A NAMPT protein-targeting degrader and its preparation method and use

Abstract

The present invention relates to a nicotinamide phosphoribosyltransferase (NAMPT) protein degradation-targeting chimeric (PROTAC) compound, a preparation method thereof, and an application in medicine, belonging to the technical field of medicine. Specifically, the compound of the present invention is shown by the general formula (I). In the general formula, E3 ligase is an E3 ubiquitin ligase ligand; Linker is a linking group. The NAMPT protein degrader with this structure can specifically recognize the NAMPT protein, effectively ubiquitinate and degrade the NAMPT protein, thereby inducing the death of tumor cells. The compound of the present invention or a pharmaceutically or physiologically acceptable salt thereof can be used to prepare a drug for cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and relates to a compound for targeting the degradation of human nicotinamide phosphoribosyltransferase (NAMPT), a preparation method thereof, and an application thereof in a medicament for preventing and / or treating cancer. Background Art

[0002] NAD + (nicotinamide adenine dinucleotide) is a coenzyme that plays a key role in many physiologically necessary processes (Ziegkel, M. Eur. J. Biochem. 267, 1550 - 1564, 2000). NAD is essential for some signal transduction pathways, including poly-ADP-ribosylation in DNA repair, mono-ADP-ribosylation in the immune system and G protein-coupled signal transduction, and the deacetylase activity of deacetylases also requires NAD (Garten, A. et al. Trends in Endocrinology and Metabolism, 20, 130 - 138, 2008). NAD is involved in processes such as cell substance, energy metabolism, protein modification, and DNA repair. When the human body is in a stress state or suffering from a disease, the content of NAD in the human body + will increase.

[0003] NAMPT (Nicotinamide Phosphoribosyltransferase), also known as PBEF (pre-B cell colony-enhancing factor) or Visfatin (visceral fat adipokine), is a rate-limiting enzyme in the salvage synthesis pathway of NAD (Nicotinamde adenine dinucleotide) in mammals.

[0004] NAMPT is widely distributed in the human body. The mechanism of action of NAMPT is different inside and outside human cells (cytoplasm, nucleus, and mitochondria). Inside the cell, NAMPT mainly regulates cell metabolism, promotes cell differentiation and maturation, promotes cell growth, and maintains the stability of various cell functions by synthesizing NAD. Outside the cell, NAMPT can not only play a role by synthesizing NMN (nicotinamide mononucleotide), but also has a non-enzymatic function, that is, NAMPT can act as a cytokine and play a role by acting on its unknown receptor, such as generating reactions mediating inflammation, oxidative stress, etc.

[0005] Studies have shown that rapidly proliferating tumor cells require more NAD and have a higher basal turnover of NAD. The content of NAMPT in tumor cells is much higher than that in normal cells. By inhibiting the expression of NAMPT in tumor cells, the growth of tumor cells can be inhibited. Increased NAMPT expression has been reported in colorectal cancer (Int. J. Cancer 101, 118 - 127, 2002), and NAMPT is involved in angiogenesis (Biochem. Biophys. Res. Commun. 357, 150 - 156, 2007). Small molecule inhibitors of NAMPT have been shown to cause a decrease in intracellular NAD + levels and ultimately induce tumor cell death (Anticancer Res. 20, 4211 - 4220, 2000) and inhibit tumor growth in xenograft models (Mol. Cancer Ther. 9, 1609 - 1617, 2010). Therefore, NAMPT has become a new target for anti-tumor drug research.

[0006] Existing NAMPT inhibitors include FK866 and CB30865. Five anti-tumor clinical trials of FK866 have been conducted, but FK866 and CB30865 have poor anti-tumor effects due to easy metabolism during use, and at the same time have relatively large side effects. Therefore, new NAMPT inhibitors are still being actively explored.

