CN109666028B - 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity - Google Patents

Abstract

The invention discloses a1, 3, 4-thiadiazole heterocyclic compound with hedgehog pathway antagonist activity, which has a structure shown in a general formula I:

the 1,3, 4-thiadiazole heterocyclic compound with the hedgehog pathway antagonist activity can block metastasis and regeneration of tumor cells through hedgehog pathway antagonism, and has an obvious anti-tumor effect.

Description

1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity

Technical Field

The invention relates to the technical field of medicine synthesis, in particular to a1, 3, 4-thiadiazole heterocyclic compound with hedgehog pathway antagonist activity.

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 novel lead compound which has high efficiency and low toxicity and selectively acts on a specific target is found by taking some key enzymes of a cell signal transduction pathway related to tumor cell differentiation and proliferation as a drug screening target, and becomes 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 typical.

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 of hedgehog (Hh) signaling pathway-hedgehog pathway has been paid more and more attention in recent years, not only because the abnormal activation of Hh signaling pathway plays a crucial role in the development process of many tumors including basal cell carcinoma, brain tumor, breast cancer, prostate cancer and some malignant tumors of digestive system, but also more importantly, Hh signaling pathway is an embryonic development pathway and plays an important role in controlling tumor stem cells, thereby controlling tumor metastasis and regeneration.

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 and, in turn, the transcriptional expression of downstream genes; in the presence of Hh signal, Hh binds to PTCH, inducing phosphorylation of multiple filament/threonine residues at the carboxy-terminal end of SMO, resulting in accumulation and activation of SMO on the cell surface, and the activated SMO forms a complex with kinesin-like molecule Costal2(Cos2) and filament/threonine kinase fused (fus), and is dissociated from microtubules to exert transcriptional activation in full-length form, finally causing activation of zinc finger-like transcription factor Gli, which enters the nucleus to cause transcription of the target gene. Thus, in the Hh signaling pathway, Hh is the start of the signaling pathway, Gli is the end of the signaling pathway as a transcription factor, Hh and SMO are agonists, and PTCH is an inhibitor, regulating the activity of the signaling pathway.

The transmembrane protein receptor SMO is a key member of an Hh signal path, is an information converter in the Hh signal path, and can convert an extracellular Hh signal into an intracellular Gli1 signal so as to start the transcription of genes in a cell nucleus and activate the Hh signal path. Functional mutation of SMO exists in the process of generation and development of most tumor cells related to the activation of Hh cellular pathway. Small molecule SMO protein antagonists specifically block the Hh signal pathway by antagonizing SMO, and the Hh signal pathway is in an inactive state in normal adults, so the antagonists do not produce side effects on other parts of the body, which is the theoretical basis of the feasibility of targeted therapy of tumors. Therefore, the SMO protein has become one of the most interesting targets in the current research and development of antitumor drugs, and the synthesis of small molecule antagonists targeting the SMO protein has also become a research and development hotspot of various pharmaceutical companies internationally.

At least 5 small molecule antagonists targeting SMO proteins are currently in clinical testing, and GDC-0449, a small molecule SMO antagonist co-developed by Genentech and Curis, was approved by the food and drug administration FDA for the treatment of advanced basal cell carcinoma patients in month 2012. The research and development of the small molecular SMO antagonist as a new antitumor drug have good application value and market prospect.

Representative small molecule SMO antagonists in clinical testing include the following:

despite their superior efficacy, vismodegib and other clinical compounds have problems with each. Such as vismodegib, which has poor physicochemical properties, low solubility, strong side effects and resistance, etc. Therefore, there is a need to provide a novel hedgehog pathway antagonist which avoids the drug resistance and side effects of the drugs in the prior art, and better blocks the metastasis and regeneration of tumor cells to treat tumors.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a1, 3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity, which can block metastasis and regeneration of tumor cells and treat tumor diseases.

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

a1, 3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity, comprising the compound and pharmaceutically acceptable salts thereof, having a structure shown in general formula I:

wherein m is 1,2,3 or 4;

n is 0 or 1;

R₁selected from hydrogen atom, halogen, methyl, trifluoromethyl, hydroxyl, sulfhydryl, ethyl, methoxyl and cyclopropyl;

R₂selected from C (O) R₄、C(O)OR₄、C(O)NR₄R₅、NR₆C(O)R₄、NR₆C(O)OR₄、NR₆C(O)NR₄R₅；

A1 is selected from unsubstituted or substituted by 1-4R₁₀Substituted five-membered, six-membered, and six-membered aromatic rings containing 1-3 heteroatoms, or A1 is unsubstituted or substituted with 1-4R₁₀Substituted by

The heteroatom is selected from O, N, S;

l is selected from the group consisting of a bond, -O-, -N-, -S-, - (CR)₇R₈)_k-、-O(CR₇R₈)_k-、-S(CR₇R₈)_k-、-NR₉(CR₇R₈)_k-、-(CR₇R₈)_kO-、-(CR₇R₈)_kS-、-(CR₇R₈)_kNR₉-、-R₇C＝CR₈-、

Wherein k is 1,2,3 or 4;

u is selected from unsubstituted or substituted by 1-2R₁₁Substituted five-or six-membered heteroaromatic and six-membered aromatic rings containing 1-3 heteroatoms selected from O, N, S;

R₄、R₅are independently selected from hydrogen atoms, unsubstituted or substituted by 1-3R₁₂C substituted by substituents_1-8Alkyl, unsubstituted or substituted by 1 to 3R₁₂C substituted by substituents_3-8Cycloalkyl radical, C_2-8Alkenyl or C_2-8Alkynyl, C_1-8Sulfonyl radical, C_1-8Sulfonamide, C_1-8Alkoxy radical, C_1-8Ester group, C_1-8Acyl radical, C_1-8Amide group, C_1-8Oxo-ureido radical, C_1-8A urea group; or R₄、R₅Selected from unsubstituted or substituted by 1-3R₁₃Phenyl ring substituted by radicals, unsubstituted or substituted by 1 to 3R₁₃5-6 membered heteroaryl ring substituted with radicals containing 1-3 heteroatoms, unsubstituted or substituted with 1-3R₁₃The 5-7 membered saturated heterocyclic ring substituted by a group containing 1-3 heteroatoms, the heteroatoms of the heteroaromatic ring and the saturated heterocyclic ring including one of N, O and SOne or more kinds;

R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃are respectively selected from hydrogen atom, deuterium atom, halogen, cyano, hydroxyl, amino, sulfydryl and C_1-6Alkoxy radical, C_1-6Alkyl radical, C_3-6A cycloalkyl group.

Preferably, the 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity as described above, wherein L is ortho-substituted with 1,3, 4-thiadiazole on U.

Preferably, the 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity as described above, wherein L is-CH₂O-、-CH(CH₃)O-、-CH₂S-、-CH₂NH-、-(CH₂)₂-, -HC ═ one of the CH-groups.

Preferably, the 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity as described above, wherein A1 is unsubstituted or substituted with 1 to 4R₁₀One of the following groups substituted with a group:

preferably, the 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity as described above, wherein U is unsubstituted or substituted with 1 to 2R₁₁One of the following groups substituted with a group:

preferably, the above-mentioned 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity, wherein R is₂Is one of the following groups:

preferably, the above-mentioned 1,3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity includes the following compounds:

the invention also proposes a pharmaceutical composition comprising as active ingredient the above compound and at least one pharmaceutically acceptable carrier or diluent.

