Transforming growth factor-beta inhibitor, preparation thereof and application of transforming growth factor-beta inhibitor in preparation of antitumor drugs

Abstract

The invention belongs to the field of anti-cancer drugs, and particularly relates to a transforming growth factor-beta inhibitor # imgabs0 #. The invention further discloses preparation of the transforming growth factor-beta inhibitor and application of the transforming growth factor-beta inhibitor in preparation of anti-tumor drugs. The inhibitor provided by the invention has excellent transforming growth factor-beta inhibitory activity, and can effectively resist tumors based on the mechanism.

Description

Technical field:

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to the field of a class of transforming growth factor-β inhibitors. Background technology:

[0002] Transforming growth factor-β (TGF-β) is an important multifunctional cytokine that participates in many cellular processes, including cell proliferation, differentiation, self-renewal, and apoptosis. It is divided into six subtypes (TGFβ1 to 6) based on its homology. TGF-β1 is a prototype cytokine of the TGF-β family and transmits signals through two highly conserved single transmembrane serine / threonine kinase receptors, namely TGF-β1 receptor (also known as ALK5) and TGF-β2 receptor. In the classic TGF-β signaling pathway, TGF-β1 dimers bind to TGF-β2 receptors and induce phosphorylation of the GS domain of type 1 receptors. TGF-β1 receptors are then phosphorylated at the carbon terminus, recruiting and phosphorylating Smad2 or Smad3. Phosphorylated Smad proteins form heterologous complexes with Smad 4 and translocate to the nucleus to regulate the expression of specific genes, thereby participating in the processes of angiogenesis, epithelial-mesenchymal transition, and extracellular matrix remodeling. In addition, TGF-β balances the production and effector functions of many immune cell types in the adaptive and innate immune systems (E. Batlle, et al., Immunity. 2019; 50: 924-940.). Recent studies have shown that TGF-β plays a central role in tumor immune evasion and adverse reactions to cancer immunotherapy. The combination of TGF-β signaling blockade and checkpoint therapy can sensitize primary tumors and metastases to anti-PD-1 / L1 therapy, thereby enhancing immune responses (S. Mariathasan, et al., Nature, 2018, 554: 544-548.).

[0003] TGF-β1 is the most common and important subtype of the TGF-β superfamily, and is also the strongest known liver fibrosis inducing factor (Yamazaki, et al., Digestive Disease, 2011, 29: 284-288). Many studies have shown that TGF-β1 and TGF-β1 receptors are highly expressed in a variety of fibrotic diseases and tumors. TGF-β1 regulates the expression of a series of genes related to fibrosis and tumor progression through the action domain TGF-βR1 (HJBaek, et al., Hepatology. 2008; 48: 1128-37.). TGF-β stimulates the expression of metastatic factors through the Smad pathway, promotes osteolytic bone metastasis caused by melanoma, and the use of TGF-β receptor inhibitor SB431542 prevents bone metastasis (Javelaud D et al., Cancer Res. 2007; 67 (5): 2317-24). The TGF-β receptor inhibitor LY2109761 inhibited tumor growth and liver metastasis and prolonged survival in the pancreatic cancer orthotopic metastasis mouse model. In the orthotopic intracranial model of glioblastoma multiforme, LY2109761 significantly inhibited tumor growth and prolonged survival, and further prolonged the survival induced by radiotherapy (Melisi D et al., Mol Cancer Ther. 2008; 7(4): 829-40; Zhang M et al., Cancer Res. 2011; 71(23): 7155-67.). The TGF-β receptor inhibitor Galuniertib inhibited tumor growth and promoted immune response in hepatocellular carcinoma, breast cancer and non-small cell lung cancer models (Herbertz S et al., Drug Des Devel Ther. 2015; 9: 4479-99.). In bladder cancer, hepatocyte growth factor upregulates epithelial-mesenchymal transition and tumor cell invasion mediated by the TGFβ pathway. In vivo experiments show that inhibiting the TGFβ receptor exhibits significant anti-tumor effects (SimWJ et al., Nat Commun. 2019; 10(1): 4349.). TGFβ1 secreted by anaplastic thyroid cancer induces polarization of M2-like macrophages and plays a key role in upregulating SNAIL and SLUG transcription factors. Targeting the TGF-β receptor has the potential to treat thyroid cancer (Jaroszewski A et al., Am J Cancer Res. 2024; 14(7): 3626-3638.). Therefore, inhibiting the TGF-β signaling pathway is considered a feasible method for treating these diseases.