[0007] Compared with the mechanism of action of traditional small molecule inhibitors that inhibit the function of target proteins by binding to the active sites of target proteins, PROTAC (targeted protein degradation conjugate) achieves the effect of disease treatment by degrading target proteins. This new mode of action has currently received extensive attention. PROTAC consists of a small molecule ligand that recognizes the target protein, a linker, and an E3 ubiquitin ligase ligand. This bifunctional molecule can degrade the target protein through the ubiquitin-proteasome pathway in vivo to exert its therapeutic effect. Since its affinity requirement for the small molecule ligand of the target protein is not high and it can exert the protein degradation function only at a very low concentration, this technology can well overcome the serious drug resistance problem of existing drugs. Summary of the Invention

[0008] The main content of the present invention is to address the efficacy and side effect problems of existing NAMPT small molecule inhibitors, and provide a NAMPT degrader based on the PROTAC strategy of targeted protein degradation, its preparation method and application, as well as the application of this type of NAMPT degrader in prevention, alleviation or treatment (such as cancers like colon cancer and gastric cancer).

[0009] The present invention first uses chemical proteomics technology to find small molecules that can specifically bind to NAMPT, serving as the target protein ligands of the compounds for targeted degradation of NAMPT described in the present invention. Subsequently, this small molecule is chemically linked to the ligand recognized by the ubiquitin ligase E3, and it is confirmed that this compound can specifically degrade intracellular NAMPT protein through the ubiquitination pathway.

[0010] To achieve the above object, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention provides a compound of formula (I)-1 and formula (I)-2 for targeted degradation of human nicotinamide phosphoribosyltransferase (NAMPT), its optical isomers, and its pharmaceutically or physiologically acceptable salts or solvates,

[0011]

[0012] wherein: in formula (I)-1 and formula (I)-2, Linker represents a linking chain; E3 Ligand represents the ligand part of the E3 ubiquitin ligase, and the ligand part of the E3 ubiquitin ligase is the cerebroside E3 ubiquitin ligase binding part (CLM) or the VHLE3 ligase binding part (VLM).

[0013] Preferably, Linker is any one of the structures shown in formula (II) or any combination thereof; wherein m represents a natural number from 1 to 10, and represents the connection point of the connecting part, and the hydrogen in formula (II) is optionally substituted by one or more halogens, cyano groups, nitro groups, amino groups, hydroxyl groups, C1-C10 alkyl groups, C1-C10 alkoxy groups, halogen-substituted C1-C10 alkyl groups, and halogen-substituted C1-C10 alkoxy groups;

[0014]

[0015] Preferably, Linker is a straight-chain or branched alkylene or alkoxy group that is optionally interrupted one or more times by one or more groups selected from the following: -(CH2) n -、-(CH2) n CO-、-NR1(CH2) n CO-、-NR2(CH2) n -、-(OCH2CH2O) n -、-(CH2CH2O) n -、-(OCH2CH2OCH2) n -、-(CH2CH2OCH2) n -、-(CH2CH2OCH2CH2) n- alkenylene, alkynylene, cycloalkylene, heteroarylene or any combination thereof; wherein n represents a natural number from 0 to 20, and R1 and R2 each independently select H or C 1-10 alkyl.

[0016] Preferably, the ligand part of the E3 ubiquitin ligase is any one of the structures of the compound shown in formula (IV)-1, wherein represents the connection point of the connection part;

[0017]

[0018] wherein X is any one of O, N or S; W is CH2 or C═O; the hydrogen in any structure of formula (IV)-1 is optionally substituted by one or more halogens, cyano, nitro, amino, hydroxyl, C1-C10 alkyl, C1-C10 alkoxy, halogen-substituted C1-C10 alkyl, halogen-substituted C1-C10 alkoxy.

