization method.

[0044] The general synthetic method for purino[1,2-a]carbazoline derivatives disclosed in this application has the following synthetic route:

[0045] ;

[0046] The structural formulas of compounds 4a~16a and 4b~16b are as follows:

[0047] .

[0048] The specific synthetic route includes: reacting tryptamine and paraformaldehyde in an acetic acid-methanol mixed solution to obtain intermediate 1; intermediate 1 undergoes a catalyst-free redox condensation reaction with ethyl 4-fluoro-3-nitrobenzoate or ethyl 3-fluoro-4-nitrobenzoate to generate fused polyhexane ring benzimidazole compounds 2a and 2b; 2a and 2b are further hydrolyzed under alkaline conditions to obtain carboxyl-containing compounds 3a and 3b; and a series of compounds 4a~16a and 4b~16b are finally obtained through subsequent amide condensation reactions.

[0049] The above synthetic route will be described in detail below with reference to specific embodiments.

[0050] Example 1: General synthetic method for compounds 4a-16a and 4b-16b

[0051] 1. Synthesis of Intermediate 1

[0052] Intermediate 1 was synthesized according to the previously reported method. 3 mmol of tryptophan was dissolved in a mixed solvent of methanol (MeOH) and acetic acid (volume ratio 1:5), and 3.6 mmol of paraformaldehyde was added. The mixture was refluxed for 6 h. After the reaction was complete, the methanol was removed from the system under reduced pressure, and a large amount of distilled water was added. The pH of the system was adjusted to 8-9 with 2 mol / L sodium hydroxide solution. At this point, a large amount of solid precipitated. This solid was filtered, and the filter cake was dried to obtain intermediate 1, which required no further purification. The product was a pale yellow solid with a yield of 97%.

[0053] 1 H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 3.86 (s, 1H), 2.98 (t, J = 5.6 Hz, 1H), 2.59 (t, J = 5.4 Hz, 1H). 13 C NMR (101MHz, DMSO-d6) δ 135.93, 134.67, 127.71, 120.62, 118.55, 117.59, 111.22,107.37, 43.84, 43.11, 22.65.

[0054] 2. General synthetic methods for compounds 2a and 2b

[0055] The synthesis of compounds 2a and 2b followed the methods reported in the literature. Intermediate 1 (1.0 g, 5.86 mmol) was added to a reaction flask with ethyl 4-fluoro-3-nitrobenzene or ethyl 3-fluoro-4-nitrobenzene (0.5 g, 2.35 mmol). The solid powder was thoroughly ground and mixed under solvent-free conditions, and the mixture was stirred and heated to 200 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, heating was stopped, and the system was allowed to cool naturally to room temperature. 20 mL of ammonia water was added to form a suspension, which was then filtered under reduced pressure. The resulting filter cake was purified by column chromatography using a dichloromethane (DCM) / tetrahydrofuran (THF) mixture (v / v 100:1) as the eluent to obtain the target product.

[0056] Compound 2a was obtained with a yield of 65% and a purity of 99.1% (HPLC). The mp value was > 300 °C, and its structural formula is as follows:

[0057]

[0058] The structure of compound 2a is confirmed as follows:

[0059] 1 H NMR (600 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.18 (s, 1H), 7.83 (d, J =8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.43 (d, J =8.2 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.55 (t, J =7.3 Hz, 2H), 4.34 (q, J = 7.0 Hz, 2H), 3.34 (t, J = 7.3 Hz, 2H), 1.35 (t, J =7.1 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 166.67, 148.15, 147.83, 138.48,135.17, 126.10, 124.64, 124.27, 124.08, 123.43, 120.23, 119.99, 118.74,114.97, 112.77, 111.98, 61.02, 42.07, 20.31, 14.79. HRMS (ESI; m / z). Calcdfor C 20 H18 N3O2, [M + H] + , 332.1399; found, 332.1401.

[0060] Compound 2b was obtained in 65% yield and with a purity of 96.5% (HPLC). The mp value was > 300 °C. Its structural formula is as follows:

[0061]

[0062] The structure of compound 2b is confirmed as follows:

[0063] 1 H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.23 ​​(d, J = 1.0 Hz, 1H), 7.91 (dd, J = 8.4, 1.4 Hz, 1H), 7.71-7.63 (m, 2H), 7.45 (d, J = 8.2 Hz, 1H),7.24 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 4.53 (t, J = 7.2 Hz,2H,), 4.35 (q, J = 7.1 Hz, 2H), 3.40-3.32 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 166.77, 147.26, 143.82, 138.81, 138.44, 126.09,124.62, 124.21, 124.05, 123.89, 120.43, 120.38, 119.94, 114.75, 112.75,110.33, 60.98, 42.13, 20.27, 14.73. HRMS (ESI; m / z). Calcd for C 20 H 18 N3O2, [M +H] + , 332.1394; found, 332.1396.

[0064] 3. General synthetic methods for compounds 3a and 3b

[0065] 2a / 2b (0.40 g, 1.21 mmol) and lithium hydroxide monohydrate (0.15 g, 3.62 mmol) were added to a reaction flask, followed by the addition of a mixed solvent of tetrahydrofuran, methanol, and water (volume ratio 4:1:1). The reaction system was refluxed and stirred for 1.5 h. After the reaction was complete, the reaction solution was concentrated by rotary evaporation to remove the organic solvent. Then, the solid precipitated by water was added to the flask, and 1 mol / L hydrochloric acid solution was added dropwise under stirring until no more precipitate was formed. The reaction mixture was filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain compound 3a / 3b.

[0066] Compound 3a was obtained with a yield of 65% and a purity of 96.8% (HPLC). The mp value was > 300 °C, and its structural formula is as follows:

[0067] .

[0068] The structure of compound 3a is confirmed as follows:

[0069] 1 H NMR (400 MHz, TFA-d) δ 8.68 (s, 1H), 8.57 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.67 - 7.59 (m, 1H), 7.43 (t, J = 7.5 Hz, 1H), 4.94 (t, J = 7.9 Hz, 2H), 3.82(t, J = 7.9 Hz, 2H). 13 C NMR (101 MHz, TFA-d) δ 171.06, 143.73, 140.65,134.26, 131.49, 128.89, 128.61, 126.88, 124.75, 123.63, 122.33, 120.14,116.23, 115.07, 113.64, 112.61, 42.56, 19.25. HRMS (ESI; m / z). Calcd forC 18 H 12 N3O2, [M + H] + , 302.0930; found, 302.0923.

[0070] Compound 3b was obtained in 65% yield with a purity of 98.1% (HPLC). The mp value was > 300 °C. Its structural formula is as follows:

[0071] .

[0072] The structure of compound 3b is confirmed as follows:

[0073] 1 H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.24 (s, 1H), 7.94 (d, J =8.2 Hz, 1H), 7.71 (dd, J = 16.9, 8.1 Hz, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.26(d, J = 7.3 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H), 4.57 (m, 2H), 3.38 - 3.03 (m,2H). 13 C NMR (101 MHz, DMSO-d6) δ 168.14, 146.51, 141.76, 138.60, 138.06,125.96, 125.36, 124.78, 124.69, 123.63, 120.44, 120.15, 119.84, 115.77,112.91, 110.60, 42.27, 20.21. HRMS (ESI; m / z). Calcd for C 18 H 14 N3O2, [M + H] + ,304.1081; found, 304.1089.

[0074] 4. General synthetic methods for compounds 4a~16a and 4b~16b

[0075] Add 3a / 3b (0.10 g, 0.33 mmol) and thionyl chloride (SOCl2, 4 mL) to the reaction flask, then add 1 drop of N,N-dimethylformamide (DMF). Reflux the mixture and stir for 1 h, then concentrate to dryness by rotary evaporation. After concentration, add the amine chain, anhydrous triethylamine (Et3N), and anhydrous N,N-dimethylformamide to the reaction residue and stir at room temperature for 1 h. Quench the reaction with 25 mL of water, then extract with dichloromethane (25 mL × 3). Combine the organic phases, wash with 20 mL of saturated brine, dry with anhydrous sodium sulfate (Na2SO4), and concentrate to dryness under reduced pressure. Purify the crude product by column chromatography using dichloromethane / methanol (20:1 v / v) to elute the target products 4a–16a and 4b–16b.

