CN109553617B - Wnt and Hedgehog signal path double inhibitor and application thereof - Google Patents

Abstract

The invention provides a dual inhibitor for Wnt and Hedgehog signal pathways and application thereof, and particularly provides a heterocyclic compound with Wnt and Hedgehog signal pathway inhibitory activity, and pharmaceutically acceptable salts thereof, wherein the heterocyclic compound has a structure shown in a general formula I:

Description

Wnt and Hedgehog signal pathway dual-inhibitor and application thereof

Technical Field

The invention relates to a heterocyclic compound with Wnt and Hedgehog signal channel inhibition activity and application thereof, belonging to the technical field of medicines.

Background

Malignant tumors are one of the most major diseases that endanger human health, with about 1090 million new cases of malignant tumors per year worldwide and about 670 million patients dying from malignant tumors per year. The research and development of the antitumor drugs have undergone great changes in recent years, the antitumor drugs commonly used in clinical treatment in the past are mainly cytotoxic drugs, and the anticancer drugs have the defects of difficult avoidance, poor selectivity, strong toxic and side effects, easy generation of drug resistance and the like. With the rapid progress of life science research in recent years, various basic processes such as signal transduction in malignant tumor cells, regulation of cell cycle, induction of apoptosis, angiogenesis, and interaction between cells and extracellular matrix have been elucidated. The key enzymes of cell signal transduction pathways related to the differentiation and proliferation of tumor cells are used as drug screening targets, and a novel lead compound which selectively acts on a specific target and has high efficiency and low toxicity is found to become an important direction for the research and development of the current antitumor drugs; the successful marketing of targeted drugs such as trastuzumab (trastuzumab), imatinib (imatinib), gefitinib (gefitinib) and erlotinib (erlotinib) is a typical example.

Metastasis and regeneration are characteristic of malignant tumors and are also a difficult problem in treating malignant tumors. Even the new generation of targeted drugs have little effect on tumor metastasis and regeneration. Based on this, the research on the Wnt signaling pathway and the Hedgehog (Hh) signaling pathway has been increasingly emphasized in recent years, not only because the abnormal activation of the Wnt signaling pathway and the Hh signaling pathway plays a significant role in the development process of many tumors including basal cell carcinoma, brain tumor, breast cancer, prostate cancer and some malignant tumors of the digestive system, but also more importantly, the Wnt signaling pathway and the Hh signaling pathway are embryonic development pathways and play an important role in controlling tumor stem cells, thereby controlling tumor metastasis and regeneration.

The discovery of the Wnt signaling pathway was first derived from studies of oncogenic viruses and drosophila developmental mechanisms. The Wnt gene was discovered in 1982 and was originally identified as a site for preferential integration of mouse mammary tumor virus, which is an oncogene that transmits proliferation and differentiation information between cells, and was then designated the Int gene (mouse Int-1 and Int-3). It was then found to belong to the same gene (orthologues) as the wingless gene (wingless) of Drosophila, and thus was named the Wnt gene in combination. To date, 19 members of the Wnt gene family have been discovered and cloned, and the signal transduction pathway mediated by the Wnt gene has been termed Wnt signaling pathway. The Wnt pathway is a very conserved signaling pathway. Members of the lower organism drosophila are highly homologous, ranging from higher mammals to lower organisms. The Wnt signaling pathway is involved in the regulation of a variety of biological processes, including embryonic growth and morphological development, tissue stabilization, balance of energy metabolism, and maintenance of stem cells (Logan et al, annu. Rev. Cell.dev.biol.,2004,20, 781-810). In recent years, stem cell studies have found that the Wnt signaling pathway plays an important role in the regulation of the maintenance of epidermal stem cells, intestinal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells, and tumor stem cells (Reya et al, nature,2005,434, 843-850).

The canonical Wnt signaling pathway is formed by the combination of the extracellular ligand Wnts protein with 7 cell transmembrane Frizzled receptor and helper receptor LRP5/6 to open the Wnt/beta-catenin signaling pathway and activate the intracytoplasmic Dsh protein, which can inhibit the activity of GSK-3 beta, a key component of degradation complex formed by APC protein, GSK-3 beta, axin, beta-catenin, etc., so that beta-catenin is not phosphorylated by GSK-3 beta to avoid the recognition and degradation of ubiquitin proteasome, and then gradually accumulated in cytoplasm (Boutros et al, mech.Dev.,1999,83,27-37 Perrimon, cell,1994,76, 781-784. When the beta-catenin accumulates in cytoplasm to a certain concentration, the beta-catenin begins to transfer to cell nucleus and is combined with transcription factors TCF/LEFs in the cell nucleus to cause the exposure of a promoter of a downstream target gene of the beta-catenin so as to activate and express, such as c-myc, cyclin-D1, survivin, gatrin, VEGF, ASEF and the like to cause abnormal cell proliferation. In normal somatic cells, most of the intracytoplasmic beta-catenin is combined with cell membrane adhesion protein E-cadherin and alpha-catenin to form a complex which participates in the regulation of cytoskeleton, maintains the adhesion of homotypic cells and prevents cell transfer, a small part of free beta-catenin is phosphorylated in cytoplasm by a degradation complex and then recognized and degraded by ubiquitin proteasomes, and the low-level state of the intracellular beta-catenin is kept, so that the Wnt signal pathway is in a closed state.