[0019] More preferably, the ligand part of the E3 ubiquitin ligase is any one of formula (IV);

[0020]

[0021] Preferably, the NAMPT-targeting degrading compounds provided by the present invention include, but are not limited to, the compounds NP1 to NP10 shown in Table 1 below:

[0022] Table 1 Structures of Preferred NAMPT-Targeting Degrading Compounds NP1 to NP10

[0023]

[0024]

[0025] In a second aspect, a pharmaceutical composition is provided, comprising compounds of formula (I)-1 and formula (I)-2 that target and degrade human nicotinamide phosphoribosyltransferase (NAMPT) as pharmaceutically active components; preferably, the pharmaceutical composition contains a second pharmaceutically active component, and the second pharmaceutically active component is at least one of ibrutinib, cyclophosphamide, doxorubicin, cytarabine, artemisinin, dihydroartemisinin, artesunate, carfilzomib, thalidomide, lenalidomide, pomalidomide, gefitinib, erlotinib, ostatin, afatinib, orlistat, nilutamide.

[0026] In a third aspect, there is provided the use of a compound of formula (I)-1 and formula (I)-2 that targets and degrades human nicotinamide phosphoribosyltransferase (NAMPT) or the above pharmaceutical composition for the preparation of a drug for diseases associated with overexpression of NAMPT protein.

[0027] Preferably, the disease associated with NAMPT protein overexpression is a non-solid tumor or a solid tumor; further preferably, the non-solid tumor is leukemia (including acute myeloid leukemia and chronic myeloid leukemia). The solid tumor is cervical cancer, breast cancer, liver cancer, or colon cancer.

[0028] Preferably, the solid tumor is a cancer caused by any one of the tumor cells of HCT-116, HeLa, HepG2, K562 and MCF-7.

[0029] Beneficial effects of the present invention:

[0030] To date, most PROTACs have been designed based on target protein inhibitors. This invention breaks with the conventional PROTAC design approach of using inhibitors as target protein ligands by using steviol, a natural food additive that has no effect on catalytic function, as a NAMPT ligand, significantly improving drug safety. The compounds of this invention exhibit significant NAMPT degradation and tumor cell proliferation inhibition, demonstrating their potential as anti-tumor drugs for the treatment of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The biotin-steviol probe enriched NAMPT in cell lysate. Figure A shows the silver staining of the biotin-steviol probe specifically enriched proteins in HCT116 cells; Figure B shows the mass spectrometry identification result of the 55 kDa band in Figure A as NAMPT; Figure C verifies the 55 kDa band in Figure A using a NAMPT antibody.

[0032] Figure 2 Steviol photoaffinity probe labels NAMPT protein in cancer cells.

[0033] Figure 3 NAMPT recombinant protein was labeled with steviol photoaffinity probe.

[0034] Figure 4 Western blot assay was used to determine the degradation activity of the compounds on NAMPT.

[0035] Figure 5 Compounds NP1 to NP10 reduced the NAD+ content in HCT116 cells. DETAILED DESCRIPTION

[0036] Example 1 Biotin-steviol probe enriches NAMPT in cell lysate.

[0037] The structural formula of Biotin-Steviol is:

[0038]

[0039] Its synthesis method refers to the literature ACS Chem. Biol. 2018, 13, 1944.

[0040] Dilute the HeLa cell lysate to a final concentration of 5 μg / μL in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2), add 10 μM biotin-steviol and incubate at room temperature for 1 hour. Subsequently, add 50 μL of streptavidin resin (GE Healthcare) and incubate at room temperature for 2 hours. Wash away the unbound proteins on the streptavidin resin, elute the proteins bound to the streptavidin resin with 1 mM steviol, concentrate the eluate to 30 μL using an ultrafiltration tube, add 10 μL of 4×SDS loading buffer (Invitrogen) and boil at 95 °C for 15 minutes, then apply all samples to a NUPAGE 4-12% Bis-tris denaturing gel (Invitrogen) and visualize by silver staining. As Figure 1 shown in A, the biotin-steviol probe successfully enriched a 56 kDa protein. After cutting this band, in-gel digestion was performed, and it was identified as human nicotinamide phosphoribosyltransferase (NAMPT, Figure 1 B) by mass spectrometry. At the same time, after separating the eluted sample through a NUPAGE 4-12% Bis-tris denaturing gel (Invitrogen), it was transferred to a PVDF membrane and verified using a NAMPT antibody. Figure 1 C shows that the 56 kDa protein enriched by the biotin-steviol probe is indeed NAMPT.