[0076] Example 2 Compound 4a

[0077] Following the general synthetic method described in Example 1 above, a yellow solid compound 4a was obtained after purification by column chromatography with a yield of 65% and a purity of 95.9% (HPLC). Its specific structural formula is as follows:

[0078] .

[0079] The structure of compound 4a is confirmed as follows:

[0080] 1 H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.87 (t, J = 5.6 Hz, 1H), 8.14 (d, J = 0.9 Hz, 1H), 7.77 (dd, J = 8.5, 1.5 Hz, 1H), 7.69 (dd, J = 13.7,8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 1H), 7.27-7.18 (m, 1H), 7.11 (t, J = 7.5Hz, 1H), 4.53 (t, J = 7.3 Hz, 2H), 3.55 (q, J = 6.3 Hz, 2H), 3.39 (d, J = 7.3Hz, 2H), 2.83 (t, J = 6.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.27,147.43, 146.51, 138.36, 135.11, 128.40, 126.13, 124.83, 124.14, 121.58,120.20, 119.90 (2C), 118.47, 114.55, 112.73, 109.95, 42.02, 36.06, 20.34,18.12. HRMS (ESI; m / z). Calcd for C 21 H 18 N5O, [M + H] + , 356.1511; found, 356.1514.

[0081] Example 3 Compound 4b

[0082] Following the general synthetic method described in Example 1 above, a yellow solid compound 4b was obtained after purification by column chromatography with a yield of 65% and a purity of 95.9% (HPLC). Its specific structural formula is as follows:

[0083] .

[0084] The structure of compound 4b is confirmed as follows:

[0085] Yellow solid with yield of 65% and purity of 95.3% (HPLC). 1 H NMR(400 MHz, DMSO-d6) δ 12.16 (s, 1H), 8.89 (t, J = 5.6 Hz, 1H), 8.22 (d, J =1.1 Hz, 1H), 7.84 (dd, J = 8.5, 1.5 Hz, 1H), 7.67 (dd, J = 8.1, 4.9 Hz, 2H), 7.45 (d, J = 8.2 Hz, 1H), 7.28 - 7.19 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 4.52(t, J = 7.2 Hz, 2H), 3.55 (q, J = 6.3 Hz, 2H), 3.37 (d, J = 6.8 Hz, 2H), 2.83(t, J = 6.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.39, 146.85, 143.78,138.34, 137.59, 128.06, 126.12, 124.84, 124.08, 122.42, 120.17, 119.95,119.89, 118.37, 114.37, 112.71, 109.98, 42.08, 36.07, 20.30, 18.11. HRMS(ESI; m / z). Calcd for C 21 H 18 N5O, [M + H] + , 356.1511; found, 356.1514.

[0086] Example 4 Compound 5a

[0087] Following the general synthetic method described in Example 1 above, a yellow solid compound 5a was obtained after purification by column chromatography with a yield of 65% and a purity of 95.7% (HPLC). Its specific structural formula is as follows:

[0088] .

[0089] The structure of compound 5a is confirmed as follows:

[0090] 1 H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.71 (t, J = 5.6 Hz, 1H), 8.11 (d, J = 1.0 Hz, 1H), 7.74 (dd, J = 8.4, 1.5 Hz, 1H), 7.70 (s, 1H), 7.69- 7.64 (m, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.29 - 7.19 (m, 1H), 7.16 - 7.05(m, 1H), 4.53 (t, J = 7.3 Hz, 2H), 3.56 (dd, J = 12.6, 6.8 Hz, 2H), 3.39 (d,J = 7.3 Hz, 2H), 2.68 - 2.53 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.09,147.38, 146.43, 138.36, 135.10, 128.65, 127.38 (q, J = 277.9 Hz), 126.01,124.84, 124.13, HRMS (ESI; m / z). Calcd for C 21 H 18 N4OF3, [M + H] + , 399.1433; found, 399.1435.

[0091] Example 5 Compound 5b

[0092] Following the general synthetic method described in Example 1 above, a yellow solid compound 5b was obtained after purification by column chromatography with a yield of 65% and a purity of 99.5% (HPLC). Its specific structural formula is as follows:

[0093] .

[0094] The structure of compound 5b is confirmed as follows:

[0095] 1 H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.71 (t, J = 5.2 Hz, 1H), 8.18 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.45 (d, J= 8.2 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 4.52 (t, J= 7.2 Hz, 2H), 3.55 (dd, J = 12.8, 6.6 Hz, 2H), 3.36 (t, J = 7.1 Hz, 2H),2.71 - 2.54 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 167.21, 146.82, 143.78,138.35, 137.51, 128.32,127.28 (q, J = 277.8 Hz), 126.13, 124.86, 124.06,122.35, 120.16, 119.87, 118.30, 114.34, 112.71, 109.93, 42.07, 33.38 (q, J=3.86 Hz), 33.09 (q, J=26.5 Hz), 20.31. HRMS (ESI; m / z). Calcd forC 21 H 18 N4OF3, [M + H] + , 399.1433; found, 399.1437.

[0096] Example 6 Compound 6a

[0097] Following the general synthetic method described in Example 1 above, a yellow solid compound 6a was obtained by column chromatography purification with a yield of 65% and a purity of 99.7% (HPLC). The specific structural formula is as follows:

[0098] .

[0099] The structure of compound 6a is confirmed as follows:

[0100] 1 H NMR (600 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.50 (s, 1H), 8.15 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 8.3 Hz, 2H), 7.46 (d, J = 8.2 Hz, 1H),7.23 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 4.53 (t, J = 7.3 Hz, 2H),3.50 (s, 3H), 3.38 (t, J = 7.3 Hz, 2H), 3.30 (s, 2H), 1.22 (s, 2H). 13C NMR(151 MHz, DMSO-d6) δ 166.95, 147.25, 146.26, 138.34, 135.03, 128.94, 126.12,124.87, 124.03, 121.74, 121.54, 119.90, 118.41, 114.39, 112.72, 109.89,71.10, 58.52, 41.99, 29.46, 20.32. HRMS (ESI; m / z). Calcd for C 21 H 21 N4O2, [M +H] + , 361.1665; found, 361.1668.

[0101] Example 7 Compound 6b

[0102] Following the general synthetic method described in Example 1 above, a yellow solid compound 6b was obtained after purification by column chromatography with a yield of 65% and a purity of 97.4% (HPLC). The mp temperature was 281.7-283.1 °C. The specific structural formula is as follows:

[0103] .

[0104] The structure of compound 6b is confirmed as follows:

[0105] 1 H NMR (600 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.55 (s, 1H), 8.23 ​​(s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.67-7.60 (m, 2H), 7.46 (d, J = 8.1 Hz, 1H), 7.23(t, J = 7.4 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 4.50 (t, J = 6.9 Hz, 2H), 3.56-3.46 (m, 4H), 3.35 (t, J = 6.8 Hz, 2H), 3.30 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.14, 146.73, 143.79, 138.36, 137.38, 128.65, 126.15, 124.93, 124.03, 122.48, 120.15, 119.83, 118.39, 114.26, 112.70, 109.78, 71.12, 58.40,42.05, 39.57, 20.32. HRMS (ESI; m / z). Calcd for C 21 H 21 N4O2, [M + H] + , 361.1665;found, 361.1656.

[0106] Example 8 Compound 7a

[0107] Following the general synthetic method described in Example 1 above, a yellow solid compound 7a was obtained by column chromatography purification with a yield of 65% and a purity of 98.6% (HPLC). The specific structural formula is as follows:

[0108] .