It is found that when Wnt gene itself or any other member factor of the pathway is changed to make it abnormally activated, it may cause tumor. For example, there are mutations in genes for the Wnt pathway, including APC, β -catenin, axin, TCF, etc., that are widely present in colon cancer patients, resulting in the overexpression of genes associated with growth (Klaus et al, nat. Rev. Cancer,2008,8, 387-398). Lozzo et al found that Wnt-5amRNA was overexpressed in all of breast, lung, prostate, and melanoma cancers, and that expression was particularly pronounced in breast Cancer, as determined by studies on more than 100 normal and tumor tissues and 10 human tumor cell lines (Lozzo et al, cancer Research,1995,55, 3495).

The abnormal activation of the Wnt pathway plays an important role in the processes of canceration of cells, tumorigenesis and tumor invasiveness, and the blocking of the abnormal Wnt signal pathway can inhibit the proliferation of tumor cells and induce the apoptosis of the tumor cells. Therefore, the Wnt signal pathway has better anti-tumor targeting effect.

The Hedgehog signaling pathway is a highly conserved intercellular signal transduction system, and is found in Drosophila in 1980, and is named Hedgehog pathway Hedgehog (Hh) because the gene mutation of the pathway of Drosophila can cause larvae to show a plurality of Hedgehog-like spikes. The Hh signal pathway consists of an Hh ligand, two transmembrane protein receptors, namely a patched membrane receptor (PTCH) and a smoothened transmembrane protein (SMO), a downstream transcription factor Gli protein and the like. PTCH and SMO are two transmembrane proteins located on the membrane of a target cell, where PTCH is a 12-transmembrane protein encoded by the cancer suppressor PTCH, a cell surface receptor, and have dual roles in sequestering and transducing Hh. SMO is a 7-transmembrane protein, is highly similar to G protein coupled receptor family in structure, and has the function of transducing Hh signals. PTCH and SMO function as receptors in Hh signaling, where PTCH is a receptor for Hh. In the absence of Hh, PTCH prevents the translocation of SMO to the cell membrane, thereby inhibiting SMO activity; thereby inhibiting the transcription expression of the downstream gene. When the Hh signal exists, the Hh is combined with PTCH to induce phosphorylation of a plurality of silk/threonine residues at the carboxyl terminal of SMO, so that the SMO is accumulated and activated on the cell surface, the activated SMO forms a complex with a kinesin-like molecule Costal2 (Cos 2) and silk/threonine kinase Fused (Fus) and super-pressure Fused (Sufu) and exerts a transcription activation effect in a full-length form, and finally, a zinc finger-like transcription factor Gli is activated, and the latter enters into the nucleus to cause the transcription of a target gene. Thus, in the Hh signaling pathway, hh is the start of the signaling pathway, and Gli is the end of the signaling pathway as a transcription factor; hh and SMO are used as agonist, PTCH is used as inhibitor, and the activity of signal channel is regulated.

The Hh signaling pathway can induce cell proliferation, a function that is important in embryonic development and tissue maintenance, but there is a link between abnormal activation of this pathway and tumors in adulthood. Approximately 25% of all patients who die from cancer have associated with aberrant activation of the Hh signaling pathway. Which comprises the following steps: basal cell carcinoma, medulloblastoma, breast cancer, pancreatic cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, rhabdomyosarcoma, prostate cancer, meningioma, and basal cell carcinoma-like syndrome, etc. (Sachin Gupta et al, ther.

The Wnt signal path and the Hedgehog signal path belong to the embryonic path, so that the two paths are in complex connection. For example, it has been shown that activation of the Wnt signaling pathway upregulates expression of the RNA-binding protein CRD-BP, stabilizing Gli1 m-RNA, thereby activating the Hedgehog signaling pathway (noubsi et al, cancer res.2009,69, 8572-8578.); further studies have shown that activation of the Hedgehog signaling pathway up-regulates the expression of sFRP-1 protein, a glycoprotein that inhibits the Wnt signaling pathway (He et al, j.biol.chem.2006,281, 35598-35602.); in addition, simultaneous activation of both Wnt and Hedgehog signaling pathways was found in mouse models of lung cancer tumor caused by smoking (Hassan et al, PLoS ONE.2006,1, e93.).

Therefore, whether directed to a condition caused by Wnt pathway malfunction, or directed to a condition caused by Hedgehog signaling pathway malfunction, or caused by both Wnt and Hedgehog signaling pathways malfunctioning, simultaneous inhibition of both the Wnt signaling pathway and the Hedgehog signaling pathway would have better therapeutic applications than inhibition of only a single signaling pathway.

