A compound as a PARP-NAMPT dual-target inhibitor and its application

Abstract

The present invention relates to a compound that acts as a PARP-NAMPT dual-target inhibitor, its preparation method, and use. The compound that acts as a PARP-NAMPT dual-target inhibitor described herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof. The present invention also relates to a preparation method and use of the compound of formula (I). The compound of the present invention has strong PARP-NAMPT enzyme inhibitory activity and significant anti-tumor activity. Therefore, the compound of the present invention can be used to prepare a therapeutic agent for treating malignant tumors and diseases related to differentiation and proliferation. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a compound serving as a PARP-NAMPT dual-target inhibitor and applications thereof. Background Art

[0002] Cancer seriously threatens human life and health. With the increasing incidence and mortality rates worldwide year by year, cancer is not only the main cause of death in humans, but also a global public health issue.

[0003] The phenomenon of "synthetic lethality" in cancer has been recognized as a reliable strategy for anticancer therapy. Targeting DNA repair defects has become a proven cancer treatment strategy. However, cancers with DNA repair defects often rely on backup DNA repair pathways, which are targetable "Achilles' heel" of cancer cells and the basis of synthetic lethality. PARP is a family of proteins involved in the repair of DNA single-strand breaks (SSBs), exerting their function by binding to sites of DNA damage. The success of poly (ADP-ribose) polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers demonstrates the synthetic lethality effect. PARP inhibitors (such as olaparib, rucaparib, niraparib, fluzopanib, pamipanib, and talazopanib) have been approved for the treatment of breast and ovarian cancer in patients with BRCA mutations.

[0004] Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the conversion of nicotinamide (NAM) to nicotinaminde mononucleotide (NMN), regulating the levels of NAD, an essential energy substance in mammalian cells. It is the rate-limiting enzyme in the NAD biosynthesis pathway and plays a crucial role in cellular physiology. Studies have shown that NAMPT is closely associated with the development and progression of tumors, making it a key new target in anti-tumor drug research. Tumor cells have high NAD consumption and metabolic rates, making them more dependent on NAD than normal cells and more susceptible to NAMPT inhibitors. In tumor cells, NAD acts as an essential coenzyme in the synthesis of various tumor-essential substances. Furthermore, NAD can significantly reduce the levels of reactive oxygen species in the environment, protecting tumor cells. NAMPT plays a crucial role in angiogenesis and inducing the production of vascular endothelial growth factor.

[0005] Studies have shown that PARP inhibitors and NAMPT inhibitors have a synergistic synthetic lethal effect, and their combined use will expand the clinical application of PARP inhibitors. However, this multi-component administration form often has defects such as complex dosage setting, drug-drug interactions, pharmacokinetic differences, and low patient compliance. Multi-target drugs can act on a single chemical molecule at multiple targets in the disease network at the same time, and can produce a synergistic effect on each target, making the total effect greater than the sum of the single effects to achieve the best therapeutic effect. It can also simplify the dosing regimen, improve patient compliance, and avoid drug-drug interactions and problems caused by different absorption, distribution, metabolism, and excretion processes between components. Therefore, the design of PARP-NAMPT dual-target inhibitors is expected to become a new strategy for the development of new anti-tumor drugs. Summary of the Invention

[0006] The first object of the present invention is to provide a compound as a PARP-NAMPT dual-target inhibitor and a pharmaceutically acceptable salt thereof.

[0007] The second object of the present invention is to provide the use of a compound as a PARP-NAMPT dual-target inhibitor in the preparation of an anti-tumor drug. Experimental results show that the compound not only has strong inhibitory activity against both PARP-1 and NAMPT, but also has significant in vitro anti-breast cancer cell proliferation activity, which is stronger than the positive drug Olaparib and is independent of the cell BRCA mutation status. Its IC 50 The values ​​are between 0.09-30.05 μM. The preferred compounds can significantly inhibit the growth of human breast cancer cell line MDA-MB-468 nude mouse transplanted tumors and have excellent in vivo tumor growth inhibitory activity.

[0008] The third object of the present invention is to provide a method for preparing the compound as a PARP-NAMPT dual-target inhibitor and a pharmaceutically acceptable salt thereof.

[0009] In order to achieve the above first purpose, the technical solution adopted by the present invention is:

[0010] A compound that acts as a PARP-NAMPT dual-target inhibitor, simultaneously targeting PARP and NAMPT, exerting a dual effect of inhibiting both PARP and NAMPT. It is characterized by having the structure represented by the following general formula (I), as well as its optical isomers, diastereoisomers, and racemic mixtures, and pharmaceutically acceptable salts thereof;

[0011]

[0012] in:

[0013] X is an oxygen atom or a sulfur atom;

[0014] R1 is any one of the following structures:

[0015] ;

[0016] R2 is any one of the following structures:

[0017] .

