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

Abstract

The present invention discloses a compound or a pharmaceutically acceptable salt thereof as a NAMPT-PDEδ dual-target inhibitor, wherein the general structural formula is selected from one of the following structures: #imgabs0# The present invention also discloses the use of the compound or a pharmaceutically acceptable salt thereof as a NAMPT-PDEδ dual-target inhibitor in the preparation of a NAMPT-PDEδ dual-target inhibitor, and the use of the compound or a pharmaceutically acceptable salt thereof as a NAMPT-PDEδ dual-target inhibitor in the preparation of an anti-tumor drug. In vitro anti-tumor activity tests of the compounds prepared by the present invention showed strong in vitro anti-tumor activity and broad-spectrum anti-tumor characteristics; NAMPT and PDEδ inhibitory activity showed that most compounds had strong inhibitory effects on both, and were dual-target inhibitors for NAMPT-PDEδ. The compounds of the present invention can be used to treat malignant tumors and diseases related to differentiation and proliferation.

Description

Technical Field

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

[0002] NAD plays a crucial role in cellular physiology. Tumor cells, characterized by rapid proliferation and elevated energy metabolism, are particularly sensitive to NAMPT, the rate-limiting enzyme in the NAD biosynthesis pathway. Therefore, NAMPT has become a popular target in cancer therapy. Currently discovered NAMPT inhibitors effectively and selectively reduce NAD levels in tumor cells, exhibiting potent tumor cell-killing effects and inhibiting the growth of xenograft tumors in mice. However, most reported small molecule NAMPT inhibitors suffer from dose-toxicity issues, limiting their further application. Among the NAMPT inhibitors currently in clinical trials are FK866 and CHS828. However, both compounds exhibit dose-limiting side effects such as thrombocytopenia and gastrointestinal symptoms, leading to stagnant research. Therefore, reducing the toxicity of NAMPT inhibitors has become a key area of ​​research. In recent years, multi-targeted NAMPT-based drugs have become a research hotspot, promising synergistic effects, enhanced efficacy, and reduced side effects. Therefore, designing and developing new dual-target inhibitors based on NAMPT and its targets with synergistic anti-cancer activity may be an effective strategy to achieve balanced regulation of multiple targets in cells, reduce toxic side effects, and improve anti-cancer efficacy.

[0003] The KRAS protein acts as a binary molecular switch. In its activated state, its guanosine triphosphate (GTP) binding site binds to GTP, recruiting effector proteins to the cell membrane and activating downstream growth and proliferation signaling pathways such as Mst1, PI3K / Akt, and MAPK, leading to cell proliferation. When the KRAS protein is in its inactive state, the KRAS-GTP complex is hydrolyzed, at which point the KRAS protein rapidly binds to guanosine diphosphate (GDP) to form a KRAS-GDP complex, thereby halting activation of downstream growth and proliferation signaling pathways. In cancerous states, KRAS mutations impair the protein's intrinsic GTPase activity, preventing its conversion from the active form GTP to the inactive form GDP. This allows the KRAS protein to remain bound to GTP and persistently activate downstream growth and proliferation signaling pathways, leading to excessive cell proliferation and cancer. In recent years, the development of targeted drugs targeting mutant KRAS proteins has been a hot topic in the medical field. However, due to its small size and flat surface, lacking the "deep pockets" that traditional small molecule drugs can bind to, and its tight binding to GTP, the KRAS protein has remained an undruggable target for many years, lacking effective treatment options. Currently, several drugs targeting the KRAS protein are in clinical trials, but progress has been slow or facing failure. Therefore, developing novel, broad-spectrum KRAS protein intervention strategies holds significant research value for cancer treatment.

[0004] The function of KRAS signaling is primarily determined by its intracellular location. Studies have shown that PDEδ plays a crucial role in regulating KRAS protein localization on the cell membrane. PDEδ, encoded by the Pde6d gene, has a hydrophobic cavity that binds to the farnesyl group of KRAS, enhancing its intracellular diffusion. PDEδ also promotes the proper localization and accumulation of KRAS on the cell membrane, playing a key role in maintaining KRAS-related signaling pathways. Therefore, targeted inhibition of PDEδ and blocking the KRAS-PDEδ protein-protein interaction can effectively interfere with KRAS signaling, broadly blocking KRAS downstream signaling pathways and exerting anti-pancreatic cancer effects. For example, small-molecule PDEδ inhibitors, such as phenyldisulfonamides and benzimidazoles, discovered through AlphaScreen screening, have demonstrated potent inhibitory activity against the KRAS-PDEδ protein-protein interaction. However, existing small-molecule PDEδ inhibitors generally suffer from low cellular activity and poor druggability, and further structural optimization has not significantly improved their metabolic stability. Furthermore, the mismatch between protein-binding activity and antitumor activity is a major challenge in the research of PDEδ small-molecule inhibitors. In particular, in vivo antitumor efficacy of PDEδ small-molecule inhibitors has not yet been reported. Improving cellular viability and in vivo efficacy is currently a bottleneck in the development of PDEδ small-molecule inhibitors. Therefore, developing more potent PDEδ inhibitors by expanding the chemical structure class may offer a new approach for the treatment of pancreatic cancer. Furthermore, the functions of the PDEδ protein remain largely unresolved, and its mechanisms of collaboration with other targets remain to be elucidated. Therefore, the development of appropriate tool molecules is needed to assist in studying the protein's synergistic functions.