However, to date, the studies on Wnt and Hedgehog signaling pathway inhibitors have focused on the study of single pathway inhibitors, such as the Wnt signaling pathway inhibitor LGK974 or the Hedgehog signaling pathway inhibitor Vismodegib, and the inventors have also previously disclosed a class of 6-5-6 tricyclic fused-framework compounds that are only Wnt signaling pathway inhibitors (bioorg. Med. Chem.2016,24,5861-5872., CN 104557862A). No compound for simultaneously inhibiting the Wnt signal pathway and the Hedgehog signal pathway is reported in the prior art.

Disclosure of Invention

In view of the defects of the prior art, the present invention aims to provide a heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity and an application thereof, which can inhibit both the Wnt signaling pathway and the Hedgehog signaling pathway, thereby improving disorders caused by the malfunction of the Wnt signaling pathway or the Hedgehog signaling pathway.

The purpose of the invention is realized by the following technical scheme:

a heterocyclic compound with Wnt and Hedgehog signaling pathway inhibition activity and pharmaceutically acceptable salts thereof have a structure shown in a general formula I:

wherein:

X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ each independently selected from N or CR ⁸ ；

R ¹ 、R ² 、R ³ Each independently selected fromHydrogen atom, C _1-6 An alkyl group which is unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atom or halo group;

R ⁴ selected from deuterium atom, halogen, cyano, C _1-6 An alkoxy group;

R ⁵ selected from hydrogen atom, deuterium atom, C _1-6 An alkyl group which is unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atom or halo group;

R ⁶ 、R ⁷ 、R ⁸ each independently selected from hydrogen atom, deuterium atom, cyano, halogen, C _1-6 Alkyl radical, C _1-6 Alkoxy, said alkyl and alkoxy being unsubstituted or substituted by 1 to 3 groups independently selected from deuterium atom or halo;

m is selected from 0,1,2,3;

n is selected from 0,1,2;

when X is present ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ Are all CH, and R ⁴ When is F, R ⁵ And cannot be methyl.

Preferably, the heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity, and pharmaceutically acceptable salts thereof, has a structure represented by formula Ia:

wherein:

R ¹ 、R ² 、R ³ each independently selected from hydrogen atom, C _1-6 An alkyl group which is unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atom or halo group;

R ⁴ selected from deuterium atom, halogen, cyano, C _1-6 An alkoxy group;

R ⁵ selected from hydrogen atom, deuterium atom, C _1-6 An alkyl group which is unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atom or halo group;

R ⁶ 、R ⁷ each independently selected from hydrogen atom, deuterium atom, cyano, halogen, C _1-6 Alkyl radical, C _1-6 Alkoxy, said alkyl and alkoxy being unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atoms or halo groups;

m is selected from 0,1,2,3;

n is selected from 0,1,2;

when R is ⁴ When is F, R ⁵ And cannot be methyl.

Preferably, the heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity and pharmaceutically acceptable salts thereof have a structure represented by formula Ib:

wherein:

X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ each independently selected from N or CR ⁸ And wherein a maximum of 4 is N (preferably a maximum of 3 is N, more preferably a maximum of 2 is N), and at least one is N;

R ¹ 、R ² 、R ³ each independently selected from hydrogen atom, C _1-6 An alkyl group which is unsubstituted or substituted with 1 to 3 groups independently selected from deuterium atom or halo group;

R ⁵ selected from hydrogen atom, deuterium atom, C _1-6 An alkyl group which is unsubstituted or substituted by 1 to 3 groups each independently selected from deuterium atom or halogeno group;

R ⁶ 、R ⁷ 、R ⁸ each independently selected from hydrogen atom, deuterium atom, cyano, halogen, C _1-6 Alkyl radical, C _1-6 Alkoxy, said alkyl and alkoxy being unsubstituted or substituted by 1 to 3 groups independently selected from deuterium atom or halo;

m is selected from 0,1,2,3;

n is selected from 0,1,2.

Preferably, the above-mentioned heterocycles have inhibitory activity on the Wnt and Hedgehog signaling pathwaysA compound, and pharmaceutically acceptable salts thereof, characterized by: x ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ Each independently selected from N or CR ⁸ And wherein a maximum of 3 are N;

preferably, the above heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity, and pharmaceutically acceptable salts thereof, are characterized in that: x ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ Each independently selected from N or CR ⁸ And wherein a maximum of 2 are N;

preferably, the heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity and pharmaceutically acceptable salts thereof are characterized by: x ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ 、X ⁶ 、X ⁷ Each independently selected from N or CR ⁸ And wherein at most 1 is N;

preferably, the above heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity, and pharmaceutically acceptable salts thereof, are characterized in that: r is ¹ 、R ² 、R ³ Is a hydrogen atom.

Preferably, the heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity and pharmaceutically acceptable salts thereof are characterized by: m and n are respectively and independently selected from 0 or 1.

Preferably, the heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity and pharmaceutically acceptable salts thereof are characterized by: r ⁶ 、R ⁷ Is a hydrogen atom.