[0018] "Pharmaceutically acceptable salts" refer to salt forms of the compounds of formula (I) that are therapeutically effective and non-toxic. Many such salts are known in the art. Cationic salts formed at any acidic group (e.g., carboxyl) or anionic salts formed at any basic group (e.g., amino) are known in the art. For example, cationic salts include salts of alkali metals (e.g., sodium and potassium) and alkaline earth metals (magnesium and calcium), as well as organic salts (e.g., ammonium salts). Anionic salts can also be conveniently obtained by treating the basic forms of (I) and (II) with the corresponding acids, such as inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-propanedioic acid, ethanesulfonic acid, toluenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, and the like. Furthermore, a skilled artisan may choose one salt over another based on factors such as solubility, stability, ease of formulation, etc. The determination and optimization of these salts are within the experience of the skilled artisan.

[0019] In the above-mentioned compound as a PARP-NAMPT dual-target inhibitor and its pharmaceutically acceptable salt, the pharmaceutically acceptable salt does not contain crystalline water, or contains one or more crystalline waters.

[0020] As used herein, the terms "optical isomer," "enantiomer," "diastereomer," "racemate," etc., define all possible stereoisomeric forms of the compounds of the present invention or physiological derivatives. Unless otherwise indicated, the chemical designations of the compounds of the present invention encompass mixtures of all possible stereochemical forms, such mixtures containing all diastereomers and enantiomers of the basic structural molecule, as well as substantially pure individual isomeric forms of the compounds of the present invention, i.e., containing less than 10%, preferably less than 5%, particularly less than 2%, and most preferably less than 1% of other isomers.

[0021] The compound of formula (I) may exist in other protected forms or derivative forms, which are obvious to those skilled in the art and are intended to be included within the scope of the present invention.

[0022] As a preferred embodiment of the present invention, the compound as a PARP-NAMPT dual-target inhibitor is preferably:

[0023] Compound A1: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

[0024] Compound A2: 1-(4-((5-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

[0025] Compound A3: 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(1-((4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonyl)piperidin-4-yl)benzamide;

[0026] Compound A4: N-(1-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperidin-4-yl)-4-(3-(pyridin-3-ylmethyl)ureido)benzenesulfonamide;

[0027] Compound A5: 1-(4-((7-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,7-diazaspiro[4.4]nonan-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

[0028] Compound A6: 1-(4-((6-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,6-diazaspiro[3.3]heptan-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

[0029] Compound A7: 1-(4-((9-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea;

[0030] Compound A8: 2-fluoro-N-(2-((N-methyl-4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonamido)ethyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide;

[0031] Compound A9: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)thiourea;

[0032] Compound A10: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide;

[0033] Compound A11: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-((4-fluoropyridin-3-yl)methyl)urea;

[0034] Compound A12: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)thieno[2,3-c]pyridine-2-carboxamide.

[0035] Their structural formulas and NMR mass spectrometry data are shown in Table 1 below:

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] The advantages of the present invention are:

[0044] 1. Compounds tested for enzyme inhibition and in vitro anti-tumor cell proliferation activity showed that the preferred compounds of the present invention not only had strong inhibitory activity against both PARP-1 and NAMPT, but also had significant in vitro anti-breast cancer cell proliferation activity. The preferred compounds also had excellent in vivo tumor growth inhibition activity.

[0045] 2. Compared with the existing technology, the present invention opens up a new path and provides a new strategy for in-depth research and development of new structural types of anti-tumor drugs. DETAILED DESCRIPTION

[0046] The present invention is described below by way of specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, the spirit and scope of the present invention being limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention. The raw materials and reagents used in the present invention are all commercially available. Example 1

[0047] Preparation of compound: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea (A1)

[0048] Synthesis route:

[0049]

[0050] Reagents and conditions: (a) triethylamine, dichloromethane, room temperature, 2 hours, yield 75%; (b) hydrogen, palladium on carbon, dichloromethane / methanol, room temperature, overnight, yield 92%; (c) triphosgene, triethylamine, dichloromethane, room temperature, 2 hours; (d) triethylamine, dichloromethane, room temperature, overnight, yield 85%; (d) trifluoroacetic acid, dichloromethane, room temperature, 2 hours, yield 97%; (e) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours, yield 65%.