[0005] Studies have found that PDEδ and NAMPT have a certain synergistic effect in regulating the homeostasis of cellular metabolism. KRAS protein mutations can lead to changes in multiple metabolic pathways, which is an important driving factor for tumor metabolic reprogramming and is crucial to the occurrence and development of pancreatic cancer. NAD is a key cofactor. It is speculated that simultaneous inhibition of both NAMPT and PDEδ targets will jointly promote the stabilization of pancreatic cancer cell metabolic pathways and produce a synergistic effect against pancreatic cancer. Based on the above theory, PDEδ inhibitor 4 and NAMPT inhibitor MS0 were tested for synergistic anti-pancreatic cancer activity (synergistic index CI <1.0), suggesting that the PDEδ and NAMPT signaling pathways are synergistic. The above evidence shows that the design of PDEδ / NAMPT dual-target inhibitors is expected to become a new strategy for the development of pancreatic cancer treatment drugs.

[0006] Studies have found that both NAMPT and PDEδ inhibitors can be divided into three parts: a head group, a linker group, and a tail group. Both share similar chain-like structural features: 1) a head group, a core group composed of a nitrogen-containing heterocycle and a hydrogen bond acceptor, mimicking the substrate structure; 2) an alkyl or aryl linker, binding to the hydrophobic cavity of the protein; and 3) a tail group directed toward the protein surface. NAMPT inhibitors have an essential active group at the head, while the tail, a non-essential active group, faces the protein surface. Similarly, PDEδ inhibitors have a non-essential active group directed toward the protein surface, and structural modifications can enhance their activity. This provides a feasible basis for the construction of PDEδ / NAMPT dual-target inhibitors. Summary of the Invention

[0007] The purpose of the present invention is to provide a compound as a NAMPT-PDEδ dual-target inhibitor.

[0008] The second object of the present invention is to provide a use of the compound as a NAMPT-PDEδ dual-target inhibitor in the preparation of a NAMPT-PDEδ dual-target inhibitor.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a compound or a pharmaceutically acceptable salt thereof as a NAMPT-PDEδ dual-target inhibitor, wherein the general structural formula is selected from one of the following structures:

[0011]

[0012] wherein X is selected from a bond, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-,

[0013]

[0014] R1 is selected from

[0015] R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkoxy (such as methoxy, ethoxy, isopropoxy, n-propoxy, tert-butoxy, n-butoxy, etc.), C1-C10 alkyl (such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, etc.);

[0016] R3 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkoxy (such as methoxy, ethoxy, isopropoxy, n-propoxy, tert-butoxy, n-butoxy, etc.), C1-C10 alkyl (such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, etc.);

[0017] R4 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkoxy (such as methoxy, ethoxy, isopropoxy, n-propoxy, tert-butoxy, n-butoxy, etc.), C1-C10 alkyl (such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, etc.);

[0018] R5 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkoxy (such as methoxy, ethoxy, isopropoxy, n-propoxy, tert-butoxy, n-butoxy, etc.), C1-C10 alkyl (such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, etc.);

[0019] R6 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkoxy (such as methoxy, ethoxy, isopropoxy, n-propoxy, tert-butoxy, n-butoxy, etc.), C1-C10 alkyl (such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, etc.);

[0020] R7 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0021] R8 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0022] R9 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0023] R 10 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0024] R 11 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine).

[0025] The pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt thereof.

[0026] The inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid; the organic acid is acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, tannic acid, citric acid, trifluoroacetic acid, malic acid or benzenesulfonate.

[0027] The pharmaceutically acceptable salt contains no crystalline water, or contains more than one crystalline water.

[0028] Preferably, the compound as a NAMPT-PDEδ dual-target inhibitor is selected from one of the following structures:

[0029]

[0030]

[0031]

[0032] The second aspect of the present invention provides a use of the compound as a NAMPT-PDEδ dual-target inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a NAMPT-PDEδ dual-target inhibitor.

[0033] The third aspect of the present invention provides a use of the compound as a NAMPT-PDEδ dual-target inhibitor or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0034] The tumor is selected from pancreatic cancer, colorectal cancer, lung cancer, etc.

[0035] The tumor cells are selected from MiaPaca-2 human pancreatic cancer cells, HCT116 human colon cancer cells, and A549 human lung cancer cells.