The invention also provides an application of the pharmaceutical composition in preparing a medicament for treating tumors mediated by a Hedgehog signal pathway, which is characterized by comprising the following steps: the tumor includes nasopharyngeal carcinoma, breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous carcinoma, thyroid cancer, sarcoma, osteosarcoma, desmoid tumor, melanoma, basal cell carcinoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric 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, myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumors, astrocytomas, medulloblastoma, schwannoma, primary neuroectodermal tumors, leukemia, melanoma, and leukemia, One or more of pituitary tumors.

The invention has the outstanding effects that: the invention provides a novel 1,3, 4-thiadiazole heterocyclic compound with hedgehog pathway antagonist activity, which can block metastasis and regeneration of tumor cells so as to treat tumor diseases.

The following detailed description of the embodiments of the present invention is provided in connection with the accompanying drawings for the purpose of facilitating understanding and understanding of the technical solutions of the present invention.

Drawings

FIG. 1 is a graph of the test results for Compound D11;

FIG. 2 is a graph of the test results for Compound D25;

FIG. 3 is a graph of the test results for Compound D26;

Detailed Description

The process of the present invention is illustrated below by means of specific examples, but the present invention is not limited thereto. 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. The solvent and the medicine are analytically pure or chemically pure; the solvent is redistilled before use; the anhydrous solvent is treated according to standard or literature methods. Column chromatography silica gel (100-200 mesh) and thin layer chromatography silica gel (GF254) are products of Qingdao oceanic plant and tobacco station chemical plant; petroleum ether (60-90 ℃)/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 Na₂SO₄And (5) drying.¹H NMR was recorded using a Varian unity INOVA 400NB and Vnmrs high resolution nuclear magnetic resonance spectrometer, TMS as an internal standard. LC-MS was recorded using an Agilent model 1100 high performance liquid chromatography-ion Trap Mass spectrometer (LC-MSD Trap), Diode Array Detector (DAD), detection wavelengths 214nm and 254nm, ion Trap Mass Spectrometry (ESI source). HPLC column is Agela Durashell C18 (4.6X 50mm, 3.5 μm); mobile phase 0.1% TFA in water: acetonitrile (from 5: 95 to 95: 5 in 6 minutes); the flow rate was 1.2 ml/min.

Example 1

This example provides a1, 3, 4-thiadiazole heterocyclic compound D1 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D1-1:

2- amino 1,3, 4-thiadiazole (10g,100mmol) and sodium bicarbonate (16g,190mmol) were added to methanol (140mL), and bromine (5.5mL,110mmol) was slowly added dropwise at 0 deg.C, followed by stirring at this temperature for 0.5 hour. Most of the methanol was removed by concentration under reduced pressure, and the reaction was diluted with water (150mL), filtered and dried under vacuum to give a brown solid (16g, 89%).¹H NMR(400MHz,DMSO-d6)δ7.51(s,2H).

2) Synthesis of intermediate D1-2:

intermediate D1-1(5.0g,27.8mmol), ethyl 4-piperidinecarboxylate (5.2g, 33.3mmol) and potassium carbonate (7.7g, 55.6mmol) were added to isopropanol (100mL) and stirred at 90 ℃ for 3 hours. After cooling to room temperature, most of the isopropanol was removed by concentration under reduced pressure, and water (50mL) was added to the reaction, filtered, and dried under vacuum to give a red solid (4.7g, 66%).¹H NMR(400MHz,CDCl₃)δ4.70(br s,2H),4.16(q,J＝7.2Hz,2H),3.77-3.72(m,2H),3.12-3.06(m,2H),2.53-2.46(m,1H),2.02-1.98(m,2H),1.89-1.79(m,2H),1.27(t,J＝7.0Hz,3H).

3) Synthesis of intermediate D1-3:

intermediate D1-2(5.0g, 19.5mmol), 2, 5-dimethoxytetrahydrofuran (3.1g, 23.4mmol) was added to acetic acid (20mL), stirred at 100 ℃ for 1 hour, concentrated under reduced pressure to remove most of the acetic acid. The reaction was made basic with saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (50mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate: 20:1 to 2:1) to give a yellow solid (3g, 50%).¹H NMR(400MHz,CDCl₃)δ7.18(t,J＝2.0Hz,2H),6.32(t,J＝2.0Hz,2H),4.17(q,J＝7.2Hz,2H),3.90-3.84(m,2H),3.25-3.19(m,2H),2.59-2.52(m,1H),2.06-2.02(m,2H),1.92-1.82(m,2H),1.27(t,J＝7.2Hz,3H).

4) Synthesis of intermediate D1-4:

dropping POCl into DMF (30mL) under ice-water bath₃(8.2mL, 88.2mmol) and stirred for 20 min. Then, intermediate D1-3(9.0g, 29.4mmol) was added and reacted at 80 ℃ for 1 hour. Cooling to normal temperature, pouring the reaction solution into 100mL of ice water, adjusting the pH value to about 8 by using saturated sodium bicarbonate aqueous solution,and then extracted with ethyl acetate (200mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate 4:1 to 1:1) to give a yellow solid (3.0g, 30%).¹H NMR(400MHz,CDCl₃)δ9.69(s,1H),7.47(s,1H),7.16-7.14(m,1H),6.44-6.42(m,1H),4.16(q,J＝7.2Hz,2H),3.95-3.89(m,2H),3.29-3.22(m,2H),2.60-2.53(m,1H),2.07-2.03(m,2H),1.92-1.82(m,2H),1.27(t,J＝7.2Hz,3H).

5) Synthesis of intermediate D1-5:

intermediate D1-4(3.0g, 9.0mmol), p-toluidine (1.15g, 10.8mmol) and acetic acid (540mg, 9.0mmol) were added to dichloromethane (100mL) at 0 deg.C and stirred at this temperature for 30 min. Add NaBH (AcO) at 0 deg.C₃(5.7g,27mmol), then brought to ambient temperature and reacted for 12 hours. The reaction mixture was washed with saturated sodium bicarbonate (50mL) and then with saturated sodium chloride (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate: 5:1 to 2:1) to give a yellow solid (2.5g, 65%).¹H NMR(400MHz,CDCl₃)δ6.96(d,J＝8.0Hz,2H),6.89(s,1H),6.60(d,J＝8.0Hz,2H),6.26(s,1H),6.21-6.19(m,1H),4.44(s,2H),4.17(q,J＝7.0Hz,2H),3.88-3.85(m,2H),3.25-3.19(m,2H),2.58-2.52(m,1H),2.21(s,3H),2.11-1.98(m,2H),1.92-1.82(m,2H),1.28(t,J＝7.0Hz,3H).

6) Synthesis of intermediate D1-6:

intermediate D1-5(2.5g,5.9mmol) was added to a mixed solvent of tetrahydrofuran (18mL) and methanol (2mL), followed by dropwise addition of 1N LiOH (18mL,18 mmol). The reaction was carried out at room temperature for 4 hours, and most of tetrahydrofuran was removed by concentration under reduced pressure, diluted with water (10mL), washed with ethyl acetate (40mL), and the aqueous phase was adjusted to pH 5-6 with 1N hydrochloric acid, whereupon a solid precipitated. Filtration and vacuum drying afforded a white solid (1g, 42%).¹H NMR(400MHz,CDCl₃)δ6.96(d,J＝8.0Hz,2H),6.89(s,1H),6.62(d,J＝8.0Hz,2H),6.26(s,1H),6.21-6.19(m,1H),4.43(s,2H),3.94-3.82(m,2H),3.28-3.21(m,2H),2.67-2.58(m,1H),2.22(s,3H),2.10-2.05(m,2H),1.94-1.84(m,2H).