[0051] (I) Preparation of Intermediate 2a: tert-Butyl 4-(4-nitrophenyl)sulfonyl)piperazine-1-carboxylate

[0052] 4-Nitrobenzenesulfonyl chloride 1 (2.2 g, 10.0 mmol) (commercially available) and 1-tert-butyloxycarbonyl-piperazine (2.2 g, 12.0 mmol) (commercially available) were dissolved in 50 mL of dichloromethane. Triethylamine (3.0 g, 30.0 mmol) was added dropwise and stirred at room temperature for 2 hours. After the reaction was completed, distilled water was added to terminate the reaction, and the mixture was extracted with dichloromethane. The organic phase was dried and concentrated, and then separated by column chromatography to obtain 3.4 g of a white solid in a 92% yield. 1 H-NMR (600 MHz, DMSO- d6) δ: 8.43 (d, J = 8.9 Hz,2H), 7.99 (d, J = 8.9 Hz, 2H), 3.43-3.35 (m, 4H), 2.97-2.89 (m, 4H), 1.32 (s,9H).

[0053] (II) Preparation of Intermediate 3a: tert-Butyl 4-(4-aminophenyl)sulfonyl)piperazine-1-carboxylate

[0054] Intermediate 2a (2 g, 5.4 mmol) was dissolved in 25 mL of a 2 / 1 dichloromethane / methanol mixture. Palladium on carbon (57 mg, 0.5 mmol) was added and stirred overnight under hydrogen. After completion of the reaction, the reaction mixture was filtered and the solvent was removed by distillation under reduced pressure to obtain 1.9 g of a white solid (95% yield). 1 H-NMR (600 MHz, DMSO- d 6) δ: 7.32 (d, J =8.6 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 6.09 (s, 2H), 3.39-3.34 (m, 4H), 2.75-2.67 (m, 4H), 1.33 (s, 9H).

[0055] (III) Preparation of Intermediate 4a: tert-Butyl 4-((4-(3-(pyridin-4-ylmethyl)ureido)phenyl)sulfonyl)piperazine-1-carboxylate

[0056] Triphosgene (131 mg, 0.4 mmol) was dissolved in 5 mL of dichloromethane and stirred at 0°C. Intermediate 3a (341 mg, 1.0 mmol) and triethylamine (146 mg, 1.4 mmol) were then dissolved in 5 mL of dichloromethane. This solution was then added to the triphosgene solution and stirred at room temperature for 2 h. 3-Methylaminopyridine (120 mg, 1.11 mmol) and triethylamine (146 mg, 1.4 mmol) were dissolved in 5 mL of dichloromethane and slowly added to the reaction mixture. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the solvent was removed by filtration and vacuum distillation. The crude product was purified by column chromatography to yield 559 mg of a white solid in 85% yield. 1 H-NMR (600 MHz, DMSO- d 6 ) d : 9.41 (s, 1H), 8.49 (d, J = 6.1 Hz, 2H), 7.64(d, J= 9.0 Hz, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 6.1 Hz, 2H), 7.01 (t, J = 5.9 Hz, 1H), 4.33 (d, J = 6.2 Hz, 2H), 3.41-3.34 (m, 4H), 2.82-2.75 (m,4H), 1.32 (s, 9H).

[0057] (IV) Preparation of Intermediate 5a: 1-(4-(piperazin-1-ylsulfonyl)phenyl)-3-(pyridin-4-ylmethyl)urea

[0058] Intermediate 4a (475 mg, 1.00 mmol) was dissolved in 10 mL of a 1 / 1 mixture of trifluoroacetic acid and dichloromethane and stirred at room temperature for 2 h. After the reaction, the solution was concentrated by vacuum distillation and purified by column chromatography to obtain 364 mg of a white solid in a 97% yield. 1 HNMR (600 MHz, DMSO- d 6 ) d : 9.67 (s, 1H), 8.72 (s, 1H), 8.59 (d, J = 6.2 Hz, 2H), 7.69 (d, J = 8.9Hz, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 5.9 Hz, 2H), 7.32 (t, J = 5.9 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.19-3.15(m, 4H), 3.08-3.01 (m, 4H).

[0059] (III) Preparation of target product A1: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea

[0060] Commercially available 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (1.49 g, 5 mmol), intermediate 5a (2.06 g, 5.5 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g, 6 mmol) were dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (1.94 g, 15 mmol) was then added and stirred at room temperature for 2 h. After the reaction, 50 mL of distilled water was added, the mixture was filtered, and the filter cake was purified by column chromatography to obtain 2.13 g of a white solid in a 65% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 8.52 – 8.45 (m, 2H), 8.19 (s, 2H), 7.94 –7.88 (m, 1H), 7.80 – 7.66 (m, 5H), 7.60 – 7.50 (m, 3H), 7.44 – 7.31 (m, 3H), 4.43 – 4.39 (m, 2H), 4.25 (d, J = 0.9 Hz, 2H), 3.31 (dd, J = 10.9, 5.6 Hz,2H), 3.29 – 3.20 (m, 3H), 3.18 (s, 4H). 13 C NMR (150 MHz, DMSO- d 6) δ 166.06,166.02, 159.51, 157.74, 156.82, 156.08, 148.67, 148.45, 142.83, 140.15,136.27, 135.40, 133.99, 133.28, 132.03, 131.89, 131.87, 131.56, 131.52,130.48, 130.43, 129.05, 128.52, 128.18, 125.91, 125.51, 124.38, 124.29,122.91, 119.63, 115.65, 115.50, 48.43, 45.88, 44.17, 38.72. ESI-MS: m / z [M+H] + :656.21. Example 2

[0061] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced with tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (commercially available). Other reactions were the same as in Example 1 to obtain Compound A2 in a yield of 52%. Example 3

[0062] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced by 4-amino-1-tert-butyloxycarbonylpiperidine (commercially available), and the rest was the same as in Example 1 to obtain Compound A3 with a yield of 55%. Example 4

[0063] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced by 1-tert-butyloxycarbonyl-4-aminopiperidine (commercially available). Other reactions were the same as in Example 1 to obtain Compound A4 in a yield of 49%. Example 5

[0064] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced with 2-Boc-2,7-diaza-spiro[4.4]nonane (commercially available). Other reactions were the same as in Example 1 to obtain Compound A5 in a yield of 51%. Example 6

[0065] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced with tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (commercially available). Other reactions were the same as in Example 1 to obtain Compound A6 in a yield of 46%. Example 7

[0066] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced with tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (commercially available). Other reactions were the same as in Example 1 to obtain Compound A7 in a yield of 38%. Example 8

[0067] The 1-tert-butyloxycarbonyl-piperazine used in step (1) of Example 1 was replaced with tert-butyl 2-(methylamino)ethylcarbamate (commercially available). Other reactions were the same as in Example 1 to obtain Compound A8 in a yield of 40%. Example 9

[0068] Preparation of compound: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)thiourea (A9)

[0069] Synthesis route:

[0070]

[0071] Reagents and conditions: (a) 1,1'-thiocarbonyldiimidazole, acetonitrile, 60°C, 4 hours, 70% yield; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours, 95% yield; (c) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours, 55% yield.

[0072] (I) Preparation of Intermediate 6: tert-Butyl 4-((4-(3-(pyridin-4-ylmethyl)thioureido)phenyl)sulfonyl)piperazine-1-carboxylate

[0073] 1,1'-Thiocarbonyldiimidazole (2.67 g, 15 mmol) and intermediate 3a (3.41 g, 10 mmol) were dissolved in 100 mL of anhydrous acetonitrile and stirred at room temperature for 2 hours. 3-Methylaminopyridine (2.16 g, 20 mmol) was then added, and the reaction mixture was heated to 60°C and allowed to react for an additional 4 hours. After completion of the reaction, the reaction mixture was concentrated and separated by column chromatography to yield 3.44 g of a white solid in a 70% yield. 1 HNMR (600 MHz, DMSO- d 6) δ 12.12 (s, 1H), 10.71 (t, J = 5.9 Hz, 1H), 8.78 (p, J = 1.2 Hz, 1H), 8.50 (dt, J = 3.8, 1.7 Hz, 1H), 7.91 (dtt, J = 7.9, 1.9, 1.0Hz, 1H), 7.77 – 7.71 (m, 2H), 7.55 – 7.49 (m, 2H), 7.33 (dd, J = 7.9, 4.3 Hz,1H), 4.72 – 4.68 (m, 2H), 3.15 (d, J = 5.6 Hz, 2H), 3.09 – 2.96 (m, 4H), 1.39(s, 5H). 13 C NMR (150 MHz, DMSO- d 6) δ 176.23, 154.85, 148.67, 148.45, 140.65,135.84, 135.40, 133.99, 128.99, 122.91, 120.41, 79.30, 48.43, 46.91, 45.84,28.27.