[0004] Drug development targeting the TGF-β signaling pathway has been carried out for many years. The TGF-βR1 inhibitors developed in the early stage usually show certain side effects such as cardiotoxicity in animal models due to their low selectivity (EF Wagner, et al., Nature Reviews Cancer. 2009; 9: 537-49). Inhibitors that have entered the clinical stage, such as Galunisertib and LY3200882, have limitations such as poor monotherapy effects. Therefore, those skilled in the art are still eager to obtain TGF-β1 inhibitors with higher selectivity and lower side effects for the treatment of liver cancer, lung cancer, pancreatic cancer, thyroid cancer, bladder cancer, glioblastoma and other cancers mediated by the TGF-β signaling pathway. Summary of the invention:

[0005] In view of the problem that the activity of existing transforming growth factor-β inhibitors is not ideal, the first purpose of the present invention is to provide a transforming growth factor-β inhibitor with a completely new structure, aiming to improve its TGF-β inhibitory activity and improve its anti-tumor activity.

[0006] The second object of the present invention is to provide a method for preparing a transforming growth factor-β inhibitor and its use in preparing a drug for inhibiting TGF-β.

[0007] The third object of the present invention is to provide an anti-tumor drug comprising the transforming growth factor-β inhibitor.

[0008] A transforming growth factor-β inhibitor, which is a compound having a structure of Formula 1 and at least one of its pharmaceutically acceptable salts, derivatives, cocrystals, crystals, crystals, and solvates;

[0009]

[0010] R1 and R2 are independently H or a cyclic group; or R1 and R2 are cyclized together to form a cyclic group, and R1 and R2 are not H at the same time;

[0011] The cyclic group is an aromatic ring, a heterocyclic aromatic group, a cycloalkyl group or a heterocyclic alkyl group; wherein the cyclic group is allowed to carry the substituent group or be combined with other cyclic groups; the substituent group includes at least one of halogen, -CN, amino, substituted amino, alkyl and alkoxy.

[0012] The innovative research of the present invention shows that by introducing an N-substituted sulfonamide at the 3-position of the pyridopyrrole mother nucleus, the compound can exhibit excellent TGF-β inhibitory activity and can effectively inhibit tumors based on this pathway.

[0013] In the present invention, the cyclic group may be an aromatic ring, a saturated ring, or a ring-free ring. The cyclic group may also be a carbocyclic ring or a heterocyclic ring, and the heteroatom in the heterocyclic ring may be O, N, or S. The cyclic group may be a monocyclic ring or a bicyclic ring or a tricyclic ring formed by merging monocyclic rings. In addition, the cyclic group may have one or more substituents.

[0014] As an optional solution, the ring group is a benzene ring, a pyridine ring, a cyclohexane, an oxacyclohexane or an oxacyclopentane.

[0015] In the present invention, the formula 1 includes at least one of formula A, formula B, and formula C;

[0016]

[0017]

[0018] The R3 to R5 are independently at least one of H, halogen, -CN, amino, substituted amino, C1 to C6 alkyl, and C1 to C6 alkoxy.

[0019] The present invention shows that in the formula A, when R3-R4 is an electron withdrawing group (further halogen, especially one is F and the other is Cl), it can show better TGF-β inhibitory activity. In the formula B, when R3-R4 are not H at the same time, especially when one is halogen and the other is alkyl, it can show better TGF-β inhibitory activity. In addition, compared with formula A, formula B, formula C is expected to obtain better TGF-β inhibitory activity.

[0020] Furthermore, the formula 1 described in the present invention comprises at least one of the following structures;

[0021]

[0022] The present invention also provides a method for preparing the transforming growth factor-β inhibitor, comprising reacting the compounds of formula 2 and formula 3 to obtain the transforming growth factor-β inhibitor;

[0023]

[0024] The present invention innovatively performs an amidation reaction on Formula 2 and Formula 3, and can synthesize the target product in a simple and efficient manner.