Preferably, the above heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity, and pharmaceutically acceptable salts thereof, are characterized in that: r ⁸ Is a hydrogen atom.

Preferably, the heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity described above, and pharmaceutically acceptable salts thereof, comprise combinations of one or more of the following structural formulas:

the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity and a pharmaceutically acceptable carrier.

The invention also provides application of the compound A, or the heterocyclic compound or the pharmaceutical composition with the Wnt and Hedgehog signal pathway inhibition activity in preparing a medicament for treating or preventing Wnt or Hedgehog signal pathway abnormality; the medicament for treating or preventing Wnt or Hedgehog signaling pathway abnormality and the pharmaceutical composition thereof comprise one or more of medicaments for treating breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous epithelial cancer, thyroid cancer, sarcoma, osteosarcoma, desmoid tumor, melanoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, stomach cancer, myeloma, lymphoma, mantle cell lymphoma, cutaneous T-cell lymphoma, chronic and non-progressive anemia, idiopathic or primary thrombocythemia, idiopathic myelofibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, liver cirrhosis, diabetic retinopathy, macroglobulinemia, leukemia, acute leukemia, chronic leukemia, lymphatic leukemia, myelogenous leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumors, astrocytomas, medulloblastoma, schwannoma, primary extraneural blastoma, pituitary tumor, and the pharmaceutical composition thereof.

The pharmaceutical composition refers to a composition which takes the heterocyclic compound with Wnt and Hedgehog signal channel inhibition activity and pharmaceutically acceptable salt or carrier thereof as components.

The invention has the outstanding effects that:

both the known Wnt signaling pathway inhibitor (LGK 974) and the Hedgehog signaling pathway inhibitor (Vismodegib) can only inhibit one of the Wnt signaling pathway and the Hedgehog signaling pathway, but cannot inhibit both the Wnt and Hedgehog signaling pathways. The heterocyclic compound provided by the invention is used as an effective antagonist of the Wnt signal channel and the Hedgehog signal channel, can effectively block the Wnt signal channel and the Hedgehog signal channel at the same time, and can be better used for treating or preventing diseases caused by the abnormality of the Wnt signal channel or the Hedgehog signal channel.

Drawings

Fig. 1 is a graph showing the results of the cytostatic test of the Wnt signaling pathway with heterocyclic compound A1 having Wnt and Hedgehog signaling pathway inhibitory activity of example 8;

figure 2 is a graph of the results of the cytostatic assay of Wnt signaling pathway with heterocyclic compound A5 having Wnt and Hedgehog signaling pathway inhibitory activity of example 8;

figure 3 is a graph of the results of the cytostatic assay of the Wnt signaling pathway with heterocyclic compound a of example 8 having Wnt and Hedgehog signaling pathway inhibitory activity;

figure 4 is a graph of the results of the cytostatic assay of the Wnt and Hedgehog signaling pathway with heterocyclic compound A1 of example 9 against the Hedgehog signaling pathway;

figure 5 is a graph of the results of the cytostatic assay of the Wnt and Hedgehog signaling pathway inhibition activity of heterocyclic compound A5 to the Hedgehog signaling pathway of example 9;

figure 6 is a graph of the results of the cytostatic assay of the Wnt and Hedgehog signaling pathway with heterocyclic compound a of example 9 against the Hedgehog signaling pathway.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

In the embodiment described below, it is preferred that,the solvent and the medicine are analytically pure or chemically pure; the solvent is redistilled before use; the anhydrous solvents are processed according to standard or literature methods. Column chromatography silica gel (100-200 mesh) and thin layer chromatography silica gel (GF 254) are products of Qingdao oceanic plant and tobacco station chemical plant; petroleum ether (60-90 ℃ C.)/ethyl acetate (v/v) were used as eluents unless otherwise specified; the color developing agent is an ethanol solution of iodine or phosphomolybdic acid; all extraction solvents are, unless otherwise stated, anhydrous Na ₂ SO ₄ And (5) drying. ¹ HNMR were recorded using a varian-400 NMR spectrometer with TMS as an internal standard. LC-MS was recorded using an Agilent model 1100 high performance liquid chromatography-ion trap Mass spectrometer (LC-MSDTap), diode Array Detector (DAD), detection wavelengths 214nm and 254nm, ion trap Mass Spectrometry (ESI Source). HPLC column is AgelaDurashellC18 (4.6X 50mm,3.5 μm); mobile phase 0.1% ₄ HCO ₃ Aqueous solution: acetonitrile (from 5 to 95 in 5 minutes; the flow rate was 1.8mL/min.