[0041] Example 2: Labeling of NAMPT protein in cancer cells with steviol photoaffinity probe.

[0042] The structural formula of the steviol photoaffinity probe (Steviol-Dayne) is:

[0043]

[0044] Its synthesis method refers to the literature ACS Chem. Biol. 2018, 13, 1944.

[0045] Human colon cancer HCT-116, breast cancer MCF-7, liver cancer HepG2, cervical cancer HeLa, and lymphoma K562 cells were all cultured in serum-free DMEM medium. Steviol-Dayne probe with a final concentration of 10 μM was added to the medium and incubated at 37 °C for 1 hour. The cells were placed on ice, irradiated with ultraviolet light at 365 nm for 15 minutes, and then lysed with lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5 mM MgCl2) and diluted to a final concentration of 5 μg / μL. After adding SDS with a final concentration of 1% to the protein sample, the "click" reaction was carried out: for each reaction, 0.2 μL of TAMRA-N3 (10 mM DMSO solution, Lumiprobe), CuSO4 (100 mM aqueous solution), THPTA (10 mM aqueous solution, Sigma), and sodium ascorbate (100 mM aqueous solution) were sequentially added to 19.2 μL of the protein sample. The sample was placed in the dark at room temperature for 1 hour. Then 5 μL of 4× SDS loading buffer (Invitrogen) was added and boiled at 95 °C for 15 minutes to end the reaction. The sample was applied to a NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and subjected to in-gel fluorescence scanning using a 4600SF imaging system (Tianneng, Shanghai). As Figure 2 shown, Steviol-Dayne can selectively label the 56 kDa NAMPT protein in various cells.

[0046] Example 3: Labeling of recombinant NAMPT protein with steviol photoaffinity probe.

[0047] The recombinant NAMPT protein was diluted to a final concentration of 0.1 μg / μL in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2) and incubated with 0.1 - 10 μM Steviol-Dayne probe at room temperature for 1 hour, followed by ultraviolet light irradiation at 365 nm on ice for 15 minutes. The subsequent "click" reaction and in-gel fluorescence scanning steps were the same as those in Example 2. As Figure 3 shown, the Steviol-Dayne probe can label the 56 kDa recombinant NAMPT protein in a concentration-dependent manner, and 1 μM probe can saturate the labeling of NAMPT protein in vitro.

[0048] Example 4: Preparation of NAMPT-targeted degrading compounds NP1 - NP10.

[0049] (1) NP1 was synthesized through the reactions shown in the following formulas (i) and (ii):

[0050]

[0051] Dissolve steviol (100 mg, 0.33 mmol) in 5 mL of anhydrous dichloromethane, and dropwise add oxalyl chloride (400 μL, 5 mmol) dissolved in 1 mL of anhydrous dichloromethane under ice bath conditions. After stirring at room temperature for 2 hours, remove all solvents under vacuum. After the product is dissolved in 5 mL of anhydrous dichloromethane again, add N,N-diisopropylethylamine (DIPEA, 50 μL, 0.5 mmol) and alkynylheptylamine (44.4 mg, 0.4 mmol) under ice bath conditions. After reacting at room temperature overnight, obtain the colorless oily product steviol-C5-yne (75.9 mg, yield 56%) by silica gel column chromatography. Dissolve steviol-C5-yne (66 mg, 0.15 mmol), thalidomide-O-C5-azide (69.3 mg, 0.18 mmol), and sodium ascorbate (89 mg, 0.45 mmol) in a mixed solution of acetonitrile (1.5 mL) and water (0.5 mL), then add copper sulfate (48 mg, 0.3 mmol) dissolved in 0.5 mL of water. After stirring at 40 °C for 30 minutes, purify by silica gel column to obtain the product NP-1 (105.3 mg, yield 88%).