[0109] The structure of compound 7a is confirmed as follows:

[0110] 1 H NMR (500 MHz, DMSO-d6) δ 12.12 (s, 1H), 8.74 (s, 1H), 8.09 (d, J =33.9 Hz, 1H), 7.72 (m, 3H), 7.45 (s, 1H), 7.24 (s, 1H), 7.11 (s, 1H), 4.52(s, 2H), 3.74 (s, 2H), 3.43 (s, 2H), 3.07 (s, 3H), 1.29 (m, 2H). 13 C NMR (126MHz, DMSO-d6) δ 167.22, 147.41, 146.47, 138.37, 135.10, 128.56, 126.12,124.84, 124.13, 121.57, 120.20, 119.90, 118.46, 114.54, 112.74, 109.91,53.54, 42.02, 41.30, 33.97, 20.35. HRMS (ESI; m / z). Calcd for C21 H 20 N4O3NaS, [M+ Na] + , 431.1154; found, 431.1154.

[0111] Example 9 Compound 7b

[0112] Following the general synthetic method described in Example 1 above, a yellow solid compound 7b was obtained after purification by column chromatography with a yield of 65% and a purity of 98.9% (HPLC). The specific structural formula is as follows:

[0113] .

[0114] The structure of compound 7b is confirmed as follows:

[0115] 1 H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.75 (s, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 7.9 Hz, 1H), 7.23 (t, J = 7.1 Hz, 1H), 7.10 (t, J = 6.9 Hz, 1H), 4.52 (t, J = 6.3 Hz, 2H), 3.73 (d, J = 5.3 Hz, 2H), 3.44 (d, J = 5.9 Hz, 2H), 3.36 (s, 3H), 3.07 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 167.32, 146.83, 143.78, 138.35, 137.55,128.22, 126.13, 124.85, 124.08, 122.41, 120.18, 119.88, 118.35, 114.36,112.71, 109.96, 53.50, 42.08, 41.28, 33.90, 20.31. HRMS (ESI; m / z). Calcd forC 21 H 21 N4O3S, [M + H] + , 409.1334; found, 409.1334.

[0116] Example 10 Compound 8a

[0117] Following the general synthetic method described in Example 1 above, a yellow solid compound 8a was obtained by column chromatography purification with a yield of 65% and a purity of 98.5% (HPLC). The mp temperature was 261.7-262.8 °C. The specific structural formula is as follows:

[0118] .

[0119] The structure of compound 8a is confirmed as follows:

[0120] 1 H NMR (600 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.54 (t, J = 5.3 Hz, 1H), 8.12 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.69-7.59 (m, 2H), 7.44 (d, J = 8.2Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.50 (t, J = 7.3Hz, 2H), 3.44-3.25 (m, 4H), 2.46-2.37 (m, 2H), 2.25 (s, 6H), 1.86-1.67 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 166.93, 147.26, 146.26, 138.38, 135.08,129.22, 126.16, 124.91, 124.09, 121.60, 120.19, 119.85, 118.37, 114.43,112.74, 109.80, 57.06, 45.10, 42.02, 38.12, 27.18, 20.36. HRMS (ESI; m / z).Calcd for C 23 H 26 N5O, [M + H] + , 338.2137; found, 338.2141.

[0121] Example 11 Compound 8b

[0122] Following the general synthetic method described in Example 1 above, a yellow solid compound 8b was obtained after purification by column chromatography with a yield of 65% and a purity of 99.7% (HPLC). The mp temperature was 285.4-286.8 °C. The specific structural formula is as follows:

[0123] .

[0124] The structure of compound 8b is confirmed as follows:

[0125] 1 H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 8.54 (t, J = 5.5 Hz, 1H), 8.18 (d, J = 1.1 Hz, 1H), 7.81 (dd, J = 8.4, 1.5 Hz, 1H), 7.64 (t, J = 8.9Hz, 2H), 7.46 (d, J = 8.2 Hz, 1H), 7.26-7.19 (m, 1H), 7.15-7.05 (m, 1H), 4.51(t, J = 7.3 Hz, 2H), 3.38-3.28 (m, 4H), 2.29 (t, J = 7.1 Hz, 2H), 2.15 (s, 6H), 1.77–1.59 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 166.95, 146.70, 143.77,138.33, 137.29, 128.93, 126.14, 124.90, 124.04, 122.37, 120.17, 119.85,118.22, 114.26, 112.71, 109.81, 57.54, 45.68, 42.06, 38.40, 27.67, 20.32.HRMS (ESI; m / z). Calcd for C 23 H 26 N5O2, [M + H] + , 388.2132; found, 388.2133.

[0126] Example 12 Compound 9a

[0127] Following the general synthetic method described in Example 1 above, a yellow solid compound 9a was obtained after purification by column chromatography with a yield of 65% and a purity of 97.1% (HPLC). The chromatography temperature was 281.1-282.2 °C. The specific structural formula is as follows:

[0128] .

[0129] The structure of compound 9a is confirmed as follows:

[0130] 1 H NMR (600 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.12 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.67 (dd, J = 11.2, 8.5 Hz, 2H), 7.46(d, J = 8.2 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 4.60-4.46 (m, 2H), 3.42-3.30 (m, 4H), 2.49-2.40 (m, 6H), 1.80-1.71 (m, 2H),1.69 (s, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 166.81, 147.23, 146.22, 138.36,135.08, 129.30, 126.15, 124.91, 124.08, 121.50, 120.18, 119.86, 118.34,114.41, 112.72, 109.76, 54.14, 54.09, 42.01, 38.62, 28.86, 23.58, 20.35. HRMS(ESI; m / z). Calcd for C 25 H 28 N5O, [M + H] + , 414.2294; found, 414.2296.

[0131] Example 13 Compound 9b

[0132] Following the general synthetic method described in Example 1 above, a yellow solid compound 9b was obtained after purification by column chromatography with a yield of 65% and a purity of 97.5% (HPLC). The mp temperature was 277.9-278.4 °C. The specific structural formula is as follows:

[0133] .

[0134] The structure of compound 9b is confirmed as follows:

[0135] 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.63 (t, J = 5.4 Hz, 1H), 8.20 (d, J = 1.1 Hz, 1H), 7.83 (dd, J = 8.4, 1.5 Hz, 1H), 7.67 (t, J = 9.1Hz, 2H), 7.49 (d, J = 8.2 Hz, 1H), 7.30 - 7.22 (m, 1H), 7.20 - 7.07 (m, 1H), 4.54 (t, J = 7.3 Hz, 2H), 3.38 (dd, J = 8.8, 5.6 Hz, 4H), 2.54 (dt, J = 4.9,2.4 Hz, 2H), 2.47 (t, J = 5.8 Hz, 4H), 1.82-1.75 (m, 2H), 1.72 (dt, J = 6.4,3.1 Hz, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 166.92, 146.70, 143.78, 138.33,137.30, 128.93, 126.14, 124.89, 124.04, 122.32, 120.17, 119.85, 118.19,114.27, 112.71, 109.80, 54.24, 54.09, 42.06, 38.71, 28.78, 23.58, 20.32. HRMS(ESI; m / z). Calcd for C 25 H 28 N5O, [M + H] + , 414.2289; found, 414.2288.

[0136] Example 14 Compound 10a

[0137] Following the general synthetic method described in Example 1 above, a yellow solid compound 10a was obtained after purification by column chromatography with a yield of 65% and a purity of 97.2% (HPLC). The mp temperature was 292.8-294.3 °C. The specific structural formula is as follows:

[0138] .

[0139] The structure of compound 10a is confirmed as follows:

[0140] 1H NMR (600 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.42 (t, J = 5.4 Hz, 1H), 8.09 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 8.3 Hz, 2H), 7.43 (d, J= 8.2 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.50 (t, J= 7.3 Hz, 2H), 3.35 (t, J = 7.2 Hz, 4H), 3.29-3.21 (m, 4H), 2.22 (t, J = 8.0Hz, 2H), 2.01-1.85 (m, 2H), 1.79-1.65 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ174.48, 166.86, 147.27, 146.27, 138.36, 135.09, 129.19, 126.15, 124.90,124.09, 121.50, 120.19, 119.88, 118.39, 114.44, 112.73, 109.78, 46.84, 42.01,37.42, 30.97, 27.48, 20.35, 18.02. HRMS (ESI; m / z). Calcd for C 25 H 26 N5O, [M +H] + , 428.2087; found, 428.2088.