7) Synthesis of final product D1:

intermediate D1-6(70mg,0.18mmol), ethylamine hydrochloride (21mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1) to give a white solid (57mg, 76%). After nmr analysis (see table 1 for data), the solid obtained was compound D1.

Example 2

This example provides a1, 3, 4-thiadiazole heterocyclic compound D2 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(80mg,0.2mmol), isopropylamine (18mg,0.3mmol), DIPEA (77mg,0.6mmol) and HATU (91mg,0.24mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1) to give a white solid (25mg, 28%). After nmr analysis (see table 1 for data), the solid obtained was compound D2.

Example 3

This example provides a1, 3, 4-thiadiazole heterocyclic compound D3 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), cyclopropylamine (15mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1) to give a yellow solid (45mg, 58%). After nmr analysis (see table 1 for data), the solid obtained was compound D3.

Example 4

This example provides a1, 3, 4-thiadiazole heterocyclic compound D4 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), isobutylamine (19mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to give a white solid (10mg, 12%). After nmr analysis (see table 1 for data), the solid obtained was compound D4.

Example 5

This example provides a1, 3, 4-thiadiazole heterocyclic compound D5 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), cyclopropylmethylamine (18mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to give a white solid (44mg, 55%). After nmr analysis (see table 1 for data), the solid obtained was compound D5.

Example 6

This example provides a1, 3, 4-thiadiazole heterocyclic compound D6 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), cyclopentylamine (22mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to give a yellow solid (50mg, 60%). After nmr analysis (see table 1 for data), the solid obtained was compound D6.

Example 7

This example provides a1, 3, 4-thiadiazole heterocyclic compound D7 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), cyclopentylamine (22mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to give a white solid (44mg, 52%). After nmr analysis (see table 1 for data), the solid obtained was compound D7.

Example 8

This example provides a1, 3, 4-thiadiazole heterocyclic compound D8 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), tetrahydropyrrole (18mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to give a white solid (50mg, 62%). After nmr analysis (see table 1 for data), the solid obtained was compound D8.

Example 9

This example provides a1, 3, 4-thiadiazole heterocyclic compound D9 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), 1-amino-2-methyl-2-propanol (23mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:1) to give a white solid (43mg, 52%). After nmr analysis (see table 1 for data), the solid obtained was compound D9.

Example 10

This example provides a1, 3, 4-thiadiazole heterocyclic compound D10 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D1-6(70mg,0.18mmol), 3-pyrrolidinol (23mg,0.26mmol), DIPEA (70mg,0.54mmol) and HATU (82mg,0.22mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:1) to give a white solid (30mg, 36%). After nmr analysis (see table 1 for data), the solid obtained was compound D10.

Example 11

This example provides a1, 3, 4-thiadiazole heterocyclic compound D11 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D11-1:

intermediate D1-4(700mg, 2.1mmol), 2-amino-5-methylpyridine (248mg, 2.3mmol) and acetic acid (126mg, 2.1mmol) were added to dichloromethane (30mL) at 0 deg.C and stirred at this temperature for 30 min. Add NaBH (AcO) at 0 deg.C₃(1.3g,6.3mmol), then moved to normal temperature, and reacted for 12 hours. The reaction mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate (30mL) and then with a saturated aqueous solution of sodium chloride (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate: 5:1 to 2:1) to give a yellow oil (620mg, 69%).¹H NMR(400MHz,CDCl₃)δ7.86(s,1H),7.28(d,J＝8.4Hz,1H),6.84(s,1H),6.47(d,J＝8.4Hz,1H),6.38(s,1H),6.23-6.21(m,1H),4.71(s,2H),4.17(q,J＝7.1Hz,2H),3.89-3.86(m,2H),3.27-3.22(m,2H),2.64-2.50(m,1H),2.16(s,3H),2.06-2.03(m,2H),1.92-1.83(m,2H),1.28(t,J＝6.8Hz,3H).

2) Synthesis of intermediate D11-2:

intermediate D11-1(400mg,0.95mmol) was added to a mixed solvent of tetrahydrofuran (10mL) and methanol (1mL), followed by dropwise addition of 1N LiOH (3mL,3 mmol). The reaction was carried out at ambient temperature for 4 hours, concentrated under reduced pressure to remove most of the tetrahydrofuran, diluted with water (10mL), washed with ethyl acetate (20mL), and the aqueous phase was adjusted to pH 5-6 with 1N HCl and finally extracted with dichloromethane (30 mL. times.5). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow oil (55mg, 14%).

3) Synthesis of final product D11:

intermediate D11-2(55mg,0.14mmol), 2-methylcyclohexylamine (23mg,0.21mmol), DIPEA (54mg,0.42mmol) and HATU (64mg,0.17mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate 1:1) to give a white solid (50mg, 73%). After nmr analysis (see table 1 for data), the solid obtained was compound D11.

Example 12

This example provides a1, 3, 4-thiadiazole heterocyclic compound D12 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D12-1:

intermediate D1-4(500mg, 1.5mmol), 3-amino-6-methylpyridine (178mg, 1.7mmol) and acetic acid (90mg, 1.5mmol) were added to dichloromethane (30mL) at 0 deg.C and stirred at this temperature for 30 min. Add NaBH (AcO) at 0 deg.C₃(954mg,4.5mmol), then transferred to normal temperature, and reacted for 12 hours. The reaction mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate (30mL) and then with a saturated aqueous solution of sodium chloride (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate: 5:1 to 2:1) to give a yellow oil (450mg, 70%).¹H NMR(400MHz,CDCl₃)δ7.97(s,1H),6.95-6.92(m,2H),6.84(s,1H),6.27(s,1H),6.21-6.19(m,1H),4.47(s,2H),4.17(q,J＝7.1Hz,2H),3.89-3.86(m,2H),3.28-3.22(m,2H),2.62-2.54(m,1H),2.40(s,3H),2.09-2.04(m,2H),1.93-1.84(m,2H),1.28(t,J＝6.8Hz,3H).

2) Synthesis of intermediate D12-2:

intermediate D12-1(450mg,1.1mmol) was added to a mixed solvent of tetrahydrofuran (10mL) and methanol (1mL), followed by dropwise addition of 1N LiOH (3mL,3 mmol). The reaction was carried out at room temperature for 4 hours, concentrated under reduced pressure to remove most of the tetrahydrofuran, diluted with water (10mL), washed with ethyl acetate (20mL), and the aqueous phase was adjusted to pH 5-6 with 1N HCl, whereupon a solid precipitated, filtered, and dried under vacuum to give a white solid (300mg, 71%).

3) Synthesis of final product D12:

intermediate D12-2(50mg,0.13mmol), 2-methylcyclohexylamine (21mg,0.19mmol), DIPEA (50mg,0.39mmol) and HATU (59mg,0.16mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate 1:1) to give a white solid (46mg, 74%). After nmr analysis (see table 1 for data), the solid obtained was compound D12.