[0074] (II) Preparation of Intermediate 7:

[0075] Intermediate 6 (4.91 g, 10 mmol) was dissolved in 100 mL of a 1 / 1 mixture of trifluoroacetic acid and dichloromethane and stirred at room temperature for 2 h. After the reaction, the solution was concentrated by vacuum distillation and purified by column chromatography to obtain 3.72 g of a white solid in a 95% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 12.12 (s, 1H), 10.71 (t, J = 5.9 Hz,1H), 8.80 – 8.76 (m, 1H), 8.50 (dt, J = 3.8, 1.7 Hz, 1H), 7.94 – 7.88 (m,1H), 7.76 – 7.71 (m, 2H), 7.55 – 7.49 (m, 2H), 7.33 (dd, J = 7.9, 4.2 Hz,1H), 4.70 (dd, J = 6.1, 1.1 Hz, 2H), 3.00 (d, J = 10.0 Hz, 2H), 2.82 – 2.72(m, 5H). 13 C NMR (150 MHz, DMSO- d 6) δ 176.23, 148.67, 148.45, 140.65, 135.84,135.40, 133.99, 128.99, 122.91, 120.41, 46.91, 43.31, 42.60.

[0076] (III) Preparation of target product A9: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)thiourea

[0077] Commercially available 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (1.49 g, 5 mmol), intermediate 7 (2.15 g, 5.5 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g, 6 mmol) were dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (1.94 g, 15 mmol) was then added and stirred at room temperature for 2 h. After the reaction, 50 mL of distilled water was added, the mixture was filtered, and the filter cake was purified by column chromatography to obtain 1.85 g of a white solid in a 55% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 12.12 (s, 1H), 11.40 (s, 1H), 10.71 (t, J = 5.9 Hz, 1H), 8.80 – 8.76 (m, 1H), 8.52 – 8.45 (m, 2H), 8.19 (dd, J = 3.8,2.5 Hz, 1H), 7.94 – 7.88 (m, 1H), 7.77 – 7.66 (m, 2H), 7.60 – 7.54 (m, 1H),7.54 – 7.49 (m, 2H), 7.38 (dd, J = 10.1, 8.3 Hz, 1H), 7.33 (dd, J = 7.9, 4.2Hz, 1H), 4.70 (dd, J = 6.0, 1.0 Hz, 2H), 4.25 (d, J = 2.0 Hz, 1H), 4.25 (s,2H), 3.30 (dt, J = 11.0, 5.7 Hz, 2H), 3.29 – 3.20 (m, 2H), 3.18 (d, J = 11.5Hz, 2H). 13 C NMR (150 MHz, DMSO- d6) δ 176.23, 166.06, 166.02, 159.51, 157.74,156.08, 148.67, 148.45, 142.83, 140.65, 135.84, 135.40, 133.99, 133.28,132.03, 131.89, 131.87, 131.56, 131.52, 130.48, 130.43, 128.99, 128.52,128.18, 125.91, 125.51, 124.38, 124.29, 122.91, 120.41, 115.65, 115.50,48.43, 46.91, 45.88, 38.72. ESI-MS: m / z [M+H] + : 672.18. Example 10

[0078] The 3-aminomethylpyridine (commercially available) used in step (iii) of Example 1 was replaced by 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine (commercially available). Other reactions were the same as in Example 1 to obtain Compound A10 in a yield of 51%. Example 11

[0079] The 3-aminomethylpyridine used in step (iii) of Example 1 was replaced by 4-fluoropyridine-3-methylamine (commercially available). Other reactions were the same as in Example 1 to obtain Compound A11 in a yield of 56%. Example 12

[0080] Prepare compound: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)thieno[2,3-c]pyridine-2-carboxamide (A12)

[0081] Synthesis route:

[0082]

[0083] Reagents and conditions: (a) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature overnight, yield 52%; (b) Trifluoroacetic acid, dichloromethane, room temperature, 2 hours, yield 95%; (c) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours, yield 57%.

[0084] (I) Preparation of Intermediate 8c: tert-Butyl 4-((4-(thiophene[2,3-c]pyridine-2-carboxamide)phenyl)sulfonyl)piperazine-1-carboxylate

[0085] Thieno[2,3-c]pyridine-2-carboxylic acid (0.89 g, 5 mmol) (commercially available), intermediate 3a (1.87 g, 5.5 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinium phosphonium hexafluorophosphate (3.12 g, 6 mmol) were dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (1.94 g, 15 mmol) was then added and stirred at room temperature overnight. After the reaction, 50 mL of distilled water was added, the mixture was filtered, and the filter cake was purified by column chromatography to obtain 1.31 g of a white solid in a 52% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 10.33 (s, 1H), 9.53 (dd, J = 3.7, 1.3 Hz, 1H), 9.15 (dd, J =4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.12 (d, J = 2.2 Hz, 1H), 7.99 –7.93 (m, 2H), 7.80 – 7.74 (m, 2H), 3.15 (d, J = 5.6 Hz, 2H), 3.09 – 2.96 (m,3H), 1.39 (s, 5H). 13 C NMR (150 MHz, DMSO- d 6) δ 158.57, 154.85, 152.54,144.79, 143.61, 139.49, 136.46, 134.31, 132.04, 128.95, 121.21, 120.14,118.23, 79.30, 48.43, 45.84, 28.27.