[0036] The present invention uses the CCK8 assay for activity testing, with a NAMPT inhibitor (MS0) and a PDEδ inhibitor (Compound 4) serving as positive control drugs. Pharmacological experiments demonstrated that the compounds of the present invention, or pharmaceutically acceptable salts thereof, exhibited in vitro antitumor activity against three tumor cell lines (MiaPaca-2 human pancreatic cancer cells, HCT116 human colon cancer cells, and A549 human lung cancer cells). The results showed that most compounds exhibited moderate or higher antiproliferative activity against all three tumor cell lines, and most compounds exhibited significantly improved activity compared to the positive drugs MS0 and Compound 4.

[0037]

[0038] The compounds of the present invention were tested for enzyme inhibition activity and in vitro antitumor activity. They showed that the compounds of the present invention had strong inhibitory activity against both NAMPT and PDEδ proteins, and had relatively strong in vitro antitumor activity. Some compounds had good inhibitory activity against MiaPaca-2, HCT116, and A549 cells, and exhibited inhibitory activity superior to that of the positive drug compounds 4 and MS0.

[0039] In vitro antitumor activity tests demonstrated that the compounds prepared by the present invention exhibited strong in vitro antitumor activity and broad-spectrum antitumor properties. NAMPT and PDEδ inhibitory activity demonstrated that most compounds exhibited strong inhibitory effects against both, representing dual-target inhibitors of NAMPT and PDEδ, such as compounds A6, B5, and B14. This invention explored the design, synthesis, and antitumor activity of dual-target inhibitors of NAMPT and PDEδ. The compounds prepared by the present invention, or their pharmaceutically acceptable salts, possess novel structural frameworks and are worthy of further research as antitumor and differentiation / proliferation-related drugs. Therefore, the compounds prepared by the present invention, or their pharmaceutically acceptable salts, can be used to prepare multi-target antitumor drugs.

[0040] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0041] The in vitro anti-tumor activity test of the compounds prepared by the present invention showed strong in vitro anti-tumor activity and broad-spectrum anti-tumor characteristics; the NAMPT and PDEδ inhibitory activity showed that most compounds had strong inhibitory effects on both, and were dual-target inhibitors for NAMPT-PDEδ. The compounds of the present invention can be used to treat malignant tumors and diseases related to differentiation and proliferation. The compound B5 prepared by the present invention has excellent molecular-level inhibitory activity (NAMPT IC 50 The value was 3.41 nM, and the PDEδIC 50 0.5 nM), and also exhibited excellent growth inhibitory activity (IC 50 all below 10 nM). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the in vivo antitumor efficacy of compound B5. DETAILED DESCRIPTION

[0043] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0044] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0045] The chemical structural formula of the compounds involved in the following examples, 1 The H-NMR and MS data are detailed in Table 1. The numbers A1-A8 and B1-B19 in Tables 1, 2, and 3 are consistent with those in the specification.

[0046] Table 1. Structural formula and NMR mass spectrometry data of the compounds of the present invention

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Example 1

[0060] Preparation of N-(4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)phenyl)nicotinamide (Compound A1)

[0061]

[0062] The first step is to prepare intermediate I: N-(4-chlorobenzyl)cyclopentylamine

[0063] Dissolve p-chlorobenzaldehyde (1.41 g, 10 mmol) in 20 mL of ultra-dry methanol, add cyclopentylamine (0.85 g, 10 mmol), add anhydrous sodium sulfate (4.2 g, 30 mmol), and react at room temperature for 12 hours. After the reaction is complete, add NaBH4 (0.19 g, 5 mmol) in an ice-water bath, transfer to room temperature and react for 2 hours. TLC monitors the reaction completion. The reaction solution is poured into water (20 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases are combined and dried over anhydrous sodium sulfate. The solvent is evaporated under reduced pressure and concentrated to obtain 1.9 g of a light yellow oil, Intermediate I; it is directly carried out to the next step without further purification, with a yield of 91%.

[0064] Step 2: Preparation of Intermediate II: N-(4-chlorobenzyl)-N-cyclopentyl-4-nitrobenzenesulfonamide

[0065] Intermediate I (1.91 g, 9.1 mmol) was dissolved in 20 mL of dichloromethane, and p-nitrobenzenesulfonyl chloride (1.68 g, 7.6 mmol) and TEA (2.5 mL, 18.2 mmol) were added. The mixture was stirred at room temperature for 2 h. After completion of the reaction, DCM (100 mL x 2) was added for extraction. The combined organic phases were washed with water (200 mL x 1) and saturated brine (200 mL x 1), dried over anhydrous sodium sulfate, and filtered. The residue was mixed and purified by silica gel column chromatography (PE / EA = 4 / 1) to obtain 2.6 g of Intermediate II as a pale yellow solid in an 86% yield. 1HNMR (600MHz, DMSO-d6) δ: 8.40 (d, 2H, J = 8.9Hz), 8.15 (d, 2H, J = 8.8Hz), 7.37-7.44 (m, 4H), 4.41 (s, 2H), 1.15-1.54 (m, 8H). 13 C NMR (150MHz, DMSO-d6) δ: 150.3, 145.8, 138.7, 132.0, 129.1, 128.7, 125.1, 59.7, 46.7, 29.1, 23.3.