[0141] Example 15 Compound 10b

[0142] Following the general synthetic method described in Example 1 above, a yellow solid compound 10b was obtained after purification by column chromatography with a yield of 65% and a purity of 97.2% (HPLC). The mp temperature was 280.5-282.3 °C. The specific structural formula is as follows:

[0143] .

[0144] The structure of compound 10b is confirmed as follows:

[0145] 1H NMR (600 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.44 (t, J = 5.4 Hz, 1H), 8.21-8.13 (m, 1H), 7.78 (t, J = 14.7 Hz, 1H), 7.62 (t, J = 8.9 Hz, 2H), 7.45-7.39 (m, 1H), 7.24-7.16 (m, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.49 (t, J = 7.2Hz, 2H), 3.39-3.30 (m, 4H), 3.29-3.19 (m, 4H), 2.21 (t, J = 8.0 Hz, 2H),1.97-1.85 (m, 2H), 1.77-1.65 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 174.48,166.95, 146.73, 143.81, 138.34, 137.35, 128.81, 126.15, 124.92, 124.03,122.34, 120.16, 119.85, 118.25, 114.26, 112.70, 109.84, 46.83, 42.07, 37.39,30.97, 27.48, 20.32, 18.01. HRMS (ESI; m / z). Calcd for C 25 H 26 N5O2, [M + H] + ,428.2087; found, 428.2089.

[0146] Example 16 Compound 11a

[0147] Following the general synthetic method described in Example 1 above, a yellow solid compound 11a was obtained by column chromatography purification with a yield of 65% and a purity of 95.6% (HPLC). The specific structural formula is as follows:

[0148] .

[0149] The structure of compound 11a is confirmed as follows:

[0150] 1H NMR (600 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.51 (t, J = 5.1 Hz, 1H), 8.11 (s, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.67 (d, J= 8.1 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.14 (s,1H), 7.11 (t, J = 7.4 Hz, 1H), 6.77 (s, 1H), 4.53 (t, J = 7.2 Hz, 2H), 3.97(t, J = 6.9 Hz, 2H), 3.40-3.36 (m, 2H), 3.33 (d, J = 6.0 Hz, 2H), 2.30 (s,3H), 2.05-1.89 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 167.17, 147.25, 146.27,144.03, 138.34, 135.04, 129.11, 126.61, 126.11, 124.86, 124.05, HRMS (ESI; m / z). Calcd for C 25 H 25 N6O, [M + H] + , 425.2090;found, 425.2094.

[0151] Example 17 Compound 11b

[0152] Following the general synthetic method described in Example 1 above, a yellow solid compound 11b was obtained after purification by column chromatography with a yield of 65% and a purity of 98.9% (HPLC). The specific structural formula is as follows:

[0153] .

[0154] The structure of compound 11b is confirmed as follows:

[0155] 1H NMR (500 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.54 (t, J = 4.9 Hz, 1H), 8.20 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.66-7.59 (m, 2H), 7.45 (d, J = 8.2Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.17-7.06 (m, 2H), 6.76 (s, 1H), 4.52 (t,J = 7.2 Hz, 2H), 3.96 (t, J = 7.0 Hz, 2H), 3.36 (t, J = 7.2 Hz, 2H), 3.32(dd, J = 12.5, 6.5 Hz, 2H), 2.29 (s, 3H), 1.98 (dd, J = 13.7, 6.8 Hz, 2H). 13 CNMR (126 MHz, DMSO-d6) δ 167.28, 146.74, 144.06, 143.77, 138.34, 137.51,137.38, 128.74, 126.54, 126.14, 124.91, 124.04, 122.44, 120.17, 119.86,118.32, 114.28, 112.70, 109.85, 43.51, 42.07, 37.11, 30.74, 20.32, 13.02.HRMS (ESI; m / z). Calcd for C 25 H 25 N6O, [M + H] + , 425.2090; found, 425.2094.

[0156] Example 18 Compound 12a

[0157] Following the general synthetic method described in Example 1 above, a yellow solid compound 12a was obtained by column chromatography purification with a yield of 65% and a purity of 99.1% (HPLC). The mp temperature was 293.5-294.7 °C. The specific structural formula is as follows:

[0158] .

[0159] The structure of compound 12a is confirmed as follows:

[0160] 1H NMR (600 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.38 (t, J = 5.3 Hz, 1H), 8.10 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.63 (d, J= 7.9 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.08 (t, J= 7.4 Hz, 1H), 4.49 (t, J = 7.3 Hz, 2H), 3.57 (s, 4H), 3.44 (dd, J = 12.6,6.4 Hz, 2H), 3.35 (t, J = 7.2 Hz, 2H), 2.58-2.43 (m, 4H), 1.16 (s, 2H). 13 CNMR (126 MHz, DMSO-d6) δ 166.94, 147.28, 146.29, 138.38, 135.09, 129.19,126.16, 124.90, 124.08, 121.59, 120.18, 119.84, 118.38, 114.43, 112.73,109.82, 66.69, 58.03, 53.82, 42.01, 37.17, 20.35. HRMS (ESI; m / z). Calcd forC 24 H 25 N5O2, [M + H] + , 416.2087; found, 416.2088.

[0161] Example 19 Compound 12b

[0162] Following the general synthetic method described in Example 1 above, a yellow solid compound 12b was obtained after purification by column chromatography with a yield of 65% and a purity of 98.8% (HPLC). The mp temperature was 298.5-299.9 °C. The specific structural formula is as follows:

[0163] .

[0164] The structure of compound 12b is confirmed as follows:

[0165] 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.44 (t, J = 5.2 Hz, 1H), 8.18 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 8.2 Hz, 2H), 7.46 (d, J= 8.2 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 4.51 (t, J= 7.2 Hz, 2H), 3.66 - 3.53 (m, 4H), 3.44 (s, 2H), 3.39-3.32 (m, 4H), 2.44 (s,4H). 13 C NMR (101 MHz, DMSO-d6) δ 167.04, 146.72, 143.76, 138.33, 137.35,128.79, 126.14, 124.89, 124.06, 122.40, 120.18, 119.86, 118.28, 114.29,112.71, 109.84, 66.70, 57.99, 53.82, 42.06, 37.14, 20.31. HRMS (ESI; m / z).Calcd for C 24 H 26 N5O2, [M + H] + , 416.2081; found, 414.2078.

[0166] Example 20 Compound 13a

[0167] Following the general synthetic method described in Example 1 above, a yellow solid compound 13a was obtained by column chromatography purification with a yield of 65% and a purity of 95.8% (HPLC). The mp temperature was 271.5-272.7 °C. The specific structural formula is as follows:

[0168] .

[0169] The structure of compound 13a is confirmed as follows:

[0170] 1H NMR (600 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.46 (s, 1H), 8.08 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 4.1 Hz, 1H), 7.63 (d, J = 3.5 Hz, 1H),7.43 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.1 Hz, 1H),4.50 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 7.3 Hz, 2H), 3.32 (d, J = 5.9 Hz, 2H), 2.43-2.24 (m, 8H), 2.15 (s, 3H), 1.77-1.62 (m, 2H), 1.20 (s, 2H). 13 C NMR (126MHz, DMSO-d6) δ 166.90, 147.24, 146.23, 138.36, 135.07, 129.28, 126.15,124.90, 124.09, 121.58, 120.19, 119.87, 118.35, 114.42, 112.73, 109.79,56.03, 54.82, 52.68, 45.70, 42.03, 38.42, 26.73, 20.36. HRMS (ESI; m / z).Calcd for C 26 H 31 N6O, [M + H] + , 443.2559; found, 443.2555.

[0171] Example 21 Compound 13b

[0172] Following the general synthetic method described in Example 1 above, a yellow solid compound 13b was obtained after purification by column chromatography with a yield of 65% and a purity of 99.0% (HPLC). The mp temperature was 275.9-277.2 °C. The specific structural formula is as follows:

[0173] .