Example 1

This example provides a heterocyclic compound A1 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

1) Synthesis of intermediate A1-2:

methyl 2-chloro-3-nitropyridine-5-carboxylate (220mg, 1mmol), phenylboronic acid (146mg, 1.2mmol), pd (dppf) Cl ₂ (59mg, 0.08mmol) and potassium phosphate trihydrate (532mg, 2mmol) were added to a mixed solvent of THF (20 mL) and water (4 mL), and reacted overnight at 70 ℃ under nitrogen. Diluted with ethyl acetate (100 mL), washed with saturated brine (50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 50. ¹ H NMR(400MHz,CDCl ₃ )δ9.40(s,1H),8.69(s,1H),7.62-7.60(m,2H),7.51-7.49(m,3H),4.03(s,3H)。

2) Synthesis of intermediates A1-3:

intermediate A1-2 (200mg, 0.77mmol) and 1, 2-bis (diphenylphosphino) ethane (368mg, 0.92mmol) were added to N, N-dimethylacetamide (5 mL) and reacted overnight at 150 ℃ under nitrogen. Diluted with ethyl acetate (100 mL), washed with saturated brine (30ml × 5), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate =10:1 to 5) to give a pale yellow solid (110mg, 62%). ¹ H NMR(400MHz,CDCl ₃ )δ9.22(s,1H),8.41(d,J＝8.4Hz,1H),8.38(s,1H),8.34(s,1H),7.61-7.58(m,1H),7.52(d,J＝8.4Hz,1H),7.39-7.35(m,1H),4.01(s,3H)。

3) Synthesis of intermediates A1-4:

intermediate A1-3 (110mg, 0.48mmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and methanol (1 mL), and then 1N LiOH (2.5mL, 2.5mmol) was added dropwise and reacted at normal temperature overnight. To the reaction mixture were added ethyl acetate (20 mL) and water (15 mL), and the aqueous phase was adjusted to pH =5-6 with 1N HCl, whereupon a solid precipitated, filtered, and dried in vacuo to give a yellow solid (80mg, 77%).

4) Synthesis of product A1:

intermediate A1-4 (40mg, 0.19mmol), intermediate A1-5 (obtained from the synthetic route of bioorg.med.chem.2016,24,5861-5872., 49mg, 0.23mmol), DIPEA (49mg, 0.38mmol) and HATU (108mg, 0.29mmol) were added to DMF (1 mL), reacted overnight at room temperature, diluted with water (20 mL), a solid precipitated, filtered, and the resulting solid was purified by column chromatography (dichloromethane: methanol =50 1 to 20) to give a white solid (55mg, 70%). After nuclear magnetic spectrum analysis (the spectrum data are shown in table 1), the obtained solid is the compound A1.

Example 2

This example provides a heterocyclic compound A2 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

1) Synthesis of intermediate A2-2:

2-chloro-3-nitropyridine (316mg, 2mmol), 4-boronAcid methyl benzoate (432mg, 2.4mmol), pd (PPh) ₃ ) ₄ (185mg, 0.16mmol) and potassium carbonate (552mg, 4 mmol) were added to a mixed solvent of 1, 4-dioxane (20 mL) and water (4 mL), and reacted overnight at 90 ℃ under nitrogen. Diluted with ethyl acetate (100 mL), washed with saturated brine (50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 20. ¹ H NMR(400MHz,CDCl ₃ )δ8.90(d,J＝4.0Hz,1H),8.22(d,J＝8.0Hz,1H),8.15(d,J＝8.0Hz,2H),7.64(d,J＝8.0Hz,2H),7.52-7.49(m,1H),3.95(s,3H)。

2) Synthesis of intermediate A2-3:

intermediate A2-2 (200mg, 0.77mmol) and DPPE (463mg, 1.16mmol) were added to DMAc (3 mL) and reacted overnight at 150 ℃ under nitrogen. Diluted with ethyl acetate (100 mL), washed with saturated brine (30ml × 5), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate =5:1 to 3) to give a yellow solid (125mg, 71%). ¹ H NMR(400MHz,DMSO-d6)δ11.72(s,1H),8.53(d,J＝4.0Hz,1H),8.29(d,J＝8.4Hz,1H),8.19(s,1H),7.99(d,J＝8.4Hz,1H),7.85(d,J＝8.0Hz,1H),7.50–7.46(m,1H),3.92(s,3H)。

3) Synthesis of intermediate A2-4:

intermediate A2-3 (125mg, 0.55mmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and methanol (1 mL), and then 1N LiOH (3mL, 3mmol) was added dropwise, followed by reaction at ordinary temperature overnight. Ethyl acetate (20 mL) and water (15 mL) were added to the reaction mixture, and the aqueous phase was adjusted to pH5-6 with 1N HCl, whereupon a solid precipitated, filtered, and dried under vacuum to give a yellow solid (65mg, 55%).

4) Synthesis of product A2:

intermediate A2-4 (35mg, 0.16mmol), intermediate A1-5 (43mg, 0.2mmol), DIPEA (41mg, 0.32mmol) and HATU (91mg, 0.24mmol) were added to DMF (1 mL), reacted overnight at room temperature, diluted with water (20 mL), then extracted with ethyl acetate (50 mL), and the organic phase was washed with brine (20mL × 4). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (dichloromethane: methanol =50:1 to 20) to obtain a white solid (15mg, 22%). After nuclear magnetic spectrum analysis (the spectrum data are shown in table 1), the obtained solid is the compound A2.