[0066] Step 3: Preparation of Intermediate III: 4-amino-N-(4-chlorobenzyl)-N-cyclopentylbenzenesulfonamide

[0067] Intermediate II (2.6 g, 6.5 mmol) was dissolved in 30 mL of DCM / MeOH (2:1), and Pd / C (0.7 g, 0.6 mmol) was added. The mixture was stirred overnight at room temperature under 1 atm of hydrogen. After completion of the reaction, the reaction solution was filtered through celite and the filter layer was rinsed with 50 mL of methanol. The filtrates were combined, the solvent was evaporated under reduced pressure, and the mixture was concentrated to obtain 2 g of Intermediate III as a white solid in a 90% yield. The mixture was carried on to the next step without further purification. 1 H NMR (600MHz, DMSO-d6) δ: 7.46 (d, 2H, J = 8.7Hz), 7.39 (s, 4H), 6.62 (d, 2H, J = 8.7Hz), 6.01(s,2H),4.24(s,2H),4.05-4.16(m,1H),1.31-1.43(m,6H),1.13-1.17(m,2H). 13 C NMR (150MHz, DMSO-d6) δ: 153.37, 139.95, 131.63, 129.40, 129.07, 128.51, 125.12, 113.30, 59.21, 46.28, 28.97, 23.43.

[0068] Step 4: Preparation of Compound A1:

[0069] Nicotinic acid (0.074 g, 0.4 mmol) was dissolved in 5 mL of oxalyl chloride to prepare the acyl chloride. After the reaction was allowed to proceed overnight, the excess oxalyl chloride was removed by spin drying, and the solid was dissolved in 2 mL of DMF. DIPEA (0.058 g, 0.48 mmol) and Intermediate III (0.1 g, 0.3 mmol) were added and allowed to react at room temperature for 2 h. After completion of the reaction, the mixture was extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography using MeOH / DCM (0-10%) to afford 14 mg of a white solid, Compound A1, in a 33.7% yield.

[0070] Example 2

[0071] The nicotinic acid used in the fourth step of Example 1 was replaced by 3-pyridineacetic acid. Other steps were the same as in Example 1 to obtain Compound A2 with a yield of 44.2%.

[0072] Example 3

[0073] The nicotinic acid used in the fourth step of Example 1 was replaced by 3-pyridinepropionic acid. Other steps were the same as in Example 1 to obtain Compound A3 with a yield of 53.5%.

[0074] Example 4

[0075] The nicotinic acid used in the fourth step of Example 1 was replaced by 3-pyridyloxyacetic acid. Other steps were the same as in Example 1 to obtain Compound A4 with a yield of 40.6%.

[0076] Example 5

[0077] The nicotinic acid used in the fourth step of Example 1 was replaced by 3-pyridine acrylic acid. Other steps were the same as in Example 1 to obtain Compound A5 with a yield of 45.2%.

[0078] Example 6

[0079] The nicotinic acid used in the fourth step of Example 1 was replaced by 2-(pyridin-3-yl)cyclopropanecarboxylic acid. Other reactions were the same as in Example 1 to obtain Compound A6 in a yield of 37.5%.

[0080] Example 7

[0081] The nicotinic acid used in the fourth step of Example 1 was replaced by thieno[2,3-b]pyridine-2-carboxylic acid. Other steps were the same as in Example 1 to obtain Compound A7 with a yield of 33.5%.

[0082] Example 8

[0083] The nicotinic acid used in the fourth step of Example 1 was replaced by thieno[2,3-c]pyridine-2-carboxylic acid. Other steps were the same as in Example 1 to obtain Compound A8 with a yield of 48.8%.

[0084] Example 9

[0085] Preparation of N-(4-chlorobenzyl)-N-cyclopropyl-4-(3-(pyridin-3-ylmethyl)ureido)benzenesulfonamide (B1)

[0086]

[0087] The cyclopentylamine used in the first step of Example 1 was replaced by cyclopropane, and the other steps were followed as in the first to third steps of Example 1 to obtain Intermediate IIIa with a yield of 78.4%.

[0088] The first step is to prepare intermediate IVa: (4-(N-cyclopentyl-N-(4-chlorobenzyl)sulfamoyl)phenyl)carbamic acid phenyl ester

[0089] Intermediate IIIa (0.36 g, 1.0 mmol) was dissolved in 20 mL of THF, and phenyl chloroformate (0.18 g, 1.2 mmol) was added. The mixture was allowed to react at room temperature for 4 h. After completion of the reaction, the mixture was extracted with EA / water (100 mL x 2). The combined organic phases were separated and purified by silica gel column chromatography (PE / EA = 3 / 1) to obtain 0.46 g of Intermediate IVa as a white solid in a 95% yield. 1 H NMR (600MHz, DMSO-d6) δ: 10.73 (s, 1H), 7.86 (d, 2H, J = 8.9Hz), 7.73 (d, 2H, J = 8.7Hz), 7.39-7.47 (m, 6H), 7. 26-7.31(m,3H),4.33(s,2H),4.17-4.23(m,1H),1.44-1.47(m,4H),1.33-1.36(m,2H),1.13-1.18(m,2H). 13 C NMR(600MHz,DMSO-d6)δ:152.08,150.74,143.26,139.52,133.93,131.78,129.97,129.82,129. 09,128.90,128.61,126.18,122.39,118.66,115.68,59.36,46.43,28.95,23.37HRMS(ESI):m / z calcd.for C 25 H 25 ClN2O4S 484.1224,found483.1156.[MH] - .