[0025] The present invention also provides the use of the transforming growth factor-β inhibitor in preparing a product for inhibiting transforming growth factor-β.

[0026] The research of the present invention shows that the structure of Formula 1 unexpectedly has an excellent effect of inhibiting transforming growth factor-β, and can be used to realize research applications of non-disease treatment and diagnosis based on this effect. It can also be used to develop pharmaceutical applications based on inhibiting growth factor-β to obtain pharmacological effects.

[0027] The product described in the present invention is an anti-tumor drug based on inhibiting growth factor-β.

[0028] Furthermore, the anti-tumor drug is an anti-tumor drug that inhibits at least one tumor cell of NIH3T3, H22, Hep3B, and CT26.

[0029] Furthermore, the anti-tumor drug is a drug for at least one of colorectal cancer, liver cancer, and brain glioma.

[0030] The present invention also provides an anti-tumor drug comprising a pharmaceutically effective amount of the transforming growth factor-β inhibitor.

[0031] The anti-tumor drug of the present invention further comprises pharmaceutically acceptable excipients.

[0032] Beneficial Effects

[0033] The present invention shows that the compound of formula 1 has excellent TGF-β inhibitory activity. Further combined control of R1 and R2 is expected to further improve the TGF-β inhibitory activity of the compound of formula 1 and obtain a better anti-tumor effect. Description of the drawings:

[0034] Figure 1 is the hydrogen spectrum of intermediate 1;

[0035] Figure 2 is the carbon spectrum of intermediate 1;

[0036] Figure 3 is the hydrogen spectrum of formula 1-A;

[0037] Figure 4 is the carbon spectrum of formula 1-A;

[0038] Figure 5 is the fluorine spectrum of formula 1-A;

[0039] Figure 6 is the hydrogen spectrum of formula 1-B;

[0040] Figure 7 is the carbon spectrum of formula 1-B;

[0041] Figure 8 is the hydrogen spectrum of formula 1-C;

[0042] Fig. 9is the carbon spectrum of formula 1-C;

[0043] Fig.10 is the fluorine spectrum of formula 1-C;

[0044] Fig.11 is the hydrogen spectrum of formula 1-D;

[0045] Fig.12 is the carbon spectrum of formula 1-D;

[0046] Fig.13 is the hydrogen spectrum of formula 1-E;

[0047] Fig.14 is the carbon spectrum of formula 1-E;

[0048] Fig.15 is the hydrogen spectrum of formula 1-F;

[0049] Fig.16 is the carbon spectrum of formula 1-F;

[0050] Fig.17 is the hydrogen spectrum of formula 1-G;

[0051] Fig.18 is the carbon spectrum of formula 1-G;

[0052] Fig.19 is the hydrogen spectrum of formula 1-H;

[0053] Fig. 20 is the carbon spectrum of formula 1-H;

[0054] Fig.21 is the hydrogen spectrum of formula 1-I;

[0055] Fig. 22 is the hydrogen spectrum of formula 1-J;

[0056] Fig.23 is the carbon spectrum of formula 1-J;

[0057] Fig.24 is the hydrogen spectrum of formula 1-K;

[0058] Fig.25 is the carbon spectrum of formula 1-K; Specific implementation method:

[0059] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0060] Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to conventional methods well known in the art and described in various references. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of the protection claimed in the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0061]

[0062] In the present invention, the raw materials of Formula 3 can be changed to obtain the desired Formula 1.

[0063] Synthesis and structural characterization of compounds

[0064] Example 1 Formula 1-A

[0065]

[0066] Step 1: Synthesis of Intermediate 1

[0067]

[0068] 1H pyrrolo[2,3-b]pyridine (2.0 g, 16.93 mmol) was added to chlorosulfonic acid (12 mL) in batches under stirring at 0 ° C. The reaction mixture was then slowly heated to 120 ° C. The reaction was monitored by TLC. The reaction mixture was poured into ice water under vigorous stirring. The precipitate was filtered, washed with water, and dried under vacuum to obtain 1.6 g of a white solid crude product with a yield of 44%. 1 H NMR (400MHz, DMSO-d6) δ12.97(s,1H),8.69(d,J=8.0Hz,1H),8.52(d,J=6.0Hz,1H),7.76(s,1H),7.59(dd,J=8.0,6.0Hz,1H). 13 C NMR (101MHz, DMSO-d6) δ138.88,137.40,135.16,128.05,124.92,122.57,116.64.

