Patent Application
20240374738
This application claims priority to Chinese Patent Application No. 2023102574885, filed Mar. 16, 2023, which is hereby incorporated by reference in its entirety.
The present disclosure belongs to the field of biomedicine, in particular to a bifunctional compound capable of degrading receptor tyrosine kinase or pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. This type of compound regulates downstream key proteins by degrading the receptor tyrosine kinase, especially discoidin domain receptors (DDRs), further plays a role in treating related diseases, and shows a prominent capability of inhibiting proliferation of tumor cells.
Receptor tyrosine kinases (RTKs) are located on cell surfaces and are receptors having highly affinity for various signaling molecules such as growth factors, cytokines, and hormones. As an important component of a tyrosine kinase family, the receptor tyrosine kinases participate in cellular signal transduction by catalyzing downstream protein tyrosine phosphorylation. There are currently over fifty known RTKs, which may be divided into twenty different subfamilies, including an epidermal growth factor receptor subfamily, a vascular endothelial growth factor receptor subfamily, a hepatocyte growth factor receptor subfamily and the like. They play a very important role in a process of controlling cell proliferation, migration, differentiation and metabolism, respectively. In terms of a protein structure, the RTKs are composed of three components, including an extracellular domain (ECD) of ligand binding sites, a hydrophobic alpha helix transmembrane domain, and an intracellular domain containing tyrosine protein kinase activity. The intracellular domain may be further divided into a near membrane domain, a tyrosine kinase domain, and a carboxyl terminal. (Trenker and Jura, Curr Opin Cell Biol. 2020, 63: 174-185.) Under a normal physiological state, inactivated receptor tyrosine kinases exist in a monomer form. When its extracellular domain binds to signaling molecules, a receptor molecule monomer dimerizes or oligomers on a cell membrane, tyrosine residues in the intracellular domain are phosphorylated, functions of the kinases are activated, and a signaling complex is formed at ends of the receptor tyrosine kinases. The phosphorylated tyrosine site of the intracellular domain becomes a binding site for downstream signal proteins in the cells. The bound downstream signal protein is activated, expands information by using different signal transduction pathways, and causes a series of biochemical reactions in the cells, or causes a comprehensive response of the cells by using multiple pathways of an information matrix, and then controls processes such as cell proliferation, migration, differentiation and apoptosis (Lemmon M A, Schlessinger J. Cell. 2010; 141:1117-34).
Dysregulation of RTK signals may lead to various abnormal states in the cells, and RTK dysregulation plays an important role in development and regulation of cancer-like cells. RTK mutations may activate a series of subordinate associated reactions, disrupt balance between cell growth/proliferation and cell death, and trigger tumor development driven by dysregulation of RTKs. In human cancer, main mechanisms leading to dysregulation of constitutive RTK activation include functional gain mutations, genome amplification, chromosome rearrangement, autocrine activation, and kinase domain duplication. Many experiments prove that the tyrosine kinase family is closely related to pathogenesis of tumors, and inhibiting the receptor tyrosine kinases is an effective measure for treating cancer. Therefore, the RTKs have become an important target for developing therapeutic means for the tumors. A wide variety of RTK inhibitors based on a small molecule compound or a monoclonal antibody have been developed as drugs for treating various tumors (Punit S. Oncogene 2021, 40(24):4079-4093). Compared with traditional chemotherapeutics, the RTK inhibitor (TKI) has the advantages of high selectivity, few side effects and the like, but such a signal transduction inhibitor can only block a part of signal pathways of tumor cells, and compensation mechanisms of other signal pathways often weaken the therapeutic effect. In addition, the application of a targeted drug TKI may cause the tumors to have drug resistance, greatly reducing the therapeutic effect. Molecular mechanisms for resistance to the tyrosine kinase inhibitors include kinase overexpression and mutation, drug uptake, drug binding, drug efflux mediated by adenosine triphosphate binding transporter, deficiency of DNA repair mechanisms, activation of aberrant signaling pathways, epigenetic modifications, and tumor microenvironment, and the like. Therefore, it is urgent to develop new methods and technological means for various diseases caused by dysregulation of the RTKs.