[0052] NP1 1 H-NMR (400 MHz, CDCl3) δ 11.07 (1H, s), 7.96 (1H, s), 7.59 (1H, s), 7.53 (1H, s), 7.50 (1H, s), 4.98 (1H, s), 4.82 (1H, s), 4.42 (1H, s), 4.46 (2H, m), 4.06 (2H, m), 3.90 (2H, m), 2.21 (3H, m), 2.18 (3H, m), 2.11 (3H, m), 2.01 (1H, m), 1.88 (5H, m), 1.78 (5H, m), 1.70 (5H, m), 1.58 (4H, m), 1.46 (2H, m), 1.23 (7H, m), 1.05 (3H, m), 0.85 (3H, s). 1313C-NMR (100 MHz, CDCl3) δ 177.24, 173.70, 168.65, 167.88, 156.11, 155.56, 140.87, 132.85, 132.65, 130.31, 121.66, 117.86, 115.01, 102.92, 68.30, 68.73, 63.20, 56.94, 53.75, 52.65, 47.42, 47.01, 43.82, 41.66, 41.34, 40.67, 39.33, 39.23, 37.99, 32.23, 32.15, 29.65, 29.37, 28.78, 28.48, 26.32, 25.22, 23.32, 21.93, 21.33, 20.44, 19.07, 15.52, 13.28. HRMS-ESI calc’d for C 45 H 61 N6O7 [M+H] + : 797.4596; Found: 797.4502.

[0053] (2) The preparation methods of the NAMPT-targeted degradation compounds NP2 - NP4 in Table 1 are the same as above.

[0054] (3) NP5 was synthesized through the reaction shown by the following formula:

[0055]

[0056] The synthesis method of the compound Steviol-PEG2-NH2 was referred to the literature (ACS Chem. Biol. 2018, 13, 1944). Thalidomide-O-C3-acid (39.6 mg, 0.11 mmol) was dissolved in 5 mL of anhydrous dichloromethane. After stirring to dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 24 mg, 0.12 mmol), 1-hydroxybenzotriazole (HOBt, 16 mg, 0.12 mmol), and N,N-diisopropylethylamine (DIPEA, 35 μL, 0.2 mmol) were added in sequence. After stirring in an ice bath for 30 minutes, Steviol-PEG2-NH2 (45 mg, 0.1 mmol) dissolved in 2 mL of anhydrous dichloromethane was slowly added. After slowly rising to room temperature, it was stirred overnight. After purification by silica gel column, 63 mg of white solid was obtained with a yield of 79.7%.

[0057] NP5: 1H-NMR(400MHz,CDCl3)δ11.08(1H,s),7.78(1H),7.50(1H),7.43(1H),6.08(1H,s),5.05(1H,s),4.94(1H,s),4.75(1H,s),3.56(5H,m),3.48(5H,m),3.40(2H,s),3.27(2H,s),2.34(2H,m),2.24(2H,m),2.12(3H,m),2.04(5H,m),1.85(4H,m),1.74(2H,m),1.60(1H,m),1.50(2H,m),1.20(2H,m),1.11(3H,s),1.00(2H,m),0.87(3H,s). 13 C-NMR(100MHz,CDCl3)δ176.60,173.54,171.90,168.68,167.80,155.64,140.27,132.88,132.68,117.89,115.32,84.07,70.20,69.62,66.85,63.24,57.57,57.02,54.69,44.33,43.62,42.18,41.26,40.86,40.22,39.38,39.00,38.04,32.63,32.43,31.93,29.70,29.40,24.82,24.42,22.70,22.31,21.26,19.36,19.11,15.37,14.16.HRMS-ESI calc’d.for C 43 H 59 N4O 10 [M+H] + :791.4226;Found:791.4235.

[0058] (4) The preparation methods of the NAMPT-targeted degrading compounds NP6, NP7, and NP8 in Table 1 are the same as that of NP5.