Example 3

This example provides a heterocyclic compound A3 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

1) Synthesis of intermediate A3-2:

4-chloro-3-nitrobenzoic acid methyl ester (300mg, 1.4mmol), pyridine-4-boric acid (258mg, 2.1mmol) and Pd (PPh) ₃ ) ₄ (127mg, 0.11mmol) and cesium fluoride (426mg, 2.8mmol) were added to a mixed solvent of isopropanol (20 mL) and water (4 mL), and reacted overnight at 90 ℃ under nitrogen. Concentrated under reduced pressure, diluted with ethyl acetate (100 mL) and washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate =3: 1) to obtain a yellow solid (220mg, 61%). ¹ H NMR(400MHz,CDCl ₃ )δ8.71(d,J＝5.2Hz,2H),8.61(s,1H),8.33(d,J＝8.0Hz,1H),7.52(d,J＝8.0Hz,1H),7.25(d,J＝5.6Hz,2H),4.01(s,3H).

2) Synthesis of intermediate A3-3:

intermediate A3-2 (200mg, 0.77mmol) and DPPE (617mg, 1.55mmol) were added to DMAc (3 mL) and reacted overnight at 150 ℃ under nitrogen. Diluted with ethyl acetate (100 mL) and washed with saturated brine (30ml × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate =1 to 1.

3) Synthesis of intermediate A3-4:

intermediate A3-3 (104mg, 0.46mmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and methanol (1 mL), followed by dropwise addition of 1N LiOH (2mL, 2mmol), and the reaction was carried out overnight at ordinary temperature. To the reaction mixture were added ethyl acetate (20 mL) and water (15 mL), and the aqueous phase was adjusted to pH =5-6 with 1N HCl, and a solid precipitated. The mixture is filtered and then is filtered,drying in vacuo afforded a yellow solid (37mg, 37%). ¹ H NMR(400MHz,DMSO-d ₆ )δ11.88(s,1H),8.99(s,1H),8.39(d,J＝4.8Hz,1H),8.34(d,J＝8.4Hz,1H),8.20(s,1H),8.18(d,J＝5.2Hz,1H),7.83(d,J＝8.4Hz,1H).

4) Synthesis of product A3:

intermediate A3-4 (67mg, 0.32mmol), intermediate A1-5 (83mg, 0.38mmol), DIPEA (82mg, 0.64mmol) and HATU (182mg, 0.48mmol) were added to DMF (2 mL) and reacted at room temperature overnight. Diluted with water (20 mL) and extracted with ethyl acetate (50 mL). The organic phase was washed with saturated brine (20ml × 4), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (dichloromethane: methanol =50:1 to 20) to obtain a yellow solid (15mg, 11%). After nuclear magnetic spectrum analysis (spectrum data are shown in table 1), the obtained solid is the compound A3.

Example 4

This example provides a heterocyclic compound A4 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

a4-1 (obtained from the synthetic route of WO2017062688A1, 46mg,0.2 mmol) and A4-2 (obtained from the synthetic route of Bioorg, med. Chem.2016,24,5861-5872, 42mg,0.2 mmol) were dissolved in DMF (2 mL) and HATU (76mg, 0.2 mmol) and DIPEA (129mg, 1.0 mmol) were added. After stirring at room temperature overnight, water (30 mL) was added, extraction was performed with ethyl acetate (30ml × 3), and the organic phases were combined, washed with saturated brine (30ml × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol =30 1) to obtain a pale yellow solid (60mg, 71%). After nuclear magnetic spectrum analysis (the spectrum data are shown in table 1), the obtained solid is the compound A4.

Example 5

This example provides a heterocyclic compound A5 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

a5-1 (obtained from the synthetic route of patent WO2017062688A1, 45mg, 0.2mmol) and A4-2 (42mg, 0.2mmol) were dissolved in DMF (2 mL) and HATU (76mg, 0.2mmol) and DIPEA (129mg, 1.0mmol) were added. After stirring at room temperature overnight, water (30 mL) was added, and extraction was performed with ethyl acetate (30ml × 3), and the organic phases were combined, washed with saturated brine (30ml × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol =30: 1) to obtain a white solid (38mg, 46%). After nuclear magnetic spectrum analysis (spectrum data are shown in table 1), the obtained solid is the compound A5.

Example 6

This example provides a heterocyclic compound A6 with Wnt and Hedgehog signaling pathway inhibitory activity, which is synthesized by the following method:

1) Synthesis of intermediate A6-2:

a6-1 (80mg, 0.5 mmol) and A4-2 (105mg, 0.5 mmol) were dissolved in DMF (2 mL) and HATU (190mg, 0.5 mmol) and DIPEA (323mg, 2.5 mmol) were added. After stirring at room temperature overnight, water (30 mL) was added, and extraction was performed with ethyl acetate (30ml × 3), and the organic phases were combined, washed with saturated brine (30ml × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol =30: 1) to obtain a white solid (160mg, 91%). ¹ H NMR(400MHz,DMSO)δ11.49(s,1H),9.19(t,J＝5.6Hz,1H),8.32(s,1H),8.22-8.15(m,2H),8.02(s,1H),7.91(d,J＝9.2Hz,1H),7.70(d,J＝8.0Hz,1H),7.53(d,J＝8.0Hz,1H),7.48-7.40(m,1H),7.22-7.16(m,1H),4.57(d,J＝5.6Hz,2H).