Discoidin domain receptors (DDRs), as a member of the receptor tyrosine kinases (RTKs), play an important role in controlling signal transduction pathways for cell proliferation and differentiation. The dysregulation of DDRs is closely related to various diseases, including cancer, neurological degeneration, chronic inflammation, and fibrosis. The DDRs can promote malignant proliferation of tumor cells and are associated with invasion and metastasis of the tumor cells. An in-depth study of DDR regulation means can open up a new pathway for prevention and treatment of diseases such as the tumors in clinical practice. According to research, members of the DDR family, including a DDR1 and a DDR2, are widely expressed in various tissues. The DDR1 is mainly expressed in epithelial cells, smooth muscle cells, fibroblasts, oligodendrocytes, and macrophages in lungs, kidneys, colon, and brain, while the DDR2 is mainly expressed in fibroblasts, myofibroblasts, smooth muscle cells, and chondrocytes in kidneys, skin, lungs, heart, and connective tissue.
The DDRs are composed of three main structural domains, namely an extracellular ligand binding region, a transmembrane region, and an intracellular tyrosine kinase-containing region. The extracellular domain is composed of a discoidin domain and a discoidin-like domain capable of binding to ligand collagen; the transmembrane domain includes an extracellular juxtamembrane domain and a transmembrane helical region, and the region may mediate collagen-independent receptor dimerization (Yeung D A, J mol Biol. 2019; 431:368-390); and the intracellular domain includes an intracellular juxtamembrane domain and a catalytic tyrosine kinase domain. In addition, the DDRs have activation mechanisms different from other RTKs, which require collagen binding stimulation to initiate downstream pathways, resulting in autophosphorylation. A process of activating the DDRs has collagen specificity, that is, the DDR1 and the DDR2 require different types of collagen activation. The DDR1 preferentially binds to collagen I-V and VIII collagen, but has a lower affinity for X-type collagen. Research has shown that the function of DDR1 is mainly achieved through three pathways, namely, a classic collagen DDR1 signaling pathway, which relies on DDR1 kinase activity and directly affects downstream molecules such as Shc, Nck2, and Shp-2; or, a non-classical DDR1 signaling pathway that rely on collagen binding but do not rely on DDR1 kinase activity; or, it can function without relying on collagen stimulation or DDR1 kinase activity
Studies found that DDR1 is expressed in lung cancer, breast cancer, brain tumor, ovarian cancer, esophageal cancer, head and neck tumor, liver cancer, testicular cancer and other tumors; and the high expression is closely related to poor tumor prognosis (Rammal H, Front Pharmacol. 2016; 7:55). For example, the expression of DDR1 in cancer cell tissues of non-small cell lung cancer patients is positively correlated with mortality rate. The expression and phosphorylation levels of DDR1 are significantly increased in lung cancer tissues. Immunohistochemical analysis of 171 cases of non-small cell lung cancer showed that the positivity rate of DDR1 in aggressive non-small cell lung cancer is as high as 61% (Yang S H. Oncol Rep. 2010; 24:311-319). Meanwhile, DDR1 promote the proliferation and growth of various malignant tumor cells. For example, in human colon cancer and breast cancer, DDR1 can upregulate the anti apoptotic protein Bcl xL by activating downstream signaling proteins Ras/Raf/ERK and PI3K/Akt pathways, thereby enabling cancer cells to survive under toxic stress (Matadag S P. Medical Chemistry Research, 2021, 30 (3): 535-551).