[0090] The second step is to prepare compound B1:

[0091] Intermediate IVa (0.33 g, 0.7 mmol) was dissolved in 20 mL of 1,4-dioxane, and triethylamine (0.21 g, 2.1 mmol) and 3-pyridinemethylamine (0.09 g, 0.86 mmol) were added. The mixture was allowed to react at 70°C for 4 h. After completion of the reaction, the mixture was extracted with EA / water (100 mL x 2) and backwashed with saturated sodium chloride (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 100:5) to obtain 141 mg of a yellow solid, Compound B1, in a 42% yield.

[0092] Example 10

[0093]

[0094] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate III to prepare intermediate IVb with a yield of 90.2%.

[0095] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVb. Other reactions were the same as in Example 9 to obtain compound B2 in a yield of 45.6%.

[0096] Example 11

[0097] The 3-pyridinemethylamine used in Example 10 was replaced by 3-pyridineethylamine. Other reactions were the same as in Example 10 to obtain Compound B3 in a yield of 36.2%.

[0098] Example 12

[0099] The 3-pyridinemethylamine used in Example 10 was replaced by 3-pyridinepropylamine. Other reactions were the same as in Example 10 to obtain Compound B4 in a yield of 26.9%.

[0100] Example 13

[0101]

[0102] The p-chlorobenzaldehyde used in the first step of Example 1 was replaced by p-methoxybenzaldehyde. The other steps were followed from the first step to the third step of Example 1 to obtain Intermediate IIIc with a yield of 88%.

[0103] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate IIIc to prepare intermediate IVc with a yield of 90%.

[0104] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVc. Other reactions were the same as in Example 9 to obtain compound B5 in a yield of 45.8%.

[0105] Example 14

[0106] The 3-pyridinemethylamine used in Example 13 was replaced by 3-pyridineethylamine. The other reactions were the same as in Example 13 to obtain Compound B6 in a yield of 34.5%.

[0107] Example 15

[0108] The 3-pyridinemethylamine used in Example 13 was replaced by 3-pyridinepropylamine. The other procedures were the same as in Example 13 to obtain Compound B7 in a yield of 30.8%.

[0109] Example 16

[0110]

[0111] The p-chlorobenzaldehyde used in the first step of Example 1 was replaced by p-fluorobenzaldehyde. The other steps were followed from the first step to the third step of Example 1 to obtain Intermediate IIId in a yield of 86%.

[0112] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate IIId to prepare intermediate IVd with a yield of 90%.

[0113] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVd. Other reactions were the same as in Example 9 to obtain compound B8 in a yield of 32.5%.

[0114] Example 17

[0115] The 3-pyridinemethylamine in Example 16 was replaced by 3-pyridineethylamine. Other reactions were the same as in Example 16 to obtain compound B9 in a yield of 24.6%.

[0116] Example 18

[0117] The 3-pyridinemethylamine in Example 16 was replaced by 3-pyridinepropylamine. Other reactions were the same as in Example 16 to obtain Compound B10 in a yield of 17.8%.

[0118] Example 19

[0119]

[0120] The cyclopentylamine used in the first step of Example 1 was replaced by cyclohexylamine, and the other steps were followed as in the first to third steps of Example 1 to obtain intermediate IIIe with a yield of 82%.

[0121] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate IIIe to prepare intermediate IVe with a yield of 95%.

[0122] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVe. The other procedures were the same as in Example 9 to obtain compound B11 in a yield of 35.4%.

[0123] Example 20

[0124] The 3-pyridinemethylamine in Example 19 was replaced by 3-pyridineethylamine. Other reactions were the same as in Example 19 to obtain Compound B12 in a yield of 33.5%.

[0125] Example 21

[0126] The 3-pyridinemethylamine in Example 19 was replaced by 3-pyridinepropylamine. Other reactions were the same as in Example 19 to obtain Compound B13 in a yield of 19.6%.

[0127] Example 22

[0128]

[0129] The p-chlorobenzaldehyde used in the first step of Example 1 was replaced by p-methoxybenzaldehyde. The intermediate IIIf was prepared according to the first to third steps of Example 1 with a yield of 78%.

[0130] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate IIIf to prepare intermediate IVf with a yield of 93%.

[0131] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVf. The other procedures were the same as in Example 9 to obtain compound B14 in a yield of 35.7%.

[0132] Example 23

[0133] The 3-pyridinemethylamine in Example 22 was replaced by 3-pyridineethylamine. Other reactions were the same as in Example 22 to obtain Compound B15 in a yield of 35.4%.