[0086] (II) Preparation of Intermediate 9c: N-(4-(piperazin-1-ylsulfonyl)phenyl)thiophene[2,3-c]pyridine-2-carboxamide

[0087] Intermediate 8c (5.02 g, 10 mmol) was dissolved in 100 mL of a 1 / 1 mixture of trifluoroacetic acid and dichloromethane and stirred at room temperature for 2 h. After the reaction, the solution was concentrated by vacuum distillation and purified by column chromatography to yield 3.82 g of a white solid in a 95% yield.1 H NMR (600 MHz, DMSO- d 6) δ 10.33 (s, 1H), 9.53 (dd, J = 3.8, 1.3Hz, 1H), 9.15 (dd, J = 4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.12 (d, J = 2.2 Hz, 1H), 7.99 – 7.93 (m, 2H), 7.80 – 7.74 (m, 2H), 3.00 (d, J = 10.0Hz, 2H), 2.82 – 2.72 (m, 5H). 13 C NMR (150 MHz, Common NMR Solvents) δ 158.57,152.54, 144.79, 143.61, 139.49, 136.46, 134.31, 132.04, 128.95, 121.21,120.14, 118.23, 43.31, 42.60.

[0088] (III) Preparation of target product A12: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)thieno[2,3-c]pyridine-2-carboxamide

[0089] Commercially available 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid (1.49 g, 5 mmol), intermediate 9c (2.21 g, 5.5 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.12 g, 6 mmol) were dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (1.94 g, 15 mmol) was then added and stirred at room temperature for 2 h. After the reaction, 50 mL of distilled water was added, the mixture was filtered, and the filter cake was purified by column chromatography to obtain 1.94 g of a white solid in a 57% yield. 1 H NMR (600 MHz, DMSO- d 6) δ 11.40 (s, 1H), 10.33 (s, 1H), 9.53 (dd, J = 3.7, 1.3 Hz, 1H), 9.15 (dd, J= 4.0, 2.1 Hz, 1H), 8.59 (d, J = 1.5 Hz, 1H),8.49 – 8.45 (m, 1H), 8.19 (dd, J = 3.5, 2.3 Hz, 1H), 8.12 (d, J = 2.1 Hz,1H), 7.98 – 7.93 (m, 2H), 7.80 – 7.71 (m, 4H), 7.71 – 7.66 (m, 1H), 7.60 –7.54 (m, 1H), 7.38 (dd, J = 10.1, 8.2 Hz, 1H), 4.25 (s, 1H), 3.30 (dt, J =11.0, 5.7 Hz, 2H), 3.27 – 3.20 (m, 2H), 3.18 (d, J = 11.5 Hz, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 166.06, 166.02, 159.51, 158.57, 157.74, 156.08, 152.54,144.79, 143.61, 142.83, 139.49, 136.46, 134.31, 133.28, 132.04, 131.89,131.87, 131.56, 131.52, 130.48, 130.43, 128.95, 128.52, 128.18, 125.91,125.51, 124.38, 124.29, 121.21, 120.14, 118.23, 115.65, 115.50, 48.43, 45.88,38.72. ESI-MS: m / z [M+H] + : 683.16. Example 13

[0090] In vitro inhibition test of PARP-1 enzyme activity by target compounds

[0091] 1. Experimental Materials

[0092] PARP1 colorimetric assay kit (Cat #80580, BPS Bioscience); PBS (Cat #21600-010, Gibco); Tween-20 (Cat #P9416, Sigma); ELISA stop buffer (Cat #C1058, Solarbio); positive drug olaparib.

[0093] 2. Measurement Method

[0094] (1) Dilute 10× PARP buffer with deionized water to obtain 1× PARP buffer. Dilute the compound stock solution to 1 μM with DMSO and set aside. Dilute the 1 μM compound solution to 10 nM with 1× PARP buffer. Wash the tray three times with 200 μL / well PBST, add 200 μL / well blocking buffer, and incubate at room temperature for 90 min. Wash the tray three more times with 200 μL / well PBST.

[0095] (2) Prepare the master mix: N wells × (2.5 µL 10× PARP buffer + 2.5 µL 10× PARP detection solution + 5 µL activated DNA + 15 µL distilled water), add 25 µL to each well.

[0096] (3) Add 5 μL / well of the diluted compound. Add the same volume of 1× PARP buffer containing 10% DMSO to the vehicle control wells and blank wells. Thaw PARP1 on ice and dilute it to 1.0 ng / ul with 1× PARP buffer. Add 20 μL of diluted PARP enzyme to the non-blank wells. Add 20 μL / well of 1× PARP buffer to the blank wells. Incubate at room temperature for 60 minutes. Wash the tray three times with 200 μL / well PBST.