Example 13

This example provides a1, 3, 4-thiadiazole heterocyclic compound D13 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D13-1:

isoamyl nitrite (2.8g,23.9mmol) and copper bromide (5.4g, 23.9mmol) were added to acetonitrile (100mL) at 0 deg.C and stirred at this temperature for 10 minutes. Then, D1-2(4.7g,18.4mmol) was further added to the reaction solution and reacted at 50 ℃ for 1 hour. The mixture was concentrated under reduced pressure, and saturated brine (50mL) was added thereto, followed by extraction with ethyl acetate (40 mL. multidot.3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate 50:1 to 5:1) to give a yellow solid (3g, 51%).

2) Synthesis of intermediate D13-2:

intermediate D13-1(8.0g,25mmol) was dissolved in a mixed solvent of THF (100mL) and methanol (10mL), followed by dropwise addition of 3N LiOH (25mL,75mmol), and stirring was carried out at room temperature for 4 hours. Dilute with water (20mL), concentrate under reduced pressure to remove most of the THF, wash with ethyl acetate (50mL), adjust the aqueous phase to pH 5-6 with 2N HCl, precipitate a solid, filter, and dry in vacuo to give a white solid (7.0g, 95%).

3) Synthesis of intermediate D13-3:

intermediate D13-2(1.0g,3.4mmol), 2-methylcyclohexylamine (576mg,5.1mmol), DIPEA (877mg,6.8mmol) and HATU (1.67g,4.4mmol) were added to DMF (10mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (100mL) and washed with saturated aqueous sodium chloride (50mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1) to give a white solid (1.0g, 75%).

3) Synthesis of intermediate D13-4:

intermediate D13-3(1.0g,2.58mmol) and hydrazine hydrate (1.3g, 25.8mmol) were added to 20mL of 1, 4-dioxane and reacted in a sealed tube at 100 ℃ for 24 hours. After cooling to ambient temperature, a solid precipitated, which was filtered and dried in vacuo to give a white solid (450mg, 51%).

4) Synthesis of intermediate D13-5:

intermediate D13-4(200mg,0.59mmol) and 4- (dimethylamino) -1, 1-dimethoxy-3-buten-2-one (112mg,0.65mmol) were added to toluene (10mL) and reacted at 100 ℃ for 1.5 hours. Concentrated under reduced pressure, and acetic acid (5mL) was added to the mixture to conduct a reaction at 100 ℃ for 30 minutes. The mixture was concentrated under reduced pressure to remove acetic acid, made alkaline with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate (20mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate 1:1) to give a yellow solid (60mg, 25%).¹H NMR(400MHz,CDCl₃)δ10.76(s,1H),7.70(s,1H),7.11(s,1H),5.48(d,J＝8.4Hz,0.7H),5.20(d,J＝8.8Hz,0.3H),4.13-4.08(m,0.7H),4.04-4.01(m,2H),3.55-3.46(m,0.3H),3.28-3.22(m,2H),2.42-2.32(m,1H),1.98-1.87(m,4H),1.74-1.50(m,5H),1.42-1.33(m,2H),1.25-1.05(m,2H),0.91(d,J＝6.4Hz,1H),0.86(d,J＝6.8Hz,2H).

4) Synthesis of final product D13:

intermediate D13-5(60mg,0.15mmol), p-toluidine (24mg,0.22mmol) and acetic acid (9mg,0.15mmol) were added to dichloromethane (10mL) and stirred at room temperature for 1 hour. Add NaBH (AcO) at 0 deg.C₃(95mg,0.45mmol), transferred to ambient temperature and reacted for 36 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (20mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 5:1 to 1:1) to give a white solid (50mg, 67%). After nmr analysis (see table 1 for data), the solid obtained was compound D13.

Example 14

This example provides a1, 3, 4-thiadiazole heterocyclic compound D14 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

intermediate D13-5(40mg,0.1mmol), p-toluidine (12mg,0.11mmol) and acetic acid (6mg,0.1mmol) were added to dichloromethane (10mL) and stirred at room temperature for 1 hour. Add NaBH (AcO) at 0 deg.C₃(63mg,0.45mmol), transferred to ambient temperature and reacted for 36 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (20mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1:2) to give a white solid (7.0mg, 14%). After nmr analysis (see table 1 for data), the solid obtained was compound D14.

Example 15

This example provides a1, 3, 4-thiadiazole heterocyclic compound D15 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D15-1:

intermediate D13-2(1.3g, 4.45mmol) and DMAP (108mg, 0.89mmol) were added to t-butanol (15mL) followed by (Boc)₂O (1.94g,8.9mmol), and stirred at room temperature for 4 hours. Concentration under reduced pressure and column chromatography (petroleum ether: ethyl acetate: 100:1 to 2:1) gave a yellow oil (1.4g, 90%).

2) Synthesis of intermediate D15-2:

intermediate D15-2(1.3g,3.7mmol) and hydrazine hydrate (3.7g, 74mmol) were added to 20mL of 1, 4-dioxane and reacted at 100 ℃ for 48 hours in a sealed tube. Cooled to room temperature, concentrated under reduced pressure, added water (20mL) and a solid precipitated, filtered and dried in vacuo to give a white solid (900mg, 80%).

3) Synthesis of intermediate D15-3:

intermediate D15-2(900mg,3mmol) and 4- (dimethylamino) -1, 1-dimethoxy-3-buten-2-one (1g,6mmol) were added to toluene (40mL) and reacted at 105 ℃ for 10 hours. Concentrated under reduced pressure, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1) to obtain a red solid (640mg, 50%).

4) Synthesis of intermediate D15-4:

intermediate D15-3(640mg, 1.5mmol) was added to acetic acid (15mL) and reacted at 100 ℃ for 3 hours, concentrated under reduced pressure to remove acetic acid, made basic with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate (30mL x 3), the organic phases combined, dried over anhydrous sodium sulfate, filtered, and concentrated to a red solid (480mg, 88%).¹H NMR(400MHz,CDCl₃)δ10.75(s,1H),7.69(s,1H),7.10(s,1H),3.97-3.82(m,2H),3.35-3.21(m,2H),2.56-2.43(m,1H),2.11-1.96(m,2H),1.90-1.81(m,2H),1.46(s,9H).

5) Synthesis of intermediate D15-5:

intermediate D13-4(200mg,0.55mmol), 2-methyl-5-aminopyridine (89mg,0.83mmol) and acetic acid (33mg,0.55mmol) were added to dichloromethane (20mL) and stirred at ambient temperature for 1 h. Add NaBH (AcO) at 0 deg.C₃(350mg,1.65mmol), brought to ambient temperature and reacted for 24 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (30mL), the organic phase was dried over anhydrous sodium sulfate, filtered,concentration and column chromatography (petroleum ether: ethyl acetate: 8:1) gave a yellow oil (150mg, 59%).

6) Synthesis of intermediate D15-6:

intermediate D15-5(150mg,0.33mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl, extracted with dichloromethane (20mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to a yellow solid (80mg, 60%).

7) Synthesis of final product D15:

intermediate D15-6(33mg,0.08mmol), 2-methylcyclohexylamine (12mg,0.11mmol), DIPEA (21mg,0.16mmol) and HATU (42mg,0.11mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate 1:1) to give a white solid (23mg, 56%). After nmr analysis (see table 1 for data), the solid obtained was compound D15.