Activated by natural type IV collagen, DDR1 can increase the expression of CD9 in breast cancer MDA-MB-231 cell line, leading to the migration of the cancer cells. DDR1 also induces the secretion and invasion of matrix metalloproteinase-2 and metalloproteinase-9 through DDR1 and Src dependent pathways, increasing their cellular expression levels. DDR1 activation plays an important role in the invasive ability and metabolic activity of breast cancer cells. Similarly, the upregulation of these two metalloproteinases caused by DDR1 is also a necessary condition for the metastasis and invasion of liver cancer cells (Lee, JH. Sci. Rep. 2018, 8, 1). In addition, in vitro and in vivo experiments have shown that DDRs are involved in the differentiation of tumor cells. Furthermore, they promote malignant cell transformation and tumor invasion and metastasis by disrupting normal signal transduction in the extracellular matrix, which prevent T cell infiltration by helping tumor cells establish a barrier around them. It has been shown that DDR1 can increase the invasion and migration ability of non-small cell lung cancer cells by promoting epithelial mesenchymal cell transformation. DDR1 can also regulate T cells CD4+ and CD8+inhibit the anti-tumor immune response, thus promoting the growth of breast cancer (Zhong, X. Oncol Rep. 2019; 42 (6): 2844-2854); DDR1 can weaken the immune system's clearance of cancer cells by affecting the tumor microenvironment. DDR1 can present a highly ordered extracellular matrix (ECM) during tumor development, affecting the infiltration of immune cells and weakening their ability to kill tumor cells. Other studies have shown that in the knockout of DDR1 mouse model, the infiltration of T cells within the tumor is improved and tumor growth is inhibited. It can be inferred that, Knockout or inhibition of DDR1 protein by DDR1 gene is a potential method to block the anti immune monitoring ability of tumors. DDR1 may become a new target for tumor immunotherapy (Sun X, Nature, 2021599 (7886): 673-678). Preclinical studies suggest that DDR1 inhibitors possess a wide range of anti-tumor activities in tumor transplantation mouse models derived from patients with non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, breast cancer and colorectal cancer.
In addition to its role in the pathogenesis of tumors, DDRs are also key mediators for the secretion of a variety of inflammatory cytokines. They are dysfunctional in a variety of inflammatory diseases, including osteoarthritis, organ fibrosis and atherosclerosis (Leitinger, B. Int. Rev. Cellmol. Biol. 2014310, 39-87). DDR1 is also associated with fibrosis of liver, kidney, lung tissue, and blood vessels. Study has confirmed that DDR1 not only directly stimulates the secretion of inflammatory factors, but also enhances the effects of other pro-inflammatory cytokines and bacterial products (Matsuyama, W. J. Immunol. 2004, 172, 2332-2340). In various kidney disease models, inhibition of DDR expression can prevent inflammatory changes and fibrosis in the kidneys. Experimental results have shown that DDR1 deficiency can effectively reduce bleomycin induced pulmonary inflammation and fibrosis (Avivi-Green, C. Am. J. Respir. Crit. Care Med. 2006, 174, 420-427). In a model of anti glomerular basement membrane glomerulonephritis with prominent inflammation and unilateral ureteral obstruction, it was found that inhibiting DDR1 expression can reduce the migration of inflammatory cells and hinder fibrosis formation (Guerrot, D Am. J. Pathol. 2011, 179, 83).
As a unique member of the RTKs family, DDRs, especially DDR1, are closely related to the occurrence and development of cancer, inflammation, fibrosis, and neurodegenerative diseases, and are considered potential important targets for intervention and treatment of these diseases. The regulation and inhibition of RTKs are usually achieved through small molecule inhibitors that antagonize intracellular kinase activity or antibody drugs that interfere with extracellular domain function. Similarly, DDR1 inhibitors can also be divided into monoclonal antibodies that act on the extracellular binding region and small molecule inhibitors that act on the intracellular kinase domain and affect downstream signaling pathways. The latter can be further divided into multi-target RTK inhibitors and selective DDR1 inhibitors. Because the structure of the kinase ATP binding region of some members of the RTKs family has high homology, especially DDRs and c-Kit, many RTK multi target kinase inhibitors exhibit certain DDR1 inhibitory activity between Bcr-Abl kinases. For example, multi-target inhibitors Imatinib, Dasatinib, and Nilotinib can inhibit collagen induced self phosphorylation of DDR1. At present, various marketed Bcr Abl inhibitors, such as Ponatinib, Bosutinib, and Bafitinib, exhibit strong DDR1 inhibitory activity. In addition, P38 MAPK inhibitor Doramapimod, the B-Raf/VGFR dual target inhibitor Sorafenib and the c-Met/VEGFR-2 dual target inhibitor Foretinib kinase inhibitor all have strong binding affinity to the DDR1 kinase domain. In recent years, a considerable number of highly selective DDR1 inhibitors based on the rational design of DDR and small molecule three-dimensional molecular structures have been discovered and developed, many of which have potential applications in tumors, neurodegenerative diseases, Alzheimer's disease, dry eye syndrome, inflammatory and fibrotic diseases (William A D Biomolecules. 2021, 11 (11): 1671). DDR1 small molecule inhibitors include different types of core structures such as urea, quinoline, isoquinoline, tetrahydroisoquinoline, 1H-indole, spirocycloindoline, and triheterospirocyclodecanone (WO2017/038870, WO2016064970A1, WO2015060373A1, CN108276388A, WO2017137334A1, WO2017/038871).