[0174] The structure of compound 13b is confirmed as follows:

[0175] 1H NMR (500 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.55 (t, J = 5.1 Hz, 1H), 8.18 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 9.4 Hz, 2H), 7.45 (d, J= 8.2 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 4.52 (t, J= 7.2 Hz, 2H), 3.55 - 3.26 (m, 8H), 2.44 (s, 3H), 2.28 (s, 3H), 1.83 - 1.64(m, 2H), 1.39 - 1.20 (m, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 166.99, 146.70,143.78, 138.33, 137.32, 128.87, 126.14, 124.91, 124.03, 122.38, 120.16,119.85, HRMS (ESI; m / z). Calcd for C 26 H 32 N6O, [M +H] + , 443.2559; found, 443.2564.

[0176] Example 22 Compound 14a

[0177] Following the general synthetic method described in Example 1 above, a yellow solid compound 14a was obtained by column chromatography purification with a yield of 65% and a purity of 96.2% (HPLC). The mp temperature was 283.9-285.6 °C. The specific structural formula is as follows:

[0178] .

[0179] The structure of compound 14a is confirmed as follows:

[0180] 1H NMR (600 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.52 (t, J = 5.3 Hz, 1H), 8.11 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.63-7.69 (m, 2H), 7.46 (d, J = 8.2Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.90-6.86 (m,2H), 6.78 (d, J = 8.3 Hz, 1H), 4.52 (t, J = 7.3 Hz, 2H), 3.74 (s, 3H), 3.72 (s, 3H), 3.53 (dd, J = 13.2, 6.8 Hz, 2H), 3.38 (t, J = 7.3 Hz, 2H), 2.84 (t,J = 7.3 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 166.89, 149.09, 147.70, 147.26,146.26, 138.37, 135.07, 132.57, 129.28, 126.16, 124.91, 124.09, 121.57,120.97, 120.19, 119.87, 118.37, 114.43, 113.05, 112.73, 112.40, 109.78,55.99, 55.83, 42.00, 41.68, 35.26, 20.35. HRMS (ESI; m / z). Calcd forC 28 H 27 N4O3, [M + H] + , 467.2083; found, 467.2086.

[0181] Example 23 Compound 14b

[0182] Following the general synthetic method described in Example 1 above, a yellow solid compound 14b was obtained by column chromatography purification with a yield of 65% and a purity of 99.0% (HPLC). The specific structural formula is as follows:

[0183] .

[0184] The structure of compound 14b is confirmed as follows:

[0185] 1 H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.56 (t, J = 5.4 Hz, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 8.9 Hz, 2H), 7.45 (d, J= 8.2 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.87 (d, J= 8.1 Hz, 2H), 6.78 (d, J = 8.1 Hz, 1H), 4.51 (t, J = 7.2 Hz, 2H), 3.73 (d, J= 7.7 Hz, 6H), 3.52 (dd, J = 13.2, 6.7 Hz, 2H), 3.36 (t, J = 7.2 Hz, 3H), 2.83 (t, J = 7.3 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 167.00, 149.05, 147.67,146.71, 143.75, 138.34, 137.33, 132.63, 128.91, 126.15, 124.92, 124.04,122.40, 120.96, 120.16, 119.86, 118.27, 114.26, 113.03, 112.70, 112.36,109.82, 55.97, 55.79, 42.06, 41.68, 35.25, 20.32. HRMS (ESI; m / z). Calcd forC 28 H 27 N4O3, [M + H] + , 467.2083; found, 467.2087.

[0186] Example 24 Compound 15a

[0187] Following the general synthetic method described in Example 1 above, a yellow solid compound 15a was obtained after purification by column chromatography with a yield of 65% and a purity of 95.7% (HPLC). The specific structural formula is as follows:

[0188] .

[0189] The structure of compound 15a is confirmed as follows:

[0190] 1 H NMR (400 MHz, CDCl3+ 1 drop CD3OD) δ 8.09 (s, 1H), 7.98 (dd, J =8.5, 1.4 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.46(d, J = 4.44 (t, J = 7.4Hz, 2H), 3.96-3.77 (m, 4H), 3.40 (t, J = 7.4 Hz, 2H), 3.24 - 3.10 (m, 4H). 13 CNMR (101 MHz, CDCl3 + 1 drop CD3OD) δ 166.97, 151.29, 147.60, 146.16, 138.16,134.21, 129.99, 127.27, 125.63, 124.82, 124.81, 124.45, 123.20, 120.48,119.39, 118.05, 115.54, 115.21, 112.52, 111.43, 66.86, 66.85, 49.10, 42.08,20.43. HRMS (ESI; m / z). Calcd for C 29 H 27 N4O3, [M + H] + , 479.2083; found, 479.2086.

[0191] Example 25 Compound 15b

[0192] Following the general synthetic method described in Example 1 above, a yellow solid compound 15b was obtained after purification by column chromatography with a yield of 65% and a purity of 96.9% (HPLC). The specific structural formula is as follows:

[0193] .

[0194] The structure of compound 15b is confirmed as follows:

[0195] 1 H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.62(d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.38(d, J = 8.5 Hz, 1H), 7.35-7.28 (m, 2H), 7.18 (t, J = 7.4 Hz, 1H), 6.95 (d, J= 8.3 Hz, 2H), 5.33 (s, 2H), 4.45 (t, J = 7.2 Hz, 2H), 3.88 (s, 5H), 3.42 (t,J = 7.2 Hz, 3H), 3.19 (s, 4H). 13 C NMR (101 MHz, CDCl3) δ 167.15, 151.19,146.80, 142.43, 138.07, 137.82, 129.82, 127.42, 125.61, 124.88, 124.63,124.60, 123.30, 120.76, 120.38, 119.29, 115.58, 114.78, 112.41, 108.81,66.82, 66.69, 49.12, 42.01, 20.34. HRMS (ESI; m / z). Calcd for C 29 H 27 N4O3, [M + H] + , 479.2083; found, 479.2081.

[0196] Example 26 Compound 16a

[0197] Following the general synthetic method described in Example 1 above, a yellow solid compound 16a was obtained by column chromatography purification with a yield of 65% and a purity of 97.9% (HPLC). Its specific structural formula is as follows:

[0198] .

[0199] The structure of compound 16a is confirmed as follows:

[0200] 1H NMR (400 MHz, CDCl3 + 1 drop CD3OD) δ 8.10 (s, 1H), 7.94 (dd, J =8.5, 1.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.46(d, J = 8.3 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 5.39(s, 1H), 4.50 (t, J = 5.7 Hz, 2H), 4.46 (t, J = 7.4 Hz, 2H), 3.62-3.50 (m,4H), 3.40 (t, J = 7.4 Hz, 2H), 2.84 (t, J = 5.7 Hz, 2H), 2.68-2.55 (m, 4H), 2.31 (s, 3H). 13 C NMR (101 MHz, CDCl3 + 1 drop CD3OD) δ 166.97, 166.30,163.22, 163.13, 147.84, 146.77, 138.12, 134.38, 125.67, 124.72, 124.39,124.15, HRMS (ESI; m / z).Calcd forC 29 H 31 N8O2, [M + H] + , 523.2570; found, 523.2571.

[0201] Example 27 Compound 16b

[0202] Following the general synthetic method described in Example 1 above, a yellow solid compound 16b was obtained by column chromatography purification with a yield of 65% and a purity of 97.9% (HPLC). Its specific structural formula is as follows:

[0203] .

[0204] The structure of compound 16b is confirmed as follows:

[0205] 1H NMR (400 MHz, CDCl3 + 1 drop CD3OD) δ 8.25 (d, J = 0.8 Hz, 1H), 7.90 (dd, J = 8.5, 1.4 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.3Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.21-7.15 (m, 1H), 7.05 (t, J = 7.5 Hz,1H), 5.32 (s, 1H), 4.40 (t, J = 5.7 Hz, 2H), 4.34 (t, J = 7.3 Hz, 2H), 3.54 -3.41 (m, 4H), 3.30 (t, J = 7.3 Hz, 2H), 2.75 (t, J = 5.7 Hz, 2H), 2.62-2.48(m, 4H), 2.35-2.14 (m, 3H). 13 C NMR (101 MHz, CDCl3 + 1 drop CD3OD) δ 167.02,164.93, 162.80, 161.96, 146.97, 142.68, 138.07, 137.97, 125.64, 124.77,124.66, 124.30, HRMS (ESI; m / z).Calcd for C 29 H 31 N8O2, [M + H] + , 523.2570; found, 523.2574.