[0069] Step 2 Synthesis of Formula 1-A

[0070]

[0071] Weigh 1H-pyrrolo[2,3-b]pyridine-3-sulfonyl chloride (0.15 g, 0.69 mmol), 4-fluoroaniline (0.09 g, 0.83 mmol), 4-dimethylaminopyridine (0.01 g, 0.07 mmol) in a heart-shaped bottle, add pyridine (3 mL), and stir at 80°C overnight. After the reaction is completed by TLC monitoring, the mixture is poured into water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated, purified by silica gel column chromatography (dichloromethane / methanol, 20:1), and recrystallized in acetonitrile to obtain a white solid 0.12 with a yield of 61%. H NMR (400MHz, DMSO-d6) δ12.59 (s, 1H), 10.16 (s, 1H), 8.33 (dd, J = 4.8, 1.6Hz, 1H), 8.1 2(dd,J=8.0,1.6Hz,1H),8.05(s,1H),7.24(dd,J=8.0,4.8Hz,1H),7.13–6.96(m,4H). 13 C NMR(101MHz,DMSO-d6)δ159.19(d,J=239.0Hz),148.40,145.02,134.81(d,J=2 .5Hz),131.94,128.01,122.42(d,J=8.1Hz),118.00,116.24,116.02,112.25. 19 FNMR(376MHz,DMSO-d6)δ-119.19.

[0072] Example 2 Formula 1-B

[0073]

[0074] The preparation method is the same as that of Example 1, except that in step 2, the precursor raw material is replaced with an equal molar amount of 4-chloroaniline to prepare a compound of formula 1-B. 1 H NMR (400MHz, DMSO-d6) δ12.65(s,1H),10.41(s,1H),8.34(dd,J=4.8,1.6Hz,1H) ,8.19(dd,J=8.0,1.6Hz,1H),8.15(s,1H),7.28–7.20(m,3H),7.15–7.09(m,2H). 13 C NMR (101MHz, DMSO-d6) δ148.45,145.09,137.59,132.27,129.40,127.97,127.92,121.15,118.09,115.94,112.10.

[0075] Example 3 Formula 1-C

[0076]

[0077] The preparation method is the same as that in Example 1, except that in step 2, the precursor material is replaced with an equimolar amount of 3-chloro-4-fluoroaniline to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.67(s,1H),10.47(s,1H),8.35(dd,J=4.8,1.6Hz,1H) ,8.17(s,1H),8.15(dd,J=8.0,1.6Hz,1H),7.30–7.18(m,3H),7.10–7.02(m,1H). 13 C NMR(101MHz,DMSO-d6)δ154.14(d,J=241.5Hz),148.44,145.17,135.88(d,J=3.0Hz),132.37,127.91 ,121.36,120.29(d,J=7.1Hz),119.98(d,J=18.5Hz),118.15,117.77(d,J=21.9Hz),115.92,111.82. 19 F NMR(376MHz,DMSO-d6)δ-122.88.

[0078] Example 4 Formula 1-D

[0079]

[0080] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 4-aminotetrahydropyran to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.52(s,1H),8.36(d,J=4.8Hz,1H),8.25(d,J=8.0Hz,1H),8.04(s,1H),7.64( d,J=7.2Hz,1H),7.26(dd,J=8.0,4.8Hz,1H),3.76–3.57(m,2H),3.25–3.10(m,3H),1.55–1.20(m,4H). 13 C NMR (101MHz, DMSO-d6) δ148.59,144.87,130.54,128.21,117.77,116.09,114.94,66.02,49.51,33.81.

[0081] Example 5 Formula 1-E

[0082]

[0083] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 4-methylaniline to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),10.05(s,1H),8.33(dd,J=4.8,1.6Hz,1H),8.18(dd,J =8.0,1.6Hz,1H),8.05(s,1H),7.23(dd,J=8.0,4.8Hz,1H),7.03–6.92(m,4H),2.12(s,3H). 13 C NMR (101MHz, DMSO-d6) δ148.41,144.95,135.96,133.13,131.89,129.85,128.08,120.19,117.94,116.06,112.54,20.69.