[0134] Example 24

[0135] The 3-pyridinemethylamine in Example 22 was replaced by 3-pyridinepropylamine. Other reactions were the same as in Example 22 to obtain compound B16 in a yield of 25.7%.

[0136] Example 25

[0137]

[0138] The p-chlorobenzaldehyde used in the first step of Example 1 was replaced by p-fluorobenzaldehyde, and the other steps were followed as in the first to third steps of Example 1 to obtain intermediate IIIg with a yield of 90%.

[0139] According to the first step ratio in Example 9, intermediate IIIa was replaced by intermediate IIIg to prepare intermediate IVg with a yield of 92%.

[0140] The intermediate IVa used in the second step of Example 9 was replaced by intermediate IVg. The other procedures were the same as in Example 9 to obtain compound B17 in a yield of 34.2%.

[0141] Example 26

[0142] The 3-pyridinemethylamine in Example 25 was replaced by 3-pyridineethylamine. Other reactions were the same as in Example 25 to obtain Compound B18 in a yield of 29.5%.

[0143] Example 27

[0144] The 3-pyridinemethylamine in Example 25 was replaced by 3-pyridinepropylamine. Other reactions were the same as in Example 25 to obtain compound B19 in a yield of 25.3%.

[0145] Example 28: In vitro enzyme inhibitory activity test of the compounds of the present invention

[0146] 1. NAMPT protein inhibitory activity of target compound

[0147] (1) Principle: NAMPT protein catalyzes the conversion of nicotinamide NAM into the product nicotinamide mononucleotide NMN; in the detection reaction, after two steps of chemical reaction, NMN is converted into a fluorescent derivative, and the maximum fluorescence signal can be detected at an excitation wavelength of 382 nm and an emission wavelength of 445 nm.

[0148] (2) Material preparation

[0149] a. Test solution: bovine serum albumin (BSA, Kingmorn) + Tris-HCl (pH 7.5) + MgCl2 (Sangong)

[0150] ATP solution (100 mM, Sangon), PRPP solution (40 mM, source leaf), DTT solution (2 mM, Sangon)

[0151] b. Compound gradient dilution solution

[0152] c. NAM solution (0.2 μM, Sangon); d. 20% acetophenone 2M KOH 88% formic acid.

[0153] (3) Enzyme activity determination method

[0154] a) 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;

[0155] b) 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 incubate at 37°C for 15 minutes;

[0156] c) After the reaction, heat at 95°C for 1 minute and then quickly cool on ice to terminate the enzyme reaction;

[0157] d) Add 10 μL each of 20% acetophenone and 2M KOH to the stopped enzyme reaction solution, quickly centrifuge, mix on a vortex mixer, and react at 0°C for 10 minutes;

[0158] e) Add 45 μL of 88% formic acid and incubate at 37°C for 10 min;

[0159] f) After cooling, transfer 85 μL of the 90 μL reaction mixture to a black flat-bottom 96-well fluorescent plate;

[0160] g) 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 9 software.

[0161] 2. Compound-probe competitive inhibition of PDEδ test

[0162] (1) Experimental materials:

[0163] PDEδ protein, buffer (PBS+charps+DMSO), 96-well black plate.

[0164] (2) Experimental methods:

[0165] The obtained compounds were tested for competitive protein binding based on the fluorescence polarization (FP) assay:

[0166] a. Protein binding constant K of fluorescently labeled Atorvastatin D1 Fluorescently labeled atorvastatin was dissolved in DMSO and diluted to 24 nM (0.5% DMSO) in PBS buffer (0.05% Chaps). The mixture was then added to a serial dilution of PDEδ protein and incubated in the dark for 3 hours at room temperature. Fluorescence anisotropy values ​​were read using a Biotek Synergy microplate reader (ex: 485 nm, em: 535 nm). The protein binding constant was fitted to the fluorescence anisotropy values ​​using Mathematica 7 (Wolfram Research Inc.).

[0167] b. Conduct compound activity assay (K D2):Select Deltazinone reported in the literature as the positive control, with a protein concentration of 40 nM and a probe of 25 nM. Then, the compound was serially diluted by half. 50 μL of the compound was added to 50 μL of the protein-probe mixture and incubated overnight at 30 °C with shaking for 13 h. Fluorescence values and anisotropy values were measured using a Biotek Synergy H2 microplate reader (excitation wavelength: 485 nm, detection wavelength: 528 nm).

[0168] (3) Result calculation: Data fitting was performed using Mathamatica 9 software (Wolfram Research Inc.).