[0206] Experimental Example 1: Detection of TDP1 and TOP1 Inhibitory Activity

[0207] This experiment tested the tyrosine DNA phosphodiesterase 1 (TDP1) inhibitory activity and topoisomerase 1 (TOP1) inhibitory activity of the compounds shown in Examples 2-27 above. The specific methods are as follows.

[0208] 1. Discovery of TDP1 inhibitory activity and binding conformation analysis of compound 3b

[0209] A TDP1 biosensor based on a hydrolyzable fluorophore-quencher coupling was developed. This study evaluated the inhibitory activity of compound 3b, based on a purino[1,2-a]carbazolinite skeleton, against TDP1. The inhibitory effect of 3b on TDP1 was quantitatively analyzed by comparing the fluorescence rise slope after the addition of 3b with that of the dimethyl sulfoxide (DMSO) control group.

[0210] The results are as follows Figure 1 As shown in Figures A and B, compound 3b exhibits real-time inhibition of the hydrolysis process of the TDP1 biosensor, demonstrating strong inhibitory activity against TDP1. Dose-response curves plotted based on 11 concentration gradients show that compound 3b has a half-maximal inhibitory concentration (IC50) for TDP1. 50 The value was 24.28 ± 0.74 μM (e.g. Figure 1 (as shown in C).

[0211] 2. TDP1 inhibitory activity and TOP1 inhibitory activity test

[0212] The inhibitory activity of TOP1 was semi-quantitatively evaluated using a DNA loosening assay, and the inhibitory activity of each compound at a concentration of 100 μM was compared with that of 50 μM topotecan (TPT, positive control).

[0213] The inhibitory activity of TDP1 was determined using a fluorescence assay. The specific experimental method included diluting the linear oligonucleotide (5′- / 5,6 FAM-aacgtcagggtcttcc-BHQ1 / -3′) to a final concentration of 105 nM using a reaction buffer (10 mM Tris-HCl, 50 mM potassium chloride, 1 mM EDTA, pH 7.5). A TDP1 dilution buffer was prepared concurrently, consisting of 200 nM TDP1, 10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, and 2 mM dithiothreitol (DTT), pH 7.5. Fluorescence detection was performed using a multi-mode microplate reader (Infinite 200 Pro, TECAN Group, Switzerland) and black 384-well plates (Costar), with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Add the following to each well sequentially: 20 μL of stock solution containing TDP1, 19 μL of buffer, 20 μL of dual-fluorescent labeled DNA strand, and 1 μL of the target compound or dimethyl sulfoxide (DMSO) at a predetermined concentration. Calculate the rate of fluorescence intensity increase over time; use the fluorescence rate increase of the DMSO group as a blank control and the fluorescence rate increase of the group without TDP1 enzyme as a positive control, and calculate the inhibition rate of the compound accordingly. In addition, set up multiple concentration gradients for each compound to determine its half-maximal inhibitory concentration (IC50). 50 The experimental results are expressed as half-maximal inhibitory concentration (IC50). 50 ± standard deviation (SD) is expressed as IC. 50 For concentrations greater than the specified concentration (μM), suramin (CAS No.: 129-46-4, Shanghai Maclean Biotechnology Co., Ltd.) was used as a positive control. EVO (CAS No.: 518-17-2, Shanghai Maclean Biotechnology Co., Ltd.) and RUT (CAS No.: 84-26-4, Shanghai Maclean Biotechnology Co., Ltd.) were used as controls.

[0214] The results are shown in Table 1.

[0215] Table 1:

[0216]

[0217] Note: ++++ indicates that the activity reaches more than 121% of topotecan, + indicates less than 40%, 0 indicates no inhibitory effect; nd indicates not detected.

[0218] The results above show that the tested compounds exhibited significant differences in TDP1 inhibitory activity: purino[1,2-a]carbazolin derivatives all showed some inhibitory activity, while a few analogs (such as 16a and 16b) showed strong inhibitory activity. Among them, 16b showed the strongest TDP1 inhibitory activity, with an IC50 of [missing information]. 50 The concentration was 1.52 ± 0.34 μM, as shown in Figure 2. Furthermore, the TOP1 inhibitory activity of this series of compounds was also tested. Data analysis showed that most compounds had no significant inhibitory effect on TOP1, suggesting that the core structure of this series of compounds may not be the dominant structure for TOP1 inhibitors. In summary, the experimental results confirm that 16b is a selective TDP1 inhibitor, and no inhibitory activity against TOP1 was detected.

[0219] Experimental Example 2: In vitro antitumor activity experiment

[0220] This study investigated the effects of compound 16b in combination with topotecan (TPT) in vitro, as well as its influence on the proliferation, migration, and invasion of HeLa cells. The specific findings include the following:

[0221] 1. Enzyme-DNA complex immunoassay (ICE) assay

[0222] Given that compound 16b possesses TDP1 inhibitory activity, this experiment employed an enzyme-DNA complex immunoassay (ICE) to investigate its effect on the formation of the topotecan (TPT)-induced topoisomerase 1 cleavage complex (TOP1cc) in HeLa cells. The specific experimental method is as follows:

[0223] HeLa cells in logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 6-well plates and cultured for 24 h. After complete cell adhesion, the medium was replaced with fresh medium containing different concentrations of the test compound, and incubation continued for 4 h. After treatment, 1 mL of DNAzol lysis buffer (Invitrogen Biosciences, USA) was added to each well to lyse the cells for 30 min; then 0.5 mL of anhydrous ethanol was added, mixed thoroughly, and the lysis buffer was transferred to a microcentrifuge tube and incubated overnight at −20 °C to precipitate. The precipitate was collected by centrifugation at 25 °C and 12000 × g for 10 min; the supernatant was discarded, and the precipitate was washed with 75% ethanol. The collected DNA was dissolved in 0.2 mL of 8 mM sodium hydroxide (NaOH) solution, and 2.5 μL of 1 M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was added to adjust the pH to 7.2; the cells were centrifuged again (25 °C, 12000 × g, 10 min), and the supernatant containing DNA was collected and quantified. Take 2 μg of DNA from each sample, dilute it with 30 μL of 25 mM sodium dihydrogen phosphate (NaH2PO4, pH 6.5), and spot it onto a nitrocellulose membrane. Block the membrane with 5% goat serum at room temperature, then add the corresponding primary antibody (1:1000 dilution) and incubate overnight at 4 °C. Wash twice with phosphate-buffered saline (PBS), then add horseradish peroxidase (HRP)-labeled secondary antibody (Wuhan Aibotek Biotechnology Co., Ltd., 1:5000 dilution) and incubate at 37 °C for 1 h. After further washing with PBS, detect the immune response signal using a high-sensitivity chemiluminescence (ECL) kit (Hefei Biosharp Co., Ltd.), and acquire images using a Tianneng 5200 chemiluminescence imaging system (Shanghai Tianneng Technology Co., Ltd.).

[0224] Experimental results are as follows Figure 3 As shown in Figure A, treatment with 50 μM TPT induces moderate levels of TOP1cc formation, while no significant TOP1cc immunospots are detected when 16b is used alone, indicating that 16b cannot directly induce TOP1cc formation. Notably, the density of TOP1cc-specific spots significantly increased after co-treatment with TPT and different concentrations of 16b (50 μM or 100 μM). This result may be attributed to the TDP1 inhibitory activity of 16b: by inhibiting the function of TDP1, it blocks the dissociation process of TPT-induced TOP1cc formation, thereby promoting TOP1cc accumulation.