[0084] Example 6 Formula 1-F

[0085]

[0086] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 4-methoxyaniline to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ8.32(dd,J=4.8,1.6Hz,1H),8.21(dd,J=8.0,1.6Hz,1H),8.13(s,1H),7.24(dd ,J=8.0,4.8Hz,1H),7.05(t,J=8.0Hz,1H),6.74–6.65(m,2H),6.49(dd,J=8.4,2.4Hz,1H),3.59(s,3H). 13 C NMR (101MHz, DMSO-d6) δ160.05,148.31,145.07,139.71,132.08,130.34,128.11,118.11,116.05,112.44,111.72,108.93,105.42,55.34.

[0087] Example 7 Formula 1-G

[0088]

[0089] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 4-(chlorodifluoromethoxy)aniline to obtain the title compound. 1H NMR (400MHz, DMSO-d6) δ12.66(s,1H),10.49(s,1H),8.34(dd,J=4.8,1.6Hz,1H),8.18(s,1H),8.14(dd,J=8.0,1.6Hz,1H),7.27–7.15(m,5H). 13 CNMR(101MHz,DMSO-d6)δ148.45,145.63,145.09,137.87,132.16,127.98,125.32(t,J=285.4Hz),122.75,120.92,118.07,115.99,112.27. 19 F NMR(376MHz,DMSO-d6)δ-24.98.

[0090] Example 8 Formula 1-H

[0091]

[0092] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 3-fluoro-4-methylaniline to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.63(s,1H),10.38(s,1H),8.34(dd,J=4.8,1.6Hz,1H),8.21(dd,J=8.0,1.6Hz,1H),8.19(s,1H), 7.25(dd,J=8.0,4.8Hz,1H),7.03(t,J=8.4Hz,1H),6.88(dd,J=11.6,2.0Hz,1H),6.82(dd,J=8.4,2.2Hz,1H),2.02(s,3H). 13 C NMR(101MHz,DMSO-d6)δ160.83(d,J=300.8Hz),148.46,145.07,137.97(d,J=10.3Hz),132.34,132.21(d,J=6.4Hz),1 27.97, 119.24 (d, J = 17.1Hz), 118.08, 115.97, 115.21 (d, J = 3.0Hz), 112.12, 106.17 (d, J = 26.0Hz), 13.83 (d, J = 2.9Hz). 19 F NMR(376MHz,DMSO-d6)δ-115.86.

[0093] Example 9 Formula 1-I

[0094]

[0095] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 3-chloro-2-aminopyridine to obtain the title compound. 1 H NMR(400MHz,DMSO-d6)δ12.61(s,1H),10.54(s,1H),8.38–8.28(m,2H),8.21(s,1 H),8.09(s,1H),7.85(d,J=8.0Hz,1H),7.27(dd,J=8.0,4.8Hz,1H),7.00(s,1H).

[0096] Example 10 Formula 1-J

[0097]

[0098] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 5-chloro-6-methyl-2-aminopyridine to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.65(s,1H),11.19(s,1H),8.35(dd,J=4.8,1.6Hz,1H),8.32(s,1H),8 .27(dd,J=8.0,1.6Hz,1H),8.09(s,1H),7.28(dd,J=8.0,4.8Hz,1H),7.11(s,1H),2.25(s,3H). 13 C NMR (101MHz, DMSO-d6) δ150.90,148.38,147.18,146.64,145.04,132.63,128.27,125.94,118.07,116.09,113.50,112.72,19.71.

[0099] Example 11 Formula 1-K

[0100]

[0101] Compared with Example 1, the only difference is that in step 2, the precursor material is replaced with an equimolar amount of 5-chloroindoline to obtain the title compound. 1H NMR(400MHz,DMSO-d6)δ12.86(s,1H),8.34(dd,J=4.8,1.6Hz,1H),8.30(s,1H),8.09(dd,J=8.0,1.6Hz,1H) ,7.54(d,J=8.4Hz,1H),7.26–7.19(m,2H),7.16–7.13(m,1H),3.95(t,J=8.4Hz,2H),2.86(t,J=8.4Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ148.27,145.24,141.22,135.25,132.67,128.32,127.88,127.55,125.76,118.47,116.81,116.17,108.91,50.58,27.55.