Although the biological activity of these DDR inhibitors has been confirmed by many experiments, like most other RTK inhibitors, these small molecule drugs rely on site occupying competition for ATP binding domains. Long-term use of them can cause cancer cell resistance. Meanwhile, RTK has both kinase catalytic function and non catalytic function in other domains. Relying solely on small molecule inhibitors for site competition cannot offset the full function of RTK. Therefore, it is urgent to find more effective means to regulate DDR1 and other RTK functions.
The use of the ubiquitin protease system (UPS) in eukaryotic cells to regulate intracellular protein levels has received great attention in the past decade. UPS can efficiently and specifically induce protein degradation for negative signal function regulation, and can degrade 80%-90% of ubiquitinated proteins in cells. It plays an extremely important role in maintaining the levels of various proteins in cells, involving almost all life activities such as regulating cell cycle, proliferation, apoptosis, metastasis, gene expression, signal transduction, etc. The ubiquitin protein, composed of 76 amino acids, is a highly conserved protein commonly found in eukaryotic cells. The ubiquitination labeling of proteins initiates the degradation of substrate proteins, which is carried out through the synergistic action of Ubiquitin activating enzyme E1, Ubiquitin binding enzyme E2, and E3 ubiquitin ligase. Firstly, ubiquitin forms a high-energy thioester bond with the carboxyl group on its C-terminal glycine and the essential cysteine thiol group on the ubiquitin activating enzyme E1, connecting to E1 and becoming an activated state of ubiquitin; Secondly, activated ubiquitin is transferred from ubiquitin activating enzyme E1 to ubiquitin binding enzyme E2; Finally, under the action of E3 ubiquitin ligase, the ubiquitin molecules connected to ubiquitin binding enzyme E2 are covalently linked to the substrate protein through isomeric peptide bonds. After ubiquitination, the substrate protein can be transported to the 26S proteasome or enter lysosomes for digestion and degradation. The specific recognition ability of E3 ubiquitin ligase towards substrate proteins determines the specificity of ubiquitin mediated protein degradation.
The Proteolytic Targeting Chimera (PROTAC) technology utilizes the intracellular ubiquitin proteasome system to degrade specific proteins. This technology will be able to combine small molecule ligands targeting specific proteins to form a bifunctional compound by connecting them with ligands of E3 ubiquitin ligase through bridge chain fragments. By optimizing the link position and bridge chain length, the ligands at both ends of the PROTAC molecule are simultaneously bound to the target protein and E3 ubiquitin ligase, forming a target protein PROTACs E3 ligase ternary complex, which promotes ubiquitination labeling of the target protein and is subsequently degraded by the protease system. Due to the characteristics of the formation of ternary complexes, the target protein ligands used in PROTAC technology do not need to have strong target binding activity, so they can target proteins that traditional inhibitors cannot be used as drugs, such as skeleton proteins and transcription factors. In addition, the overall degradation of target proteins helps to overcome the problem of resistance to small molecule inhibitors. PROTAC molecules undergo repeated cyclic reactions through a catalytic mechanism. The degradation induced by PROTAC is event driven, not site occupying driven. After forming a complex and completing ubiquitin transfer, the drug dissociates and transfers to the next target in an enzymatic manner. To a certain extent, it is possible to maintain effective drug activity at low doses. The technical difficulties of PROTAC lie in the relationship between target protein degradation and generation rate, the ability of target protein to bind ubiquitin, the conformations and sites of target protein ligands and E3 ubiquitin ligase ligands, the length and composition of bridge chains, and the concentration that affect the formation and stability of ternary complexes. Therefore, regulation is more challenging. Among the over 600 known E3 ubiquitin ligases, only a limited number are applied to the design of PROTAC compounds, including CRBN, VHL, MDM2 and CIAP1 classes. These E3 ubiquitin ligases endow substrates with specificity to achieve target protein ubiquitination. The Von Hippel Lindau (VHL) tumor suppressor factor in common E3 ubiquitin ligases is composed of extension B and C Cul2 and Rbx1 are composed, and their main substrate is hypoxia inducible factor 1 (HIF-1). The ligand study of E3 ubiquitin ligase VHL revealed the crystal structure of its complex, confirming that small molecule compounds can mimic the main substrate of transcription factor HIF-1 (CN108601764A). During the application process, The binding affinity between VHL ligands and E3 ubiquitin ligase is relatively weak, which can easily lead to incomplete degradation of target proteins and cause off target effects. Another important E3 ligase, Cereblon (CRBN), is a protein encoded by the human CRBN gene. The CRBN homologous genes are highly conserved, demonstrating their importance in physiology. Cereblon and damaged DNA binding protein 1 (DDB1) Cullin-4A (CUL4A) and Cullin-1 regulator (ROC1) form the E3 ubiquitin ligase complex. This complex can ubiquitinate a series of proteins. Considering the binding ability of the phthalimide structural unit to CRBN, this structural unit is often used as an E3 ligase recruiting ligand to hijack CRBN for the degradation of target proteins. So far, there are many experimental drugs developed using PROTAC technology, including nuclear protein receptor degrading agents, kinase degrading agents, transcription factor degrading agents, etc. PROTACs targeting RTK can block kinase matrix signaling, preventing inactivated kinases from transmitting oncogenic gene signals through their skeletal functions, leading to sustained loss of RTK function. The existing RTK degradation agents can degrade kinases including wild-type or mutant EGFR HER2, c-MET, etc. The present invention designs and develops degradation agents targeting tyrosine kinase receptors with unique structures, particularly PROTAC degradation agents targeting DDR1. These compounds can treat related diseases by degrading abnormal tyrosine kinase receptors, thus having significant clinical significance.
The present invention is firstly to solve the technical problems of providing a bifunctional compound for degrading receptor tyrosine kinases or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorphic form thereof, wherein such compounds degrade specific receptor tyrosine kinases, particularly discoidin domain receptors (DDRs), regulating downstream key proteins and further playing a therapeutic role in related diseases, and demonstrating outstanding ability to inhibit tumor cell proliferation;
The present invention is secondly to solve the technical problems of providing a preparation method and application of a bifunctional compound for degrading receptor tyrosine kinase.
Specifically, the bifunctional compound of claim 1, wherein the X part is selected from following structures as shown in X1 to X6, wherein a zigzag line represents a position where the X part is connected with the L part through covalent bonds;
Specifically, the bifunctional compound of claim 1 or 2, wherein the Y part is a VHL ligand, and the Y part is selected from following structures as shown in Y1 to Y3, wherein an asterisk (x) position represents a position where the Y part is connected with the L part through covalent bonds;
Specifically, the bifunctional compound of claim 1 or 2, wherein the Y part is a ligand of E3 ligase Cereblon, and the Y part is selected from a piperidine-2,6-diketone compound, thalidomide or its derivatives, lenalidomide or its derivatives, or pomalidomide or its derivatives.
Specifically, the bifunctional compound of claim 4, wherein the Y part has following structures as shown in Y4 to Y8, wherein an asterisk (x) position represents a position where the Y part is connected with the L part through covalent bonds;
6. The bifunctional compound of any one of claims 1 to 5, wherein the L part is a covalent bond, and directly connects the X part with the Y part;
The L part is selected from structures as shown below, wherein a zigzag line represents a position where the L part is connected with the X part through covalent bonds, and an asterisk (x) position represents a position where the L part is connected with the Y part through covalent bonds;
Specifically, the bifunctional compound of any one of claims 1 to 6, wherein the compound is selected from any one of the following structures:
In order to facilitate understanding of the content of the present invention, further detailed explanations will be provided below based on specific embodiments of the present invention and in conjunction with the accompanying drawings,
The following further illustrates the present invention in conjunction with embodiments and their accompanying drawings. These embodiments are only for more detailed and specific instructions and should not be understood as limiting the present invention in any form. The present invention can be implemented in various ways limited and covered by the claims.
Although many materials and operating methods used in the present invention are well-known in the art, the present invention is still described in as much detail as possible. In the following text, unless otherwise specified, the materials and operating methods used are well-known in the art. Unless otherwise defined, all terms used in the present invention (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The synthesis of the target compound of the present invention adopts the method shown in the reaction flowchart. The structure and purity of the product were confirmed by nuclear magnetic resonance, mass spectrometry, and liquid chromatography. Unless otherwise specified, the starting materials used in the synthesis method for preparing the compounds of the present invention are commercially available or can be prepared according to known methods in the art or described herein.