[0169] The experimental results showed that most of the compounds of the present invention exhibited good NAMPT and PDEδ inhibitory activities, all at the nanomolar level. The results are shown in Table 1. Most of the prepared amide-based Series A molecules had poor inhibitory activities against NAMPT and PDEδ. For example, A1 was inactive against NAMPT, but the extension of the carbon chain length improved the inhibitory activity against NAMPT (Compound A3 vs A2). In addition, when the cyclopentane was replaced with cyclopropane or cyclohexane, the activities against both NAMPT and PDEδ were lost simultaneously. This indicates that the size of the cycloalkane has a greater impact on the activity of PDEδ. Subsequently, while maintaining the number of carbon atoms between the pyridine ring and the amide bond greater than 3 and replacing different pyridine heterocyclic acids, the results showed a decrease in activity.

[0170] Most of the target molecules in Series B exhibited excellent dual-target inhibitory activities against NAMPT and PDEδ. By extending the carbon chain length, the results showed that the inhibitory activity of the compound against NAMPT gradually increased (e.g., Compound B2 < B3 < B4); when changing the size of the substituted ring, the inhibitory activity against NAMPT decreased significantly as the ring became smaller, and when using a cyclohexyl substitution, the inhibitory activity against PDEδ decreased, while changing the carbon chain length had no obvious effect on the activity.

[0171] Table 1. Inhibitory activities of target compounds against NAMPT and PDEδ

[0172]

[0173] Example 29: In vitro anti-tumor activity test of the compounds of the present invention

[0174] The CCK8 method was used to test the in vitro anti-tumor activity of the synthesized compounds of the present invention.

[0175] (1) Experimental materials: cell lines (human pancreatic cancer MiaPaca-2 cells, human colon cancer HCT116 cells, human lung cancer A549 cells), culture medium (DMEM high glucose medium, F12K medium, cytiva), phosphate buffered saline (PBS, cytiva), penicillin-streptomycin mixture (double antibody, cytiva), CCK8 test kit (Yishen), fetal bovine serum (FBS, Yazyme), horse serum (Kingmorn).

[0176] (2) Instrument: Gen5 operating software, Bioteck Synergy2 multifunctional microplate reader.

[0177] (3) Preparation of culture medium:

[0178] MiaPaca-2 cell culture medium: 90% DMEM + 9.75% FBS + 1% double antibody + 0.25% horse serum

[0179] HCT116 cell culture medium: 90% DMEM + 10% FBS + 1% double antibody

[0180] A549 cell culture medium: 90% F12K + 10% FBS + 1% double antibody (0.25% horse serum)

[0181] CCK8 test solution preparation: 90% DMEM basal medium + 10% CCK-8 test kit reagent

[0182] (4) Experimental methods:

[0183] a) Observe the status of cells in the culture dish. Digest the cells in the logarithmic growth phase with 0.25% trypsin, collect them by pipetting, centrifuge and discard the supernatant. Add 2-4 mL of fresh culture medium, pipette evenly, and count the cells using a cell counter.

[0184] b) Seed 6,000 cells / well in a 96-well plate, add PBS (100 μL) around the plate, and culture in a cell incubator at 37°C, 5% CO2 for 24 hours.

[0185] c) Add the prepared test compound (100 μL) at different concentrations and set up triplicate wells;

[0186] d) Place the cells in a cell incubator and culture for 48-72 hours. Remove the culture medium and add the prepared CCK8 test solution (100 μL).

[0187] e) After incubation at 37°C for 15-30 minutes, measure the OD value of the sample at 452 nm using a microplate reader;

[0188] Table 2. In vitro antitumor activity results of target compounds IC50 (μM)

[0189]

[0190]

[0191] The experimental results show that the compound of the present invention has a broad spectrum of anti-tumor activity, and its IC 50 The values ​​were between 0.001 and 10 μM. Compared with the combined administration group, most compounds showed superior or equivalent anti-proliferative activity, and the in vitro anti-tumor activity was basically consistent with the inhibitory activity of the compounds against NAMPT-PDEδ.

[0192] The activity data of the compounds prepared by the present invention were analyzed and the following conclusions were drawn: (1) The overall activity of the compounds prepared by the present invention against MiaPaca-2 and A549 cells was better than that against HCT116 cells; (2) Compared with the amide A series compounds, the urea-containing B series compounds were more active; (3) In the compounds prepared by the present invention, when the distance between the amide bond and the pyridine ring was less than 3 carbon atoms, the activity was almost lost; (4) When the inactive essential group hydrophobic ring was replaced by rings of different sizes, the activity was in the order of five-membered ring ≈ six-membered ring > three-membered ring; (5) When the substituent was an electron-withdrawing group (fluorine substitution, chlorine substitution), the activity was slightly weaker than that of the electron-donating group (methoxy group); in addition, when the distance between the urea group and the pyridine group was changed, there was no significant difference in the activity.

[0193] Based on the above results, compound B5 has good molecular level inhibitory activity (NAMPT IC 50 The value was 3.41 nM, and the PDEδIC 50 In view of the balanced and efficient dual-target inhibitory effect and excellent in vitro anti-tumor activity exhibited by compound B5, compound B5 was selected for subsequent biological activity evaluation.