Example 16

This example provides a1, 3, 4-thiadiazole heterocyclic compound D16 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D16-1:

intermediate D15-4(200mg,0.55mmol), 2-methyl-5-aminopyridine (104mg,1.1mmol) and acetic acid (33mg,0.55mmol) were added to 1, 2-dichloroethane (15mL) and stirred at 70 ℃ for 4 h. Add NaBH (AcO) at 0 deg.C₃(350mg,1.65mmol), brought to ambient temperature and reacted for 16 h. Diluting with dichloromethane (30mL), washing with saturated aqueous sodium bicarbonate solution (30mL), drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography (petroleum oil)Ether: ethyl acetate 20:1 to 1:1) gave a yellow oil (130mg, 53%).¹H NMR(400MHz,CDCl₃)δ8.08(s,1H),7.54(s,1H),7.37-7.33(m,1H),6.56-6.53(m,1H),6.46(s,1H),6.41(d,J＝7.6Hz,1H),5.84-5.77(m,1H),4.94(d,J＝6.4Hz,2H),3.91-3.88(m,2H),3.28-3.22(m,2H),2.53-2.40(m,1H),2.05-2.01(m,2H),1.91-1.77(m,2H),1.46(s,9H).

2) Synthesis of intermediate D16-2:

intermediate D16-1(130mg,0.29mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl, extracted with dichloromethane (20mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid (110mg, 98%).

3) Synthesis of final product D16:

intermediate D16-2(40mg,0.1mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (26mg,0.2mmol) and HATU (49mg,0.13mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:2) to give a white solid (32mg, 64%). After nmr analysis (see table 1 for data), the solid obtained was compound D16.

Example 17

This example provides a1, 3, 4-thiadiazole heterocyclic compound D17& D22 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D17-1:

intermediate D15-4(236mg, 0.65mmol) was dissolved in 10mL of methanol, sodium borohydride (49mg,1.3mmol) was added at 0 deg.C, and stirred at this temperature for 1 hour. Saturated brine (20mL) was added, concentrated under reduced pressure to remove most of the methanol, extracted with ethyl acetate (20mL × 2), the organic phases combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid (232mg, 99%).

2) Synthesis of intermediate D17-2:

intermediate D17-1(100mg,0.27mmol) and triethylamine (137mg, 1.37mmol) were dissolved in dichloromethane (10mL), methanesulfonyl chloride (156mg, 1.37mmol) was added dropwise at ordinary temperature, and the reaction was carried out at ordinary temperature for 3 hours. Dilute with dichloromethane (20mL) and wash with saturated aqueous sodium bicarbonate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 5:1 to 1:1) to give a yellow solid (111mg, 90%).

3) Synthesis of intermediates D17-3 and D22-1:

intermediate D17-2(220mg, 0.5mmol), 2-hydroxy-5-methylpyridine (65mg, 0.6mmol) and cesium carbonate (245mg, 0.75mmol) were added to DMF (3mL) and reacted at 60 ℃ for 12 h. Diluted with ethyl acetate (50mL), washed with saturated aqueous sodium chloride (30mL × 5), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:10) to give two intermediates D17-3 and D22-1.

D17-3(189mg, 83%), yellow oil,¹H NMR(400MHz,CDCl₃)δ7.56(s,1H),7.38(s,1H),7.21(d,J＝8.8Hz,1H),6.55(d,J＝9.2Hz,1H),6.34(s,1H),5.65(s,2H),3.91-3.87(m,2H),3.28-3.22(m,2H),2.51-2.45(m,1H),2.05(s,3H),2.01-1.97(m,2H),1.90-1.80(m,2H),1.46(s,9H).

d22-1(39mg, 17%), white solid,¹H NMR(400MHz,CDCl₃)δ7.92(s,1H),7.59(s,1H),7.40(d,J＝8.4Hz,1H),6.72(d,J＝8.4Hz,1H),6.44(s,1H),5.85(s,2H),3.90-3.87(m,2H),3.26-3.20(m,2H),2.53-2.41(m,1H),2.24(s,3H),2.03-1.99(m,2H),1.89-1.77(m,2H),1.46(s,9H).

3) synthesis of intermediate D17-4:

intermediate D17-3(189mg,0.41mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl and a solid precipitated, filtered and dried in vacuo to give a white solid (152mg, 91%).

4) Synthesis of intermediate D22-2:

intermediate D22-1(39mg,0.08mmol) was dissolved in dichloromethane (3mL), and trifluoroacetic acid (3mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl, extracted with dichloromethane (20mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid (31mg, 91%).

5) Synthesis of final product D17:

intermediate D17-4(40mg,0.1mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (26mg,0.2mmol) and HATU (50mg,0.13mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:10) to give a white solid (32mg, 64%). After nmr analysis (see table 1 for data), the solid obtained was compound D17.

6) Synthesis of final product D22:

intermediate D22-2(30mg,0.08mmol), 2-methylcyclohexylamine (13mg,0.12mmol), DIPEA (21mg,0.16mmol) and HATU (38mg,0.1mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate 1:1) to give a white solid (30mg, 80%). After nmr analysis (see table 1 for data), the solid obtained was compound D22.

Example 18

This example provides a1, 3, 4-thiadiazole heterocyclic compound D18 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D18-1:

intermediate D15-4(150mg,0.41mmol), 3-aminopyridine (58mg,0.62mmol) and acetic acid (49mg,0.82mmol) were added to dichloromethane (20mL) and stirred at ambient temperature for 1 h. Add NaBH (AcO) at 0 deg.C₃(261mg,1.23mmol), transferred to ambient temperature and reacted for 16 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (30mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 10:1 to 1:1) to give a yellow oil (80mg, 43%).

2) Synthesis of intermediate D18-2:

intermediate D18-1(80mg,0.18mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl, extracted with dichloromethane (20mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to a yellow solid (45mg, 64%).

3) Synthesis of final product D18:

intermediate D18-2(45mg,0.12mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (31mg,0.24mmol) and HATU (57mg,0.15mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:2) to give a white solid (22mg, 39%). After nmr analysis (see table 1 for data), the solid obtained was compound D18.

Example 19

This example provides a1, 3, 4-thiadiazole heterocyclic compound D19 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D19-1:

intermediate D15-4(200mg,0.55mmol), 2-amino-5-methylpyrimidine (120mg,1.1mmol), trifluoroacetic acid (188mg, 1.65mmol) and triethylhydrosilane (191mg, 1.65mmol) were added to acetonitrile (20mL) and reacted at 80 ℃ for 12 hours in a sealed tube. Concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate, and extracted with ethyl acetate (20mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:1) to give a white solid (210mg, 83%).¹H NMR(400MHz,CDCl₃)δ8.11(s,2H),7.52(s,1H),6.42(s,1H),6.19-6.16(m,1H),4.99(d,J＝6.8Hz,2H),3.89-3.86(m,2H),3.26-3.21(m,2H),2.50-2.45(m,1H),2.10(s,3H),2.04-2.00(m,2H),1.89-1.80(m,2H),1.46(s,9H).

2) Synthesis of intermediate D19-2:

intermediate D19-1(210mg,0.46mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl and a solid precipitated, filtered and dried in vacuo to give a white solid (146mg, 79%).

3) Synthesis of final product D19:

intermediate D19-2(40mg,0.1mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (26mg,0.2mmol) and HATU (49mg,0.13mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:2) to give a white solid (42mg, 84%). After nmr analysis (see table 1 for data), the solid obtained was compound D19.