General synthesis of compounds: In view of the present disclosure or through the explanatory methods shown in the following general scheme, prepare the disclosed compounds using methods known to those skilled in the art. If necessary, suitable protective groups can be used in the synthesis in either of the general schemes. It should be understood that the described implementation plans and examples are not intended to limit the scope of this disclosure, and the claims presented herein are intended to cover all implementation plans and examples, whether or not explicitly presented herein.
This embodiment was used to prepare the synthetic compound TPD005305 by the following synthetic route:
Compound 1 (20 g, 139 mol) was added to a 250 mL single-necked vial and triethyl orthoformate (70 mL) was heated to 100° C. and stirred for 2 hours. The reaction solution was reduced to room temperature, filtered by adding methyl tert-butyl ether, the filter cake was drenched with methyl tert-butyl ether and dried to give compound 2 (23 g, yellow solid), calculated yield: 82.7%.
Compound 2 (5 g, 25 mol), methyl 5-amino-2-methoxybenzoate (4.5 g, 25 mmol) and isopropanol (50 mL) were added to a 100 mL single-necked vial and stirred for 3 hours at room temperature. The reaction solution was filtered and the filter cake was eluted with methyl tert-butyl ether and dried to give compound 3 (8 g, yellow solid), calculated yield: 95%.
Diphenyl ether (80 mL) was added to a 100 mL single-necked flask and heated up to 240° C. compound 3 (8 g, 23.9 mmol) was added in one portion and stirred for 30 min, the reaction solution was reduced to room temperature, filtered by addition of Methyl tert-butyl ether, and the filter cake was drenched with Methyl tert-butyl ether and dried to give compound 4 (4.5 g, brown solid) in crude form.
In a 100 mL single-necked flask was added phosphorus oxychloride (50 mL), compound 4 (4.5 g, crude), warmed to 100° C. and stirred for 2 hours, the reaction solution will be to room temperature, concentrated to remove phosphorous trichloride. Add methyl tert-butyl ether filtration, the filter cake was washed with methyl tert-butyl ether, dried to obtain compound 5 (1 g, brown solid), calculated yield: two steps 16.7%.
In a 100 mL single-necked vial, compound 6 (2 g, 6.4 mmol), compound 5 (1.6 g, 6.4 mmol), cesium carbonate (4.2 g, 12.7 mmol), and N,N-dimethylformamide (20 mL) were added and warmed to 100° C. with stirring overnight. The reaction solution was reduced to room temperature, poured into water, extracted by adding ethyl acetate (30 mL×3), and the organic phase was concentrated by washing with water and drying. The crude product was purified by column chromatography to give compound 7 (1 g, yellow solid), calculated yield: 29.8%.
In a 100 mL three-necked flask, lithium aluminum hydride (144 mg, 3.8 mmol), anhydrous tetrahydrofuran (10 mL), and a tetrahydrofuran solution (5 mL) of compound 7 (1 g, 1.9 mmol) was added dropwise at 0° C. under nitrogen protection. After dropwise addition, the reaction solution was stirred at room temperature for 1 h. The reaction solution was quenched with sodium sulfate decahydrate and filtered, the filter cake was washed with dichloromethane:methanol (20:1), and the filtrate was concentrated to obtain compound 8 (500 mg, yellow solid), calculated yield: 52.6%.
To a 50 mL three-necked vial was added compound 8 (500 mg, 1 mmol), N,N-dimethylformamide (10 mL) and sodium hydride (200 mg, 5 mmol) under nitrogen protection. Stirred at room temperature for 30 min and compound 2-(2-bromoethoxy)tetrahydro-2H-pyran (250 mg, 1.2 mmol) was added and stirred overnight at room temperature. The reaction solution was quenched by pouring into ammonium chloride solution, extracted with dichloromethane (20 mL×3) and the organic phase was dried and concentrated to give compound 9 (230 mg, yellow solid), calculated yield: 36.6%.