[0097] (4) Dilute streptavidin-HRP 1:50 with blocking buffer, add 50 µl of diluted streptavidin-HRP to each well, and incubate at room temperature for 30 minutes. Wash the plate three times with 200 µl / well PBST, add 100 µl / well HRP colorimetric substrate at room temperature for 20 minutes, add 100 µl / well 2M sulfuric acid, and read the OD 450 nm on a microplate reader.

[0098] The experimental results (Table 2) showed that these compounds all exhibited good PARP1 inhibitory activity, among which compounds A1, A2 and A9 showed inhibitory activity comparable to that of the positive control drug olaparib.

[0099] Example 14

[0100] In vitro inhibition test of NAMPT enzyme activity by target compounds

[0101] 1. Experimental Materials

[0102] Test solution: bovine serum albumin (BSA, Kingmorn) + Tris-1HCl (pH 7.5) + MgCl2 (Sangon), ATP solution (100 mM, Sangon), PRPP solution (40 mM, Yuanye), DTT solution (2 mM, Sangon); compound gradient dilution solution; NAM solution (0.2 μM, Sangon); 20% acetophenone 2M KOH 88% formic acid; positive drug FK866.

[0103] 2. Measurement Method

[0104] (1) Add 20 μL of freshly prepared test solution and 0.5 μL of each concentration gradient compound solution to a 96-well plate and let it stand at room temperature for 5 minutes.

[0105] (2) Add 4.5 μL of NAM solution to the 96-well plate and add double-distilled water to the blank control group; mix well and react at 37°C for 15 min.

[0106] (3) After the reaction is complete, heat at 95°C for 1 min and then quickly cool on ice to terminate the enzyme reaction.

[0107] (4) Add 10 μL each of 20% acetophenone and 2M KOH to the terminated enzyme reaction solution, centrifuge quickly, mix well on a vortex mixer, and react at 0°C for 10 min.

[0108] (5) Add 45 μL of 88% formic acid and incubate at 37°C for 10 min.

[0109] (6) After cooling, transfer 85 μL of the 90 μL reaction system to a black flat-bottom 96-well fluorescent plate.

[0110] (7) NAMPT protein catalyzes the conversion of nicotinamide NAM into the product nicotinamide mononucleotide NMN. In the detection reaction, NMN is converted into a fluorescent derivative through a two-step chemical reaction, with the maximum fluorescence signal detected at an excitation wavelength of 382 nm and an emission wavelength of 445 nm. The fluorescence values ​​at excitation light of 382 nm and emission light of 445 nm were measured using a microplate reader, and the inhibitory activity of the compounds against NAMPT was calculated using GraphPad Prism software.

[0111] The experimental results show that most of the compounds of the present invention exhibit good NAMPT inhibitory activity, all at the nanomolar level. The results are shown in Table 3. Different linkers have a significant effect on the enzyme inhibitory activity of the compounds. With the exception of compounds A4, A7, and A8, the activities of the compounds are much stronger than the positive control drug FK866.

[0112] Example 15

[0113] In vitro antitumor activity test of target compounds (MTT assay)

[0114] 1. Experimental Materials

[0115] MTT, PRMI1640 culture medium, fetal bovine serum, 96-well plate, CO2 constant temperature incubator, BIO-TEK Uquant multi-function microplate reader, human breast cancer cells (MDA-MB-231), human breast cancer cells (MDA-MB-436) and human breast cancer cells (MDA-MB-468), positive control drug Olaparib.

[0116] 2. Experimental Methods

[0117] (1) Inoculate cells. Prepare a single cell suspension using culture medium containing 10% fetal bovine serum. Inoculate 5,000 cells per well in a 96-well plate with a volume of 100 μL per well and culture overnight.

[0118] (2) Preparation of test compound solutions: In a sterile oven, dilute the DMSO stock solution of the compound with culture medium to five test concentrations, with two-fold dilutions between adjacent concentrations.

[0119] (3) Add compound solutions of different concentrations to a 96-well plate that has been cultured overnight, adding 100 μL to each well, and adding three replicates for each concentration. Because the surrounding area has an edge effect and is easily contaminated with bacteria, no cells or compounds are added, and 100 μL of culture medium is added as a blank. Set up another 100% well, that is, add cells and 100 μL of culture medium without compound, and incubate in a 37°C constant temperature incubator for 48 hours.