Example 20

This example provides a1, 3, 4-thiadiazole heterocyclic compound D20 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D20-1:

intermediate D15-4(150mg,0.41mmol), 2-amino-5-methylpyrazine (89mg,0.82mmol) and acetic acid (25mg,0.41mmol) were added to 1, 2-dichloroethane (15mL) and stirred at 70 ℃ for 4 hours. Add NaBH (AcO) at 0 deg.C₃(261mg,1.23mmol), transferred to ambient temperature and reacted for 16 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (30mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 10:1 to 1:1) to give a yellow oil (120mg, 64%).¹H NMR(400MHz,CDCl₃)δ7.85(s,1H),7.82(s,1H),7.53(s,1H),6.45(s,1H),5.95-5.92(m,1H),4.90(d,J＝6.4Hz,2H),3.90-3.87(m,2H),3.29-3.23(m,2H),2.52-2.46(m,1H),2.34(s,3H),2.04-2.01(m,2H),1.89-1.81(m,2H),1.46(s,9H).

2) Synthesis of intermediate D20-2:

intermediate D20-1(120mg,0.26mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl and a solid precipitated, filtered and dried in vacuo to give a yellow solid (86mg, 81%).

3) Synthesis of final product D20:

intermediate D20-2(40mg,0.1mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (26mg,0.2mmol) and HATU (49mg,0.13mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:2) to give a white solid (34mg, 68%). After nmr analysis (see table 1 for data), the solid obtained was compound D20.

Example 21

This example provides a1, 3, 4-thiadiazole heterocyclic compound D21 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D21-1:

intermediate D15-4(200mg,0.55mmol), 3-amino-6-methylpyridazine (120mg,1.1mmol) and acetic acid (33mg,0.55mmol) were added to toluene (20mL) and stirred at 130 ℃ for 6 h. Dichloromethane (10mL) and NaBH (AcO) were added at 0 deg.C₃(350mg,1.65mmol), brought to ambient temperature and reacted for 16 h. Saturated aqueous sodium bicarbonate (10mL) was added and the mixture was concentrated under reduced pressure to remove toluene. Water (20mL) was added for dilution, followed by extraction with dichloromethane (20mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:2) to give a yellow solid (150mg, 59%).¹H NMR(400MHz,CDCl₃)δ7.54(s,1H),6.97(d,J＝8.8Hz,1H),6.60(s,1H),6.56(d,J＝8.8Hz,1H),5.92(t,J＝6.0Hz,1H),5.03(d,J＝6.8Hz,2H),3.90-3.87(m,2H),3.29-3.23(m,2H),2.56-2.43(m,4H),2.04-2.02(m,2H),1.90-1.81(m,2H),1.46(s,9H).

2) Synthesis of intermediate D20-2:

intermediate D21-1(150mg,0.33mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl, extracted with dichloromethane (20mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to a yellow solid (110mg, 83%).

3) Synthesis of final product D21:

intermediate D21-2(40mg,0.1mmol), 2-methylcyclohexylamine (17mg,0.15mmol), DIPEA (26mg,0.2mmol) and HATU (49mg,0.13mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed (dichloromethane: methanol 50:1 to 25:1) to give a white solid (24mg, 48%). After nmr analysis (see table 1 for data), the solid obtained was compound D21.

Example 22

This example provides a1, 3, 4-thiadiazole heterocyclic compound D23 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D23-1:

intermediate D15-4(300mg, 0.83mmol) and 2, 5-lutidine (267mg, 2.49mmol) were added to acetic anhydride (5mL) and stirred at 140 ℃ for 48 h. The mixture was made basic with saturated aqueous sodium bicarbonate, extracted with ethyl acetate (50mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and chromatographed (petroleum ether: ethyl acetate 4:1) to give a yellow solid (150mg, 40%).¹H NMR(400MHz,CDCl₃)δ8.41-8.35(m,2H),7.62(s,1H),7.56(d,J＝8.0Hz,1H),7.47(d,J＝8.8Hz,1H),7.26–7.24(m,1H),6.73(s,1H),3.95-3.86(m,2H),3.30-3.21(m,2H),2.53-2.45(m,1H),2.34(s,3H),2.08-1.98(m,2H),1.91-1.80(m,2H),1.46(s,9H).

2) Synthesis of intermediate D23-2:

intermediate D23-1(150mg,0.33mmol) was dissolved in dichloromethane (10mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl and a solid precipitated, filtered and dried in vacuo to give a yellow solid (110mg, 83%).

3) Synthesis of final product D23:

intermediate D23-2(105mg,0.26mmol), 2-methylcyclohexylamine (45mg,0.4mmol), DIPEA (67mg,0.52mmol) and HATU (128mg,0.34mmol) were added to DMF (4mL) and stirred at ambient temperature for 12 hours. The reaction was diluted with ethyl acetate (100mL) and washed with saturated aqueous sodium chloride (40mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (ethyl acetate) to give a yellow solid (120mg, 92%). After nmr analysis (see table 1 for data), the solid obtained was compound D23.

Example 23

This example provides a1, 3, 4-thiadiazole heterocyclic compound D24 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

compound D23(25mg, 0.05mmol) and 10% Pd/C (25mg) were added to methanol (10mL), stirred under hydrogen for 24 hours, filtered, the filtrate concentrated, and column chromatographed (ethyl acetate) to give a white solid (18mg, 71%). After nmr analysis (see table 1 for data), the solid obtained was compound D24.

Example 24

This example provides a1, 3, 4-thiadiazole heterocyclic compound D25 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D25-1:

intermediate D15-4(480mg,1.3mmol), 2-amino-5-methylpyridine (286mg,2.6mmol) and acetic acid (79mg,1.3mmol) were added to dichloromethane (25mL) and stirred at room temperature for 1 hour. Add NaBH (AcO) at 0 deg.C₃(840mg,3.9mmol), brought to ambient temperature and reacted for 48 hours. Dichloromethane (50mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (40mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 10:1 to 5:1) to give a yellow oil (400mg, 66%).¹H NMR(400MHz,CDCl₃)δ7.86(s,1H),7.53(s,1H),7.25(d,J＝8.4Hz,1H),6.47(s,1H),6.40(d,J＝8.4Hz,1H),6.31(br s,1H),4.92(s,2H),3.95-3.82(m,2H),3.33-3.17(m,2H),2.53-2.41(m,1H),2.04-2.00(m,2H),1.90-1.80(m,2H),1.46(s,9H).

2) Synthesis of intermediate D25-2:

intermediate D25-1(400mg,0.88mmol) was dissolved in dichloromethane (20mL), and trifluoroacetic acid (5mL) was added thereto, stirred at room temperature for 12 hours, concentrated under reduced pressure, adjusted to pH 8-9 with saturated aqueous sodium bicarbonate solution, and washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 5-6 with 1N HCl and a solid precipitated, filtered and dried in vacuo to give a white solid (190mg, 54%).¹H NMR(400MHz,DMSO-d6)δ7.77(s,1H),7.66(s,1H),7.25(d,J＝7.2Hz,1H),6.79(s,1H),6.53(d,J＝8.4Hz,1H),6.33(s,1H),4.82(d,J＝6.0Hz,2H),3.88-3.74(m,2H),3.26-3.17(m,2H),2.61-2.54(m,1H),2.08(s,3H),2.00-1.87(m,2H),1.72-1.55(m,2H).

3) Synthesis of final product D25:

intermediate D25-2(50mg,0.12mmol), (1S,2R) -2-methylcyclohexylamine hydrochloride (23mg,0.15mmol), DIPEA (48mg,0.37mmol) and HATU (71mg,0.19mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:2) to give a white solid (40mg, 64%). After nmr analysis (see table 1 for data), the solid obtained was compound D25.