[0225] 2. MTT assay and combined index determination

[0226] This experiment further investigated the effects of different concentrations of 16b combined with different doses of TPT on the survival rate of HeLa cells. The specific experimental methods are as follows:

[0227] Logarithmically growing cells were seeded at a density of 5000 cells / well in 96-well plates and incubated at 37 °C in a 5% CO2 saturated humidity incubator for 16 h. The test compounds were prepared as stock solutions in dimethyl sulfoxide (DMSO) and serially diluted with culture medium to six concentrations as required by the experiment. The final DMSO volume fraction in all treatment groups was ≤0.1%. All cells were purchased from the China Center for Type Culture Collection (CCC) in Wuhan.

[0228] After the cells had fully adhered to the culture medium, the original medium was discarded and replaced with medium containing the corresponding compound. A negative control group was added to medium containing the same final concentration of DMSO (≤0.1%) but without the test compound; a positive control group was also set up simultaneously. After incubating the cells for another 48 h, 20 μL of MTT solution (2.5 mg / mL) was added to each well, and the cells were incubated at 37 ℃ for 4 h. The medium was carefully aspirated, and 100 μL of DMSO was added to each well to dissolve the formazan crystals; the solution turned purple. The optical density (OD) value at 570 nm was measured using a multi-mode microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated by fitting the relationship between cell inhibition rate and drug concentration. 50 ).

[0229] In the combination drug experiment, culture media containing different concentrations of the test compounds were prepared in advance. Topotecan (TPT) was then further diluted to different concentrations to obtain combination culture media containing both drugs. The cell inhibition rate was detected using the same method described above. The combination index (CI) was calculated using CompuSyn 1.0 software (ComboSyn, Inc., USA) to assess the synergistic effect of the drugs, and dose reduction index (DRI) analysis was performed. The criteria for this method were: CI < 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect.

[0230] The expression results of the enzyme-DNA complex after combined use are shown in Figure 3B. TPT alone inhibited HeLa cell proliferation in a dose-dependent manner; however, the addition of 16b significantly enhanced TPT-induced cytotoxicity, and this enhancement was concentration-dependent. The quantitative verification results of the synergistic effect of 16b and TPT are shown in Figure 3C. At most tested concentrations of 16b, the CI values ​​for the combined use were between 0.3 and 1.0, confirming the synergistic effect of the combined drug administration. Specifically, a strong synergistic effect (CI < 0.3) was detected when 2 μM or 4 μM of 16b was combined with 0.1 μM or 0.2 μM of TPT, respectively. These CI values ​​corroborate the cell viability experiment results, confirming that the enhanced cytotoxicity after the combined use of TPT and 16b stems from a genuine synergistic effect, rather than a simple additive effect.

[0231] To further evaluate the clinical translational potential of the TPT / 16b combination therapy, this study conducted a dose reduction index (DRI) analysis. The results are as follows: Figure 3 As shown in D, this displays the logs of TPT and 16b when using combination therapy. 10 All (DRI) values ​​were positive, indicating that both drugs could achieve good dose reduction. Specifically, at high effect fractions (Fa = 0.5678~0.5726, corresponding to approximately 57% inhibition of HeLa cell proliferation), the logarithmic effect of TPT was... 10 The DRI values ​​reached a peak of 1.540–1.816, indicating that the combination therapy regimen brought the strongest dose-saving effect to TPT.

[0232] 3. Colony Formation Experiment

[0233] To evaluate the long-term antiproliferative effect of topotecan (TPT) in combination with compound 16b on HeLa cells, a colony formation assay was conducted. The specific experimental method is as follows:

[0234] HeLa cells were seeded at a density of 500 cells / well in 6-well plates and allowed to adhere statically. Once the cells had fully adhered and formed a small number of colonies, the medium was replaced with fresh medium containing the specified concentration of the compound, and cultured for another 9 days, changing the medium every 3 days. After culture, the cells were washed with phosphate-buffered saline (PBS), fixed with methanol for 10 min, and then stained with 0.1% methanol crystal violet for 30 min. After washing and drying, the culture plates were photographed against a white background to assess colony formation.

[0235] The results are shown in Figures A and B of Figure 4. Representative staining images and microscopic observations reveal that untreated HeLa cells formed large and dense cell colonies. TPT monotherapy only slightly reduced the size and density of these colonies. Treatment with 2 μM or 4 μM 16b alone did not significantly alter the morphology and distribution of the cell colonies compared to the control group. Notably, when TPT was used in combination with 16b (2 μM or 4 μM), both the number and size of HeLa cell colonies were significantly reduced, indicating a synergistic inhibitory effect on cell colony formation. Quantitative analysis further validated these findings: the relative colony formation rates in the experimental groups treated with 2 μM TPT in combination with 2 μM and 4 μM 16b were 0.50 and 0.33, respectively, both significantly lower than the TPT monotherapy group (relative colony formation rate = 0.75).

[0236] 4. Scratch healing test

[0237] To evaluate the effect of topotecan (TPT) in combination with compound 16b on HeLa cell migration, this invention conducted a scratch healing assay, the specific experimental methods of which are as follows:

[0238] HeLa cells were loaded at 5 × 10⁻⁶ 5 Cells were seeded at a density of cells / well in 6-well plates. After a dense monolayer of cells formed, uniform straight scratches were made on the monolayer using a 10 μL pipette tip. Exfoliated cells were washed with culture medium, and then fresh culture medium containing the test compound was added. Images of the scratched areas were acquired at 0 h, 24 h, 48 h, and 72 h post-drug administration. Suspended cells were aspirated and fresh culture medium was replaced before imaging to ensure a clear field of view.

[0239] The results of the scratch healing assay are shown in Figures 4C and 4D. The results indicate that TPT monotherapy significantly reduced the scratch healing rate of HeLa cells, while 16b monotherapy did not show a significant inhibitory effect on scratch healing. Importantly, the combination of TPT and 16b significantly inhibited HeLa cell scratch healing compared to TPT monotherapy: the scratch healing rate at 72 h was 0.46% in the group treated with 2 μM TPT and 2 μM 16b; the synergistic inhibitory effect was even more significant in the group treated with 2 μM TPT and 4 μM 16b, with a scratch healing rate of only 0.28% at 72 h, significantly lower than the healing rate of the TPT monotherapy group (0.54%) at the same time point. These data suggest that the combination of 16b and TPT can effectively block the migration process of HeLa cells.

[0240] 5. Transwell invasion experiment

[0241] To evaluate the effect of the combination of topotecan (TPT) and compound 16b on the invasive ability of HeLa cells, the present invention conducted a Transwell invasion assay, the specific experimental method of which is as follows:

[0242] Matrigel (Corning, USA) was diluted 1:8 with serum-free medium. 50 μL of the diluted Matrigel was added to the upper chamber of a 24-well Transwell chamber and incubated at 37 °C for 5 h to allow gelation. 2 × 10⁻⁶ μL of the diluted Matrigel was then added to the upper chamber of a 24-well Transwell chamber. 4 One cell was resuspended in 200 μL of serum-free medium and seeded into the upper chamber; 500 μL of medium containing 20% ​​fetal bovine serum (FBS) was added to the lower chamber. After incubation for 48 h, uninvaded cells on the surface of the upper chamber were gently wiped away with a cotton swab. Cells that had invaded the lower chamber through the matrix gel were fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet, and observed under a microscope with the membrane side of the chamber facing upwards.

[0243] Transwell invasion test results are as follows Figure 4 As shown in Figures E and F, treatment with different concentrations of TPT or 16b alone only showed a weak inhibitory effect on the invasive ability of HeLa cells. In stark contrast, the combination of TPT and 16b produced a significant synergistic inhibitory effect, which increased with increasing 16b concentration. Specifically, in the experimental group treated with 2 μM TPT and 4 μM 16b, the relative invasive number of HeLa cells was only 0.185, a decrease of approximately 81.5% compared to the blank control group. These results indicate that the combination of TPT and 16b can significantly weaken the invasive ability of HeLa cells.

[0244] Experimental Example 3: In vivo antitumor activity experiment

[0245] This experiment used HeLa cervical cancer xenograft mice to investigate the antitumor effect of TPT combined with 16b. The specific experiment is as follows.