[0059] (5) NP9 was synthesized through the reaction shown by the following formula:

[0060]

[0061] The VHL ligand (43 mg, 0.1 mmol) was dissolved in 5 mL of anhydrous dichloromethane. N,N-Diisopropylethylamine (DIPEA, 35 μL, 0.2 mmol) was added, and the mixture was stirred in an ice bath. Succinic anhydride (11 mg, 0.11 mmol) dissolved in 2 mL of anhydrous dichloromethane was slowly added. After slowly warming to room temperature, the mixture was stirred overnight. The product VHL-acid (47 mg) was obtained by silica gel column purification with a yield of 88.5%. The synthesis method of the compound Steviol-PEG2-NH2 was referred to the literature (ACS Chem. Biol. 2018, 13, 1944). VHL-acid (27 mg, 0.05 mmol) was dissolved in 5 mL of anhydrous dichloromethane. After stirring to dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 12 mg, 0.06 mmol), 1-hydroxybenzotriazole (HOBt, 8 mg, 0.06 mmol), and N,N-diisopropylethylamine (DIPEA, 18 μL, 0.1 mmol) were added in sequence. After stirring in an ice bath for 30 minutes, Steviol-PEG2-NH2 (23 mg, 0.05 mmol) dissolved in 2 mL of anhydrous dichloromethane was slowly added. After slowly warming to room temperature, the mixture was stirred overnight. The final product NP9, a white solid (43 mg), was obtained by silica gel column purification with a yield of 90.1%.

[0062] NP9: 1 1H-NMR (400 MHz, CDCl3) δ 9.07 (1H, s), 7.80 (2H, m), 7.46 (2H, m), 6.12 (1H, s), 5.01 (1H, s), 4.72 (1H, s), 4.38 (2H, s), 4.22 (1H, s), 4.07 (1H, m), 3.66 (6H, m), 3.49 (6H, m), 3.42 (2H, s), 3.25 (2H, s), 2.45 (3H, m), 2.33 (3H, m), 2.11 (3H, s), 2.03 (2H, m), 1.85 (4H, m), 1.74 (2H, m), 1.60 (1H, m), 1.50 (2H, m), 1.20 (2H, m), 1.12 (3H, s), 1.01 (2H, m), 0.94 (9H, s), 0.88 (3H, s). 1313C-NMR (100 MHz, CDCl3) δ 176.14, 173.35, 173.25, 172.20, 166.88, 155.74, 152.67, 150.21, 141.39, 137.65, 129.52, 127.32, 107.23, 83.87, 80.54, 70.85, 69.22, 68.32, 66.24, 58.57, 57.92, 57.82, 57.15, 54.64, 44.54, 43.62, 43.36, 42.78, 41.45, 40.88, 40.25, 39.45, 39.07, 38.12, 36.60, 35.26, 32.54, 31.82, 32.74, 32.14, 29.50, 25.76, 24.22, 22.54, 22.12, 19.24, 19.01, 15.25, 14.26, 12.15. HRMS-ESI calc’d. for C 52 H 77 N6O9S [M+H] + : 961.5476; Found: 961.5562.

[0063] (6) The preparation method of the NAMPT-targeted degrading compound NP10 in Table 1 is the same as above. Example 5 Test for the anti-proliferation activity of the NAMPT-targeted degrading compound in inhibiting cancer cell proliferation

[0064] As shown in Table 2, the compounds NP1-NP10 in the examples showed obvious anti-proliferation activities against five kinds of tumor cells, and their 24-hour half-maximal growth inhibitory GI 50 were all at the low numerical μM level, while the parent compound steviol had no activity. In summary, the compounds in the examples of the present invention have significant anti-tumor cell proliferation effects.

[0065] Table 2. 24-hour GI values of compounds NP1-NP10 in inhibiting the proliferation of five kinds of tumor cells 50 Value (μM)

[0066]

[0067]

[0068] As shown in Table 2, the compounds in the examples showed obvious anti-proliferation activities against five kinds of tumor cells, especially B-cell lymphoma K562 cells and HCT116 colon cancer cells. Among them, the GI 50 value of compound NP8 reached 0.14 μM. In summary, the compounds in the examples of the present invention have significant anti-tumor cell proliferation effects.