Example 25

This example provides a1, 3, 4-thiadiazole heterocyclic compound D26 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D26:

intermediate D25-2(50mg,0.12mmol), (1R,2S) -2-methylcyclohexylamine hydrochloride (23mg,0.15mmol), DIPEA (48mg,0.37mmol) and HATU (71mg,0.19mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate: 2:1 to 1:2) to give a white solid (43mg, 69%). After nmr analysis (see table 1 for data), the solid obtained was compound D26.

Example 26

This example provides a1, 3, 4-thiadiazole heterocyclic compound D27 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D27:

intermediate D25-2(40mg,0.1mmol), 2-methylaniline (13mg,0.12mmol), DIPEA (26mg,0.2mmol) and HATU (57mg,0.15mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:2) to give a white solid (34mg, 69%). After nmr analysis (see table 1 for data), the solid obtained was compound D27.

Example 27

This example provides a1, 3, 4-thiadiazole heterocyclic compound D28 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D28:

intermediate D25-2(40mg,0.1mmol), 3-amino-2-methylpyridine (13mg,0.12mmol), DIPEA (26mg,0.2mmol) and HATU (46mg,0.12mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 hours. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:3) to give a white solid (15mg, 30%). After nmr analysis (see table 1 for data), the solid obtained was compound D28.

Example 28

This example provides a1, 3, 4-thiadiazole heterocyclic compound D29 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D29:

intermediate D25-2(40mg,0.1mmol), 4-amino-3-methylpyridine (13mg,0.12mmol), DIPEA (26mg,0.2mmol) and HATU (46mg,0.12mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:3) to give a white solid (30mg, 61%). After nmr analysis (see table 1 for data), the solid obtained was compound D29.

Example 29

This example provides a1, 3, 4-thiadiazole heterocyclic compound D30 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D30:

intermediate D25-2(40mg,0.1mmol), 3-amino-4-methylpyridine (13mg,0.12mmol), DIPEA (26mg,0.2mmol) and HATU (46mg,0.12mmol) were added to DMF (1mL) and stirred at ambient temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:3) to give a white solid (10mg, 20%). After nmr analysis (see table 1 for data), the solid obtained was compound D30.

Example 30

This example provides a1, 3, 4-thiadiazole heterocyclic compound D31 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

synthesis of final product D31:

intermediate D25-2(40mg,0.1mmol), 2-amino-3-methylpyridine (13mg,0.12mmol), DIPEA (26mg,0.2mmol) and HATU (46mg,0.12mmol) were added to DMF (1mL) and stirred at room temperature for 12 h. The reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:3) to give a white solid (11mg, 22%). After nmr analysis (see table 1 for data), the solid obtained was compound D31.

Example 31

This example provides a1, 3, 4-thiadiazole heterocyclic compound D32 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D32-1:

intermediate D13-1(320mg, 1mmol), 2-formylphenylboronic acid (180mg, 1.2mmol), tetrakis (triphenylphosphine) palladium (92mg, 0.08mmol) and cesium fluoride (304mg, 2mmol) were added to a mixed solvent of ethanol (20mL) and water (4mL) and reacted at 90 ℃ for 4 hours under nitrogen. Concentrating under reduced pressure, adding saturated saline (30mL), extracting with ethyl acetate (30mL × 2), mixing organic phases, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography (petroleum ether: ethyl acetate 4:1) to obtain a crude productYellow solid (240mg, 69%).¹H NMR(400MHz,CDCl₃)δ10.53(s,1H),8.04(d,J＝7.6Hz,1H),7.65-7.52(m,3H),4.17(q,J＝7.1Hz,2H),4.02-3.99(m,2H),3.36-3.29(m,2H),2.64-2.55(m,1H),2.09-2.04(m,2H),1.96-1.86(m,2H),1.28(t,J＝7.2Hz,3H).

2) Synthesis of intermediate D32-2:

intermediate D32-1(150mg,0.43mmol), 2-amino-5-methylpyridine (93mg,0.86mmol) and acetic acid (26mg,0.43mmol) were added to 1, 2-dichloroethane (15mL) and stirred at 70 ℃ for 4 hours. Add NaBH (AcO) at 0 deg.C₃(275mg,1.29mmol), brought to ambient temperature and reacted for 16 h. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (30mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate ═ 2:1) to give a yellow oil (75mg, 39%). LC-MS (M/z) 438.2[ M + H⁺].

3) Synthesis of intermediate D32-3:

intermediate D32-2(75mg,0.17mmol) was added to a mixed solvent of tetrahydrofuran (10mL) and methanol (1mL), followed by dropwise addition of 1N LiOH (1mL,1.02 mmol). The reaction was carried out at room temperature for 12 hours, concentrated under reduced pressure to remove most of the tetrahydrofuran, diluted with water (10mL), washed with ethyl acetate (20mL), and the aqueous phase was adjusted to pH 5-6 with 1N hydrochloric acid and finally extracted with dichloromethane (20 mL. times.4). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid (30mg, 42%).

4) Synthesis of final product D32:

intermediate D32-3(30mg,0.07mmol), 2-methylcyclohexylamine (12mg,0.1mmol), DIPEA (18mg,0.14mmol) and HATU (35mg,0.09mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (40mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (ethyl acetate) to give a white solid (8mg, 21%). After nmr analysis (see table 1 for data), the solid obtained was compound D32.

Example 32

This example provides a1, 3, 4-thiadiazole heterocyclic compound D33 having hedgehog signaling pathway antagonistic activity, which is synthesized by the following method:

1) synthesis of intermediate D33-1:

1-methylpyrazole (2g,24mmol) was added to anhydrous tetrahydrofuran (30mL) and n-butyllithium (2.5M, 11mL,29mmol) was slowly added dropwise at-70 ℃ under nitrogen, and stirred at this temperature for 1 hour. Then a solution of DMF (2.1g, 29mmol) in tetrahydrofuran (5mL) was added slowly dropwise. After the completion of the dropwise addition, the temperature was slowly raised to room temperature, and the reaction was carried out at room temperature for 12 hours. To the reaction solution was added saturated aqueous sodium chloride (30mL), followed by extraction with dichloromethane (100mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (dichloromethane) to give a yellow oil (2.1g, 78%).

2) Synthesis of intermediate D33-2:

intermediate D33-1(2g, 18mmol) was added to DMF (14mL), and a solution of NBS (3.6g, 20mmol) in DMF (12mL) was slowly added dropwise at 0 deg.C, followed by stirring at room temperature for 12 hours. To the reaction solution was added 2M NaOH (20mL) and stirred for 10 minutes, then saturated brine (100mL) was added, extraction was performed with ethyl acetate (200mL), and the organic phase was finally washed with saturated brine (100mL × 4). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate: 10:1) to give a white solid (1.9g, 55%).¹H NMR(400MHz,CDCl₃)δ9.89(s,1H),7.51(s,1H),4.16(s,3H).