2) Synthesis of product A6:

a6-2 (50mg, 0.14mmol) and 4-pyridineboronic acid (21mg, 0.17mmol) were dissolved in dioxane (2 mL) and water (0.5 mL), and Pd (dppf) Cl was added ₂ (5mg, 0.007mmol), dppf (4mg, 0.007mmol) and potassium phosphate (59mg, 0.28mmol). NitrogenThe reaction mixture was replaced with nitrogen five times under a gas blanket, heated to 100 ℃ and reacted for 12 hours. After cooling to room temperature, concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol =30: 1) to give a gray solid (30mg, 53%). After nuclear magnetic spectrum analysis (spectrum data are shown in table 1), the obtained solid is the compound A6.

Example 7

This example provides a heterocyclic compound a having Wnt and Hedgehog signaling pathway inhibitory activity, which was synthesized by literature methods (bioorg.med.chem.2016, 24, 5861-5872.).

Table 1 lists the analytical structures and spectral data of the compounds obtained in examples 1 to 7.

TABLE 1

Example 8

In this example, the inhibitory activity of the heterocyclic compounds A1 to A6 having Wnt and Hedgehog signaling pathway inhibitory activities prepared in examples 1 to 7 and the inhibitory activity of compound a on the Wnt signaling pathway were measured.

LWnt3A cells (CRL-2647, ATCC) were cultured in DMEM medium (Gibico) containing 10% fetal bovine serum (Hyclone). HEK293STF stable clone cells (HEK 293 cells transfected with "Super-TopFlash" TCF fluorescent reporter plasmid) were cultured in complete medium (DMEM medium containing 4 mML-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 6. Mu.g/mL blasticidin and 10% fetal bovine serum). LWnt3A cells and HEK293STF stable clone cells were harvested when cultured to 90% confluence and mixed at a ratio of 1. 100 μ L/well of the mixed cell culture solution was added to a 96-well plate to give a final cell concentration of 12000 cells/well, followed by further culture for 24 hours. The test compound was diluted stepwise with DMSO and then diluted to a desired concentration with DMEM medium. mu.L of the compound solution was added to the aforementioned 96-well plate containing the cell culture solution, followed by incubation at 37 ℃ for 48 hours. Finally, 50. Mu.L of luciferase solution (Brigh-Glo, promega) was added to each well and shaken at room temperature for 5 minutes. Enzyme label for luminescence signalMeasurement by a PHERAStarFS (BMG). Testing the synthesized compound, and when the compound is not added, the LWnt3A cells secrete Wnt3A protein to activate Wnt signal path and express fluorescent protein, wherein the fluorescent signal value is set as 100%; the test compounds were added at different concentrations, and the inhibitory activity of the compounds at different concentrations on the Wnt signaling pathway differed, and when the Wnt signaling pathway was completely inhibited, the fluorescence signal value was set to 0%. The fluorescence signal value is used as the ordinate, the concentration of the compound to be detected is used as the abscissa to make a graph, and the concentration of the compound when the fluorescence signal value is 50%, namely the half inhibition concentration IC, can be obtained from the graph ₅₀ The value is obtained. IC (integrated circuit) ₅₀ The lower the value, the higher the activity of the heterocyclic compound. The inhibition results of A1 and A5 and compound A on the Wnt signal pathway are shown in the graphs of FIG. 1, FIG. 2 and FIG. 3; the test patterns for the remaining compounds were essentially the same. The overall results are shown in table 2 (results of experiments for determining the inhibitory ability of heterocyclic compounds A1-A6 and compound a on Wnt and Hedgehog signaling pathways). Wherein LGK974 and Vismodegib are control compounds, LGK974 is a known Wnt signaling pathway inhibitor, currently in secondary clinics; vismodegib is a known Hedgehog signaling pathway inhibitor that has been approved by the FDA in the united states for marketing in 2012.

Example 9

In this example, the inhibitory activity of the heterocyclic compounds A1 to A6 and compound a having Wnt and Hedgehog signaling pathway inhibitory activities prepared in examples 1 to 7 on the Hedgehog signaling pathway was measured.

NIH3T3 cells were cultured in DMEM (11965, gibico) containing 10% FBS (Hyclone). The GRE-firefly luciferin plasmid was obtained by implanting eight-fold amplified cell transcription factor GLI-1 response elements into the MCS. The monoclonal was verified by recombinant sunick hedgehog pathway protein and small molecule agonist SAG with the structural formula shown below. The verified select clones were used to detect hedgehog pathway signals.