[0069] Example 6 Western Blot Experiment to Determine the Degradation Activity of Compounds against NAMPT

[0070] Inoculate colon cancer cells HCT116 at 5×10 5 cells / well into a 6-well cell culture plate, with the volume of the culture medium in each well being 2 mL, and culture for 24 hours. Add compounds NP1 - NP10 at different concentration gradients (0 μM, 0.1 μM, 1 μM, 10 μM) and culture for 12 hours. Collect the cells, add lysis buffer to collect proteins, and adjust the concentration of the lysis buffer to 5 mg / mL. Take 15 μL of the cell lysate, add 5 μL of 4×SDS loading buffer (Invitrogen), and after boiling at 95°C for 15 minutes, apply all the samples to a NUPAGE 4 - 12% Bis-tris denaturing gel (Invitrogen) for separation, then transfer to a PVDF membrane, and develop using NAMPT antibody. The results are as Figure 4 shown. It can be observed from the Western-Blot experiment that the example compounds NP1 - NP10 showed significant concentration-dependent degradation activity in HCT116 cells. The example compound NP2 had significant NAMPT degradation at 0.1 μM, and compounds NP1, NP3, NP5, and NP9 had significant NAMPT degradation at 1 μM; compounds NP4, NP6, NP7, NP8, and NP10 had significant NAMPT degradation at 10 μM, and the parent compound steviol had no degradation activity.

[0071] Example 7 Determination of the Effect of Compounds on the Intracellular NAD + Content

[0072] Seed HCT-116 cells in a 96-well plate and starve the cells with serum-free medium for 12 hours. After adding the test compound or DMSO to the cells and incubating for 24 hours, add 100 μL of 1 M HClO4 and lyse on ice for 30 minutes. After centrifugation at 14000 g, take 20 μL of the supernatant and add 40 μL of 1 M K2CO3 to neutralize on ice. Subsequently, add 10 μL of the neutralized sample to 90 μL of the reaction solution (50 mM Tris-HCl pH 7.5, 3% ethanol, 1.66 mM phenazine ethosulfate, 0.42 mM thiazolyl blue, 90 μg / mL alcohol dehydrogenase), incubate at 37°C for half an hour, and then measure the NAD + content by absorbance at 570 nm.

[0073] The quantitative results are as Figure 5 shown. It can be seen from the results in the figure that the effect of the example compounds on the NAD + content was significantly correlated with the degradation effect on NAMPT. NP1, NP2, NP3, NP5, and NP9 on NAD+ The effect of reducing the content is significantly stronger than that of the other five compounds. The parent compound steviol has no effect on the intracellular NAD + content.

[0074] In summary, the example compounds of the present invention have significant NAMPT degradation and cell proliferation inhibition effects, indicating that the example compounds of the present invention have the potential to treat tumors as anti-tumor drugs.

[0075] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A compound for targeted degradation of human nicotinamide phosphoribosyltransferase (NAMPT), its optical isomers, and its pharmacologically or physiologically acceptable salts, any one of which is selected from the structures of NP1 to NP10:

2. A pharmaceutical composition comprising the compound for targeted degradation of human nicotinamide phosphoribosyltransferase (NAMPT) described in claim 1 as a pharmaceutically active ingredient.

3. The composition according to claim 2, characterized in that: The pharmaceutical composition contains a second pharmaceutically active ingredient, and the second pharmaceutically active ingredient is at least one of ibrutinib, cyclophosphamide, doxorubicin, cytarabine, artemisinin, dihydroartemisinin, artesunate, carfilzomib, thalidomide, lenalidomide, pomalidomide, gefitinib, erlotinib, ostatin, afatinib, orlistat, nilutamide.

4. Use of the compound for targeted degradation of human nicotinamide phosphoribosyltransferase (NAMPT) described in claim 1 or the pharmaceutical composition described in claim 2 or 3 for the preparation of a drug for a disease related to overexpression of NAMPT protein.

5. The use according to claim 4, wherein: The diseases related to overexpression of NAMPT protein are non-solid tumors and solid tumors; the non-solid tumor is leukemia, and the solid tumors are cervical cancer, breast cancer, liver cancer or colon cancer.

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