3) Synthesis of intermediate D33-3:

intermediate D33-2(700mg, 3.7mmol), pinacol diboron (1.1g, 4.4mmol), potassium acetate (1.1g,11.1mmol) and Pd (dppf) Cl₂(190mg,0.26mmol) was added to 1, 4-dioxane (25mL) and reacted at 105 ℃ for 12 hours under nitrogen. Concentrating under reduced pressure, adding saturated saline (30mL), extracting with ethyl acetate (40 mL. times.2), mixing organic phases, drying over anhydrous sodium sulfate, filtering, and concentrating to obtain crude product (2) as black oil.9g)。

4) Synthesis of intermediate D33-4:

intermediate D13-1(320mg, 1mmol), intermediate D33-3 (30%, 1.18g, 1.5mmol), tetrakis (triphenylphosphine) palladium (81mg, 0.07mmol) and cesium fluoride (304mg, 2mmol) were added to a mixed solvent of ethanol (20mL) and water (4mL) and reacted at 90 ℃ for 12 hours under nitrogen. Concentrated under reduced pressure, added with saturated brine (30mL), extracted with ethyl acetate (30mL × 2), the organic phases combined, dried over anhydrous sodium sulfate, filtered, concentrated and chromatographed (petroleum ether: ethyl acetate 1:1) to give a yellow solid (257mg, 73%).¹H NMR(400MHz,DMSO-d6)δ10.52(s,1H),7.66(s,1H),4.22(s,3H),4.16(q,J＝7.2Hz,2H),3.98-3.95(m,2H),3.33-3.27(m,2H),2.64-2.52(m,1H),2.07-2.04(m,2H),1.94-1.85(m,2H),1.27(t,J＝6.8Hz,3H).

5) Synthesis of intermediate D33-5:

intermediate D33-4(224mg,0.64mmol), 2-amino-5-methylpyridine (138mg,1.28mmol) and acetic acid (38mg,0.64mmol) were added to 1, 2-dichloroethane (20mL) and stirred at 70 ℃ for 4 hours. Add NaBH (AcO) at 0 deg.C₃(407mg,1.92mmol), warmed to room temperature and reacted for 16 hours. Dichloromethane (30mL) was added for dilution, followed by washing with saturated aqueous sodium bicarbonate (40mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (petroleum ether: ethyl acetate: 1 to 1:4) to give a yellow oil (187mg, 66%).¹H NMR(400MHz,CDCl₃)δ7.87(s,1H),7.52(s,1H),7.15(d,J＝7.6Hz,1H),6.38(s,1H),6.04(s,1H),4.78(d,J＝6.4Hz,2H),4.20-4.14(m,5H),3.96-3.92(m,2H),3.29-3.24(m,2H),2.60-2.54(m,1H),2.13(s,3H),2.07-2.04(m,2H),1.93-1.84(m,2H),1.27(d,J＝7.0Hz,3H).

6) Synthesis of intermediate D33-6:

intermediate D33-5(187mg,0.42mmol) was added to a mixed solvent of tetrahydrofuran (10mL) and methanol (1mL), followed by dropwise addition of 1N LiOH (2.5mL,2.5 mmol). The reaction was carried out at room temperature for 12 hours, concentrated under reduced pressure to remove most of the tetrahydrofuran, diluted with water (10mL), washed with ethyl acetate (20mL), and the aqueous phase was adjusted to pH 5-6 with 1N hydrochloric acid and finally extracted with dichloromethane (20 mL. times.4). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid (150mg, 86%).

4) Synthesis of final product D33:

intermediate D33-6(50mg,0.12mmol), 2-methylcyclohexylamine (20mg,0.18mmol), DIPEA (31mg,0.24mmol) and HATU (60mg,0.16mmol) were added to DMF (1mL) and stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate (40mL) and washed with saturated aqueous sodium chloride (30mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (ethyl acetate) to give a white solid (23mg, 37%). After nmr analysis (see table 1 for data), the solid obtained was compound D33.

Table 1 examples 1-33 analytical structure and spectral data were obtained for the compounds.

Example 33

This example biologically tests the compounds obtained in examples 1 to 32, with the vismodegib successfully marketed in 2012 as a standard control:

NIH3T3-GRE-Luc luciferase reporter gene detection experiment

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 selected clones were used to detect hedgehog pathway signaling.

NIH3T3 cells expressing GRE-firefly hormone were maintained in intact culture. When analytical testing 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. 10 nMSG 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. 96-well plates were shaken gently for 5 minutes at room temperature. The light signal is recorded by a plate reader. The activity of the compounds was calculated from their blocking of the luminescence signal, and the synthesized compounds were tested and the results are given in the following table:

table 2 results of the assay for the inhibitory ability of heterocyclic compounds D1-D33 on the Hedgehog signaling pathway:

the results show that 33 compounds of Hedgehog signaling pathway all have inhibitory activity, wherein the compounds of D13, D14, D15, D16, D18, D22, D24, D25 and D26 are superior to or equal to the inhibitory activity of vismodegib, and have good Hedgehog pathway antagonism effect.

Therefore, the novel 1,3, 4-thiadiazole heterocyclic compound with hedgehog pathway antagonist activity can block metastasis and regeneration of tumor cells through hedgehog pathway antagonism, thereby treating tumor diseases.

The invention has various embodiments, and all technical solutions formed by adopting equivalent transformation or equivalent transformation are within the protection scope of the invention.

Claims (6)

1. A1, 3, 4-thiadiazole heterocyclic compound having hedgehog pathway antagonist activity, having a structure represented by general formula I:

wherein the content of the first and second substances,

m is 1,2,3 or 4;

n is 0 or 1;

R₁selected from hydrogen, halogen, methyl, trifluoromethyl, hydroxyl, mercapto, ethyl, methoxy and cyclopropyl;

R₂is one of the following groups:

a1 is selected from unsubstituted or substituted by 1-4R₁₀Substituted five-and six-membered heteroaromatic rings containing 1 to 3 heteroatoms selected from O, N and S, or A1 is unsubstituted or substituted by 1 to 4R₁₀Substituted by

L is-CH₂O-、-CH(CH₃)O-、-CH₂S-、-CH₂NH-、-(CH₂)₂-and-HC ═ CH-groups;

u is selected from unsubstituted or substituted1-2R₁₁Substituted five-or six-membered heteroaromatic and six-membered aromatic rings containing 1-3 heteroatoms selected from O, N and S;

R₁₀、R₁₁are respectively selected from hydrogen atom, deuterium atom, halogen, cyano, hydroxyl, amino, sulfydryl and C_1-6Alkoxy radical, C_1-6Alkyl and C_3-6A cycloalkyl group;

l and 1,3, 4-thiadiazole are in an ortho-substitution relationship on U.

2. The 1,3, 4-thiadiazole heterocyclic compound according to claim 1, wherein: a1 is unsubstituted or substituted by 1 to 4R₁₀One of the following groups substituted with a group:

3. the 1,3, 4-thiadiazole heterocyclic compound according to claim 1, wherein: u is unsubstituted or substituted by 1-2R₁₁One of the following groups substituted with a group:

4. the following 1,3, 4-thiadiazole heterocyclic compounds:

5. a pharmaceutical composition comprising as active ingredient a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or diluent.

6. Use of a compound according to any one of claims 1-4 or a composition according to claim 5 for the manufacture of a medicament for the treatment of a Hedgehog signaling pathway mediated disease, wherein: the disease includes nasopharyngeal carcinoma, breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous carcinoma, thyroid cancer, sarcoma, osteosarcoma, desmoid tumor, melanoma, basal cell carcinoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric 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, myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumors, astrocytomas, medulloblastoma, schwannoma, primary neuroectodermal tumors, leukemia, melanoma, and leukemia, One or more of pituitary tumors.

Images