NIH3T3 cells expressing GRE-firefly hormone were maintained in intact culture. When assay detection is required, cells are added to a 96-well plate, eventually containing about fifteen thousand cells per well. 96-well plates were incubated for 48 hours. Test compounds were serially diluted with DMSO and assay buffer. 10nM SAG as hedgehog pathway agonist. Then 100 microliters of assay buffer containing the test compound and agonist were carefully added to the 96-well plate containing the cells and incubated at 37 degrees celsius for 48 hours.

After 48 hours of incubation, 40 microliters of firefly luciferase was added to each well. The 96-well plate was shaken gently at room temperature for 5 minutes. The light signal is recorded by the plate reader. The activity of the compound is calculated by its blocking of the luminescence signal. Testing the synthesized compound, and when only SAG is added and no compound is added, completely activating a Hedgehog signal path by the SAG to express a fluorescent protein, wherein the fluorescence signal value is set to be 100%; and adding SAG and the tested compounds with different concentrations simultaneously, wherein the compounds with different concentrations have different inhibitory activities on a Hedgehog signal path, and when the Hedgehog signal path is completely inhibited, the fluorescence signal value is set to be 0%. The fluorescence signal value is used as the ordinate, the concentration of the compound to be detected is used as the abscissa to make a graph, and the concentration of the compound when the fluorescence signal value is 50%, namely the half inhibition concentration IC, can be obtained from the graph ₅₀ The value is obtained. IC (integrated circuit) ₅₀ The lower the value, the higher the activity of the heterocyclic compound. The graphs of the inhibition results of A1 and A5 and the compound A on the Hedgehog signal path are shown in FIG. 4, FIG. 5 and FIG. 6; the test patterns for the remaining compounds were essentially the same. The overall results are shown in table 2 (results of experiments for determining the inhibitory ability of heterocyclic compounds A1-A6 and compound a on Wnt and Hedgehog signaling pathways). Wherein LGK974 and Vismodegib are control compounds, LGK974 is a known Wnt signaling pathway inhibitor, currently in secondary clinics; vismodegib is a known Hedgehog signaling pathway inhibitor that has been approved by the FDA in the united states for marketing in 2012.

TABLE 2 results of experiments for determining the inhibitory potency of heterocyclic compounds A1-A6 and Compound A on Wnt and Hedgehog signaling pathways

As can be seen from table 2 above, the known Wnt signaling pathway inhibitor (LGK 974) and Hedgehog signaling pathway inhibitor (Vismodegib) can only inhibit one of the Wnt signaling pathway and Hedgehog signaling pathway, but cannot inhibit both Wnt and Hedgehog signaling pathways. The heterocyclic compound provided by the invention is used as an effective antagonist of the Wnt signal channel and the Hedgehog signal channel, can effectively block the Wnt signal channel and the Hedgehog signal channel at the same time, and can be better used for treating or preventing diseases caused by the abnormality of the Wnt signal channel or the Hedgehog signal channel.

Claims (6)

1. The following heterocyclic compounds having Wnt and Hedgehog signaling pathway inhibitory activity, or pharmaceutically acceptable salts thereof:

2. a pharmaceutical composition comprising a therapeutically effective amount of the heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of the heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the manufacture of a medicament for treating or preventing Wnt and Hedgehog signaling pathway abnormalities; the medicament for treating or preventing Wnt and Hedgehog signaling pathway abnormality comprises a medicament for treating one or more of breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous carcinoma, thyroid cancer, sarcoma, osteosarcoma, desmoid tumor, melanoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, myeloma, lymphoma, chronic and non-progressive anemia, idiopathic or primary thrombocythemia, idiopathic myelofibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, liver cirrhosis, diabetic retinopathy, macroglobulinemia, leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumor, schwannoma, primary neuroectoblastoma, pituitary tumor, and a pharmaceutical composition thereof.

4. The use according to claim 3,

the lymphoma is mantle cell lymphoma or cutaneous T cell lymphoma;

the leukemia is acute leukemia, chronic leukemia, lymphatic leukemia or myeloid leukemia;

the brain tumor is astrocytoma or medulloblastoma.

5. Use of a heterocyclic compound having Wnt and Hedgehog signaling pathway inhibitory activity of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2 in the manufacture of a medicament for treating or preventing an abnormality of the Hedgehog signaling pathway; the medicament for treating or preventing the Hedgehog signaling pathway abnormality comprises medicaments for treating one or more of breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous epithelial cancer, thyroid cancer, sarcoma, osteosarcoma, desmoid tumor, melanoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, myeloma, lymphoma, chronic and non-progressive anemia, idiopathic or primary thrombocytosis, idiopathic myelofibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, cirrhosis of liver, diabetic retinopathy, macroglobulinemia, leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumor, schwannoma, primary neuroectoblastoma and pituitary tumor, and pharmaceutical compositions thereof.

6. The use according to claim 5,

the lymphoma is mantle cell lymphoma or cutaneous T cell lymphoma;

the leukemia is acute leukemia, chronic leukemia, lymphatic leukemia or myeloid leukemia;

the brain tumor is astrocytoma or medulloblastoma.

Images