[0102]

[0103] Pharmacological tests:

[0104] Example 12 Cell proliferation assay

[0105] The effect of the compounds of the present invention on the proliferation of mouse liver cancer cells H22 was tested using in vitro cell evaluation. The detection method used in this assay is the CCK8 method, the basic principle of which is that the reagent contains a water-soluble tetrazolium salt WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt], which is reduced to a highly water-soluble yellow formazan product by dehydrogenase in the cell under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells. Therefore, this property can be used to directly perform cell proliferation and toxicity analysis.

[0106] The determination procedure is as follows:

[0107] (1) Cell plating:

[0108] H22 cells in the logarithmic growth phase were taken, digested and resuspended, and then counted. The cells were inoculated in a 96-well plate at a density of 4,000 cells per well. The plate was placed in a 37° C., 5% CO 2 incubator for 72 hours, and then the compound of the present invention was added for treatment;

[0109] (2) Compound treatment:

[0110] The compounds of the present invention are configured for cell therapy, and the final concentrations of the compounds (in the above cases) are 100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.23 μM, 0.41 μM, 0.13 μM, and 0 nM. The well plate was placed in a 37° C., 5% CO2 incubator for 72 hours. Only the medium without cells was added as the blank group; the concentration of the compound in the control group was 0 nM;

[0111] (3) CCK8 detection:

[0112] After 72 hours of cell culture, 10 μL of CCK8 detection solution was added to each well, and the cells were placed in a 37°C, 5% CO2 incubator for another 2 hours. The detection value of each well was read on a multifunctional microplate reader, and the absorbance was measured at 450 nm using a microplate reader;

[0113] (4) Data analysis:

[0114] The inhibition ratio was calculated based on the OD value readings, inhibition percentage = (1-(drug group value-blank group value) / (control group value-blank group value))*100%. The log (inhibitor) of GraphPad Prism was used to fit the response-variable slope to quantify the curve and calculate the IC50 value of the compound inhibiting cell proliferation.

[0115] H22 tumor cell antiproliferative activity results

[0116] Table 1

[0117]

[0118]

[0119] As can be seen from Table 1, for H22 tumor cells, compounds of Formula 1-C, Formula 1-J and Formula 1-K can show excellent performance, followed by compounds of Formula 1-D, Formula 1-E, Formula 1-H, etc.

[0120] Example 13 Cell luciferase assay evaluation

[0121] NIH3T3 mouse fibroblasts engineered to express a Smad2 / 3-luciferase responsive promoter were used. The basic principle is that after the cells are stimulated by TGF-β1 cytokine, the phosphorylation level of Smad2 / 3 downstream of the TGF-β1 receptor increases. The chemiluminescent signal intensity detected by the Bright-glo luciferase assay system is proportional to the Smad2 / 3 phosphorylation level. Therefore, this experiment can reflect the degree of inhibition of the TGF-β1 receptor.

[0122] (1) Cell plating:

[0123] NIH3T3-Smad2 / 3-Luc cells in the logarithmic growth phase were taken, digested and resuspended, and then counted, and the cell density was inoculated in a 96-well plate, with 5,000 cells in each well, and the plate was placed in a 37° C., 5% CO2 incubator with reduced serum culture, and the compound of the present invention was added for treatment;

[0124] (2) Compound treatment:

[0125] The compounds of the present invention are configured for cell therapy, the final concentrations of the compounds (the above cases) are 100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.23 μM, 0.41 μM, 0.13 μM, 0 nM, and the final concentration of TGFβ1 in each well is 2.5 ng / mL. The well plate is placed in a 37°C, 5% CO2 incubator for 24 hours. Only the medium without cells is added as a blank group; the concentration of the compound in the control group is 0 nM;

[0126] (3) Fluorescence detection:

[0127] Remove the cell supernatant, add 100 μL Glo Lysis Buffer to each well, shake slowly, and lyse at room temperature for 5 minutes. Then add 100 μL Bright-glo luciferase assay system to each well, incubate at room temperature for 5 minutes, shake for 2 minutes, transfer 100 μL of supernatant to a 96-well plate with a white bottom, and detect the chemiluminescent signal. The detection condition is 1 second.