[0246] 1. Experimental Methods

[0247] (1) Construction of nude mouse xenograft tumor model

[0248] Female BALB / C-nu / nu nude mice aged 4-5 weeks and weighing 15-18 g were randomly divided into 6 groups, with 6 mice in each group. HeLa cells in the logarithmic growth phase were collected by trypsin digestion, centrifugation, and resuspended in serum-free DMEM medium to a concentration of 5 × 10⁻⁶. 7Cells / mL were injected subcutaneously into the right forelimb axilla of each nude mouse. After approximately 3 weeks of standard feeding, when the tumor volume grew to approximately 120 mm³, the drug was administered intraperitoneally every 2 days to establish a nude mouse xenograft tumor model.

[0249] (2) Drug administration

[0250] The test compound was dissolved in a mixture of DMSO, polyethylene glycol 300 (PEG300), Tween 80, and physiological saline (5:40:5:50, volume ratio). Topotecan (TPT) was administered at a dose of 0.3 mg / kg, and compound 16b was administered at two different doses (5 mg / kg and 10 mg / kg). Tumor volume and mouse body weight were continuously monitored and recorded during administration.

[0251] After the experiment, mice were euthanized by cervical dislocation, and the tumors were dissected and weighed. The tumor volume was calculated using the formula: Tumor volume = (short diameter)² × long diameter / 2. The tumor tissue was fixed in 4% paraformaldehyde solution, then sequentially dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. After trimming the paraffin blocks, the sections were cooled at −20 °C to a thickness of 3–5 μm. The sections were then spread in 40 °C warm water, retrieved onto glass slides, and dried at 60 °C for 2 h before subsequent hematoxylin-eosin (HE) staining.

[0252] 2. Experimental Results

[0253] The statistical results of body weight changes in mice in each group during the drug administration period are as follows: Figure 5 As shown in Figure A, administration of topotecan (TPT) at a dose of 0.3 mg / kg resulted in a significant decrease in body weight in mice, indicating that TPT has some toxicity to mice. In contrast, treatment with two different doses (5 mg / kg and 10 mg / kg) of compound 16b only resulted in a slight decrease in body weight in mice, suggesting that 16b has low inherent toxicity. Notably, no further decrease in body weight was observed after co-administration of 16b and TPT, indicating that 16b has good biocompatibility and is a well-tolerated drug.

[0254] Growth curve analysis of tumor volume changes over time, such as Figure 5As shown in Figure B, the tumor volume in the blank control group continued to increase rapidly throughout the experimental period; the 0.3 mg / kg TPT monotherapy group showed a moderate tumor-suppressing effect, with the tumor growth rate significantly slower than that of the blank control group. However, the tumor volume change trends in the 5 mg / kg and 10 mg / kg 16b monotherapy groups were not significantly different from those in the blank control group, and no significant tumor-suppressing activity was observed. In stark contrast, when TPT was combined with 5 mg / kg and 10 mg / kg 16b, respectively, the tumor growth rate in mice was significantly inhibited over time. Furthermore, the tumor images and tumor weight measurements at the experimental endpoint showed that the tumor weight in the TPT and 16b combined treatment groups was significantly lower than that in the blank control group (e.g., ...). Figure 5 (As shown in C and D in the figure), and the difference was statistically significant. These results collectively confirm that the combination of TPT and 16b exerts a superior tumor-suppressive effect compared to monotherapy or no treatment.

[0255] The results of hematoxylin-eosin (HE) staining analysis are as follows: Figure 5 As shown in E, the tumor cells in the blank control group were densely arranged, and the area of ​​the tumor parenchyma was large; while in the combined treatment groups of TPT with 5 mg / kg and 10 mg / kg 16b, extensive focal necrosis areas (eosinophilic deep-stained areas) appeared in the tumor tissue, and a small number of inflammatory cells were infiltrated in the stroma, suggesting that the drug can induce apoptosis of tumor cells.

[0256] In summary, this invention provides a purino[1,2-a]carbazolin derivative, its preparation method, and its applications. Specifically, this invention is the first to discover that the purino[1,2-a]carbazolin derivative compound 3b exhibits moderate inhibitory activity against TDP1. Through structure-based modification of 3b, a series of derivatives were synthesized, among which 16b became the strongest TDP1 inhibitor, with an IC50 value of [missing information]. 50 The effective value was 1.52 ± 0.34 μM. 16b and TPT synergistically enhanced the antitumor effect in HeLa cells. This combination significantly enhanced TPT-induced DNA damage, induced apoptosis in a dose-dependent manner, caused S-phase cell cycle arrest, and inhibited cell migration, invasion, and colony formation. In in vivo experiments, the combination of TPT and 16b also showed a significant inhibitory effect on mouse cervical cancer xenografts. These results highlight that 16b, as a promising TDP1 inhibitor, exhibits potent synergistic antitumor activity when combined with TPT, thus providing a new therapeutic strategy for cervical cancer treatment.

[0257] The above is a detailed description of the preferred embodiments of this application, but this application is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A compound having the structure shown in Formula I, or a stereoisomer, solvate, metabolite, pharmaceutically acceptable salt, cocrystal, or prodrug thereof: ； In the formula, R1 and R2 are each independently selected from hydrogen, hydroxyl, carboxyl, substituted or unsubstituted C1-C6 alkyl, -CONH-R3, -COO-R4; in, In the -CONH-R3, R3 is a substituted or unsubstituted C1-C5 alkyl group, and the substituent can be any one of cyano, halogen, alkyl C1-C5 alkoxy, C1-C5 alkyl sulfonyl, di(C1-C5 alkyl)amino, nitrogen-containing heterocyclic group, aryl, and C1-C5 heteroaryl. In the -COO-R4, R4 is selected from any one of substituted or unsubstituted aryl C1-C5 alkyl groups and nitrogen-containing saturated heterocyclic C1-C5 alkyl groups.

2. The compound according to claim 1, characterized in that: In the -CONH-R3, the nitrogen-containing heterocyclic group includes any one of pyrrolidinyl, oxopyrrolidinyl, morpholinyl, C1–C4 alkylpiperazinyl, C1–C4 alkyl-substituted imidazolyl, and C1–C4 alkoxyphenyl.

3. The compound according to claim 1, characterized in that: In the -COO-R4, R4 is a substituted or unsubstituted aryl C1-C5 alkyl group, wherein the substituent group may be any one of morpholino, piperazine, pyrrolidinyl, piperidinyl, pyrimidinyl, or pyrimidinyl.

4. The compound according to claim 1, characterized in that: In the -COO-R4, R4 is a nitrogen-containing saturated heterocyclic C1-C5 alkyl group, and the nitrogen atom of the nitrogen-containing saturated heterocyclic group may optionally be replaced by an aminopyrimidinyl group or a C1-C4 alkylpyrimidinyl group.

5. The compound according to any one of claims 1 to 4, characterized in that: R1 and R2 are each independently selected from hydrogen or any of the following groups; 、 、 、 、 、 、 、 、 、 、 、 、 ； Where n or m is independently selected from any positive integer from 1 to 6.

6. A method for preparing a compound having the structure shown in Formula I as claimed in any one of claims 1 to 5, characterized in that: The compound shown in Formula 3 was mixed with an amine chain, triethylamine, and N,N-dimethylformamide, and purified after an amide condensation reaction. ； In the formula, R1 and R2 are independently selected from H or -COOH.

7. A pharmaceutical composition, characterized in that: The active ingredient comprises a compound having the structure shown in Formula I according to any one of claims 1 to 5, or a stereoisomer, solvate, metabolite, pharmaceutically acceptable salt, cocrystal, or prodrug thereof.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition also contains topotecan.

9. The use of a compound having the structure shown in Formula I as claimed in any one of claims 1 to 5, or a stereoisomer, solvate, metabolite, pharmaceutically acceptable salt, cocrystal, or prodrug thereof, in the preparation of a tyrosine DNA phosphodiesterase 1 inhibitor.

10. The use of a compound having the structure shown in Formula I as claimed in any one of claims 1 to 5, its stereoisomers, solvates, metabolites, pharmaceutically acceptable salts, eutectics or prodrugs, or the pharmaceutical composition of any one of claims 7 to 8 in the preparation of an antitumor drug.

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