[0120] (4) Staining: Add 10 μL MTT solution (5 mg / mL, prepared with PBS) to a 96-well plate for staining. After incubation for 4 hours, centrifuge at 2500 rpm for 10 minutes, then aspirate the culture medium from the wells with a spray gun, add 150 μL DMSO, and shake on a shaking plate for 5-10 minutes to fully dissolve the formazan. Measure the OD value of each well at 570 nm using an enzyme-labeled instrument.

[0121] Inhibition rate (%) = (average OD value of 100% wells - average OD value of compound wells) / (average OD value of 100% wells - average OD value of blank wells) × 100%. Based on the inhibition rate values ​​of each concentration, linear regression was performed to calculate the drug concentration that inhibited cell growth by 50%, i.e., IC 50 .

[0122] The experimental results (Table 4) showed that these target compounds had significant anti-tumor cell proliferation activity, which was stronger than the positive drug Olaparib and independent of the cell BRCA mutation status. 50 The values ​​ranged from 0.09 to 30.05 μM, among which compounds A1, A9 and A10 were the most active potential compounds.

[0123] Example 16

[0124] In vivo anti-tumor effects of preferred compounds

[0125] Based on the above experimental results, the in vivo antitumor activity of compounds A1 and A9 was tested in a nude mouse xenograft model of the human breast cancer cell line MDA-MB-468. Compounds A1 and A9 were administered at a dose of 50 mg / kg via intraperitoneal injection twice daily for 14 consecutive days. The results (Table 5) show that both compounds A1 and A9 exhibited excellent in vivo inhibitory activity, with tumor inhibition rates of 78.95% and 73.68%, respectively, significantly exceeding the 30.53% achieved by the olaparib control group at the same dose. Furthermore, no mice died or experienced significant weight changes during the dosing period, indicating that compounds A1 and A9 exhibit low in vivo toxicity and warrant further investigation.

[0126]

Claims

1. A compound as a PARP-NAMPT dual-target inhibitor, characterized in that: Having the structure represented by the following general formula (I), and its pharmaceutically acceptable salts; ; in: X is an oxygen atom or a sulfur atom; R1 is any one of the following structures: ； R2 is any one of the following structures: ； "Pharmaceutically acceptable salts" refer to salt forms of the compounds of formula (I) which are therapeutically effective and non-toxic.

2. The compound as a PARP-NAMPT dual-target inhibitor according to claim 1 is as follows: Compound A1: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea; Compound A2: 1-(4-((5-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea; Compound A3: 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-N-(1-((4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonyl)piperidin-4-yl)benzamide; Compound A4: N-(1-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperidin-4-yl)-4-(3-(pyridin-3-ylmethyl)ureido)benzenesulfonamide; Compound A5: 1-(4-((7-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,7-diazaspiro[4.4]nonan-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea; Compound A6: 1-(4-((6-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-2,6-diazaspiro[3.3]heptan-2-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea; Compound A7: 1-(4-((9-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)urea; Compound A8: 2-fluoro-N-(2-((N-methyl-4-(3-(pyridin-3-ylmethyl)ureido)phenyl)sulfonamido)ethyl)-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzamide; Compound A9: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-(pyridin-3-ylmethyl)thiourea; Compound A10: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide; Compound A11: 1-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)-3-((4-fluoropyridin-3-yl)methyl)urea; Compound A12: N-(4-((4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)sulfonyl)phenyl)thieno[2,3-c]pyridine-2-carboxamide.

3. The method for preparing the compound as a PARP-NAMPT dual-target inhibitor according to claim 2, characterized in that: Synthesis method of compounds A1-A8: ； Reagents and conditions: (a) triethylamine, dichloromethane, room temperature, 2 hours; (b) hydrogen, palladium on carbon, dichloromethane / methanol, room temperature, overnight; (c) triphosgene, triethylamine, dichloromethane, room temperature, 2 hours; triethylamine, dichloromethane, room temperature, overnight; (d) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (e) 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours; Synthesis method of compound A9: ； Reagents and conditions: (a) 1,1'-thiocarbonyldiimidazole, acetonitrile, 60°C, 4 hours; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours; Synthesis of compounds A10 and A11: ； Reagents and conditions: (a) triphosgene, triethylamine, dichloromethane, room temperature; triethylamine, dichloromethane, room temperature, overnight; (b) trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours; Synthesis method of compound A12: ； Reagents and conditions: (a) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature overnight; (b) Trifluoroacetic acid, dichloromethane, room temperature, 2 hours; (c) 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 hours.

4. Use of the compound as a PARP-NAMPT dual-target inhibitor according to claim 1 in the preparation of an anti-tumor drug; the tumor is breast cancer.

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