[0128] (4) Data analysis:

[0129] The inhibition ratio was calculated based on the OD value readings, and the inhibition percentage = (1-(drug group value-blank group value) / (control group value-blank group value))*100%. The log (inhibitor) of GraphPad Prism was used to fit the response-variable slope to quantify the curve and calculate the IC of the compound. 50 value.

[0130] Cell luciferase activity results

[0131] Table 2

[0132] Example <![CDATA[NIH3T3-Smad2 / 3-LucIC 50 (μM)]]> Example 1 (Formula 1-A) 1.5 Example 2 (Formula 1-B) 1.9 Example 3 (Formula 1-C) 0.16 Example 4 (Formula 1-D) 0.21 Example 5 (Formula 1-E) 2.4 Example 6 (Formula 1-F) 3.2 Example 7 (Formula 1-G) 2.9 Example 8 (Formula 1-H) 0.15 Example 9 (Formula 1-I) 1.2 Example 10 (Formula 1-J) 0.18 Example 11 (Formula 1-K) 0.16 Comparison 1 >50 Comparison 2 >50

[0133] It can be seen from Table 2 that for NIH3T3 mouse fibroblasts, compounds of Formula 1-C, Formula 1-D, Formula 1-H, Formula 1-J and Formula 1-K can show excellent performance, followed by compounds of Formula 1-A, Formula 1-B, Formula 1-E, Formula 1-F, Formula 1-G, Formula 1-I, etc.

[0134] It can be seen from Examples 12 to 13 that compounds of Formula 1-C, Formula 1-J and Formula 1-K can have good universal inhibitory effects on different tumor cells.

Claims

1. A transforming growth factor-β inhibitor, characterized in that At least one of a compound having the structure of Formula 1 and a pharmaceutically acceptable salt, derivative, cocrystal, crystal, crystal, or solvate thereof; R1 and R2 are independently H or a cyclic group; or R1 and R2 are cyclized together to form a cyclic group, and R1 and R2 are not H at the same time; The cyclic group is an aromatic ring, a heterocyclic aromatic group, a cycloalkyl group or a heterocyclic alkyl group; wherein the cyclic group is allowed to carry the substituent group or be combined with other cyclic groups; the substituent group includes at least one of halogen, -CN, amino, substituted amino, alkyl and alkoxy.

2. The transforming growth factor-β inhibitor according to claim 1, characterized in that The ring group is a benzene ring, a pyridine ring, a cyclohexane, an oxacyclohexane or an oxacyclopentane.

3. The transforming growth factor-β inhibitor according to claim 1, characterized in that The formula 1 includes at least one of formula A, formula B, and formula C; The R3 to R5 are independently at least one of H, halogen, -CN, amino, substituted amino, C1 to C6 alkyl, and C1 to C6 alkoxy.

4. The transforming growth factor-β inhibitor according to claim 1, characterized in that The formula 1 comprises at least one of the following structures; 5. A method for preparing a transforming growth factor-β inhibitor according to any one of claims 1 to 4, characterized in that: The compounds of formula 2 and formula 3 are reacted to obtain the transforming growth factor-β inhibitor; 6. Use of the transforming growth factor-β inhibitor according to any one of claims 1 to 4 in the preparation of a product for inhibiting transforming growth factor-β.

7. The use according to claim 6, characterized in that The product is an anti-tumor drug based on the inhibition of growth factor-β.

8. The use according to claim 7, characterized in that The anti-tumor drug is an anti-tumor drug that inhibits at least one tumor cell among NIH3T3, H22, Hep3B and CT26.

9. The use according to claim 8, characterized in that The anti-tumor drug is a drug for at least one of colorectal cancer, liver cancer and brain glioma.

10. An anti-tumor drug, characterized in that: A pharmaceutically effective amount of the transforming growth factor-β inhibitor according to any one of claims 1 to 4; Preferably, it further comprises a pharmaceutically acceptable excipient; Preferably, it has a pharmaceutically acceptable dosage form.

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