[0194] Example 30 In vivo antitumor activity of preferred compounds

[0195] According to the results of in vitro anti-tumor experiments and the structural characteristics of the compounds, human pancreatic cancer MiaPaca-2 cells were selected as a nude mouse transplant tumor model, compound B5 was used as the research object, and MS0, compound 4 and the combination of MS0 and compound 4 were used as positive control drugs. A blank control group, a B5 high-dose group (30 mg / kg), a B5 low-dose group (20 mg / kg), a PDEδ inhibitor compound 4 group (20 mg / kg), a NAMPT inhibitor MS0 group (10 mg / kg), and a compound 4 and MS0 combination group (compound 4 20 mg / kg, compound MS0 10 mg / kg) were set up. Nude mice were purchased from Shanghai Yuanchuang Biotechnology Co., Ltd. (weighing 18-20 g). The MiaPaca-2 cell suspension was subcutaneously implanted into the right axillary area of ​​the mouse. When the implanted tumor grew and reached a volume of about 100-300 mm 3 The mice were randomly divided into groups (6 per group) after starting the drug administration. Compound B5 was suspended in 0.5% carboxymethyl cellulose and administered by intraperitoneal injection at a dose of 20 mg / kg and 30 mg / kg for 21 consecutive days. In addition, MS0 (10 mg / kg), compound 4 (20 mg / kg), compound 4+MS0 or B5 (20 mg / kg, 30 mg / kg) were treated intraperitoneally every day for 21 days, and the blank control group mice received 0.5% carboxymethyl cellulose. Tumor volume was monitored by measuring the length and width with a caliper, and TV was calculated using the following formula = 1 / 2 × a × b 2 Where a is the tumor length and b is the width. Tumor volume and body weight were monitored every 2 days during the dosing process. Mice were sacrificed on day 21 after dosing, and tumors in each group were removed and recorded for analysis.

[0196] The experimental results are as follows Figure 1 As shown, Figure 1The figure is a schematic diagram of the in vivo anti-tumor efficacy results of compound B5. A is a schematic diagram of the results of the construction of the MiaPaca-2 cell in vivo xenograft mouse model, illustrating the successful construction of the MiaPaca-2 pancreatic cancer xenograft model; B is a schematic diagram of the growth curve of nude mice with MiaPaca-2 pancreatic cancer xenografts. The results show that a daily intraperitoneal injection of 20 mg / kg B5 resulted in a tumor growth inhibition (TGI) of 59.4%. Compared with the blank control group, this anti-tumor activity was superior to that of 10 mg / kg MS0 (TGI: 13.0%) or 20 mg / kg compound 4 (TGI: 26.8%) alone, and superior to the combination of compound 4 and MS0 (TGI: 44.1%). Notably, when compound B5 was administered at a dose of 30 mg / kg (TGI: 65.9%), the antitumor effect was further enhanced, significantly inhibiting tumor size and weight. Figure C is a schematic diagram of changes in mouse body weight during treatment, showing no significant weight loss in any group, confirming that the compound did not cause significant adverse reactions in the mice. Figure D is a schematic diagram of changes in major organ (heart, liver, spleen, lung, and kidney) and tumor weights in all groups, demonstrating that compound B5 significantly reduced tumor weight in the group treated with compound B5, with no significant effects on other major organs. Figure E is a schematic diagram of tumor tissue dissection. Data are expressed as mean ± SD, n = 3. *P < 0.05 and **P < 0.01, compared with the control group. In summary, compound B5, as a potent NAMPT-PDEδ dual inhibitor, exhibits potent antitumor activity against pancreatic tumors without significant toxicity.

[0197] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof as a NAMPT-PDEδ dual-target inhibitor, characterized in that: The general structural formula is selected from one of the following structures: wherein X is selected from -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, R1 is selected from R2 is selected from hydrogen, halogen, C1-C10 alkoxy, C1-C10 alkyl; R3 is selected from hydrogen, halogen, C1-C10 alkoxy, C1-C10 alkyl; R4 is selected from hydrogen, halogen, C1-C10 alkoxy, C1-C10 alkyl; R5 is selected from hydrogen, halogen, C1-C10 alkoxy, C1-C10 alkyl; R6 is selected from hydrogen, halogen, C1-C10 alkoxy, C1-C10 alkyl; R7 is selected from hydrogen, halogen; R8 is selected from hydrogen, halogen; R9 is selected from hydrogen, halogen; R 10 is selected from hydrogen and halogen; R 11 Selected from hydrogen and halogen.

2. The compound as a NAMPT-PDEδ dual-target inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound as a NAMPT-PDEδ dual-target inhibitor is selected from one of the following structures:

3. Use of the compound as a NAMPT-PDEδ dual-target inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of a NAMPT-PDEδ dual-target inhibitor.

4. Use of the compound as a NAMPT-PDEδ dual-target inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of an anti-tumor drug, characterized in that: The tumor is selected from pancreatic cancer, colorectal cancer, and lung cancer.

5. The use according to claim 4, characterized in that The tumor cells are selected from MiaPaca-2 human pancreatic cancer cells, HCT116 human colon cancer cells, and A549 human lung cancer cells.

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