Compound 9 (1.2 g, 1.9 mmol), methanol (10 mL), 2N hydrochloric acid (10 mL) were added to a 50 mL single-necked vial and stirred at room temperature for 2 hours. The reaction solution was poured into water and sodium carbonate solution was adjusted pH=7-8, the filter cake was dried to give compound 10 (700 mg, yellow solid), calculated yield: 90%.
Compound 10 (400 mg, 0.73 mmol), triethylamine (148 mg, 1.5 mmol), dichloromethane (10 mL) were added to a 100 mL triple-necked vial, methylsulfonyl chloride (126 mg, 1.1 mmol) was added dropwise at 0° C., and stirred for 2 hr at room temperature after addition. The reaction solution was poured into water, extracted with ethyl acetate (10 mL×3), and the organic phase was dried and concentrated to obtain compound core 1 (400 mg, yellow liquid) as crude.
A tetrahydrofuran solution (150 mL) of compound 11 (15 g, 74.6 mmol), ethyl acrylate (8.96 g, 89.6 mol) and potassium tert-butanolate (12.5 g, 112 mmol) was stirred overnight at room temperature, the reaction solution was quenched with 1N HCl (100 mL) and extracted with ethyl acetate (50 mL×2). The combined organic phases were washed with water (50 mL) and dried over a column with anhydrous sodium sulfate to give compound 12 (6.5 g, 30%).
To a methanolic solution of compound 12 (6.5 g, 22.6 mmol) (60 mL) was added sodium hydroxide (1.8 g, 45.3 mmol) and stirred at room temperature overnight. The reaction solution was spun dry, diluted with water, adjusted pH=6 with 1N hydrochloric acid, extracted with ethyl acetate (30 mL×3), and the combined organic phases were dried over anhydrous sodium sulfate and spun dry to give compound 13 (2 g, 32%).
In a 100 mL single-necked vial, compound 13 (150 mg, 0.55 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (257 mg, 0.55 mmol, 1-hydroxybenzotriazole (111.4 mg, 0.82 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (158 mg, 0.82 mmol), N,N-diisopropylethylamine (248 mg, 1.9 mmol), and dichloromethane (5 mL) were stirred overnight at room temperature. The reaction solution was washed and dried and concentrated, and the crude product was purified by preparative plate to give compound 14 (230 mg, yellow solid), calculated yield: 61.2%.
In a 100 mL single-necked vial, compound 14 (230 mg, 0.34 mmol), hydrochloric acid/ethyl acetate (2N, 5 mL), was added and stirred at room temperature for 30 min. The reaction solution was concentrated to give compound 15 (200 mg, yellow solid), calculated yield: 96%.
Compound 15 (200 mg, 0.32 mmol), core 1 (200 mg, 0.32 mmol), sodium iodide (97 mg, 0.64 mmol), potassium carbonate (89 mg, 0.64 mmol), N,N-dimethylformamide (10 mL) were added to a 100 mL single-necked vial and stirred for 3 hours at 80° C. The reaction solution was reduced to room temperature, poured into water and extracted with ethyl acetate (10 ml×3). The organic phase was washed with water and dried and concentrated, and the crude was purified by high pressure preparation to give the compound TPD005305 (50 mg, yellow solid), calculated yield: 14%. LCMS (ESI) m/z calcd. for C60H69FN8O10S [M+H]+ 1112.5; found 1112.7. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005313 by the following synthetic route:
A solution of compound core 1 (200 mg, 0.32 mmol), compound 2 (109.8 mg, 0.48 mmol), sodium iodide (97 mg, 0.64 mmol) and potassium carbonate (88.6 mg, 0.67 mmol), synthesized according to the method steps in Example 1, in DMF (5 ml), was stirred at 100° C. overnight. After completion of the reaction the reaction solution was added to (20 ml) of ice water and extracted with EA (10 ml×2), the organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude compound 3 as a yellow solid (200 mg, 82.6% yield).
Compound 3 (200 mg, 0.26 mmol) was dissolved in DCM (5 ml), TFA (5 ml) was added and the reaction was carried out at room temperature for 2 hours. Spin drying of the reaction solution gave compound 4 as a yellow solid (160 mg, 92.4% yield).
Compound 4 (80 mg, 0.12 mmol) and compound 4-1 (40.4 mg, 0.15 mmol) were dissolved in NMP (5 ml), DIEA (31 mg, 0.24 mmol) was added at room temperature and the reaction solution reacted at 100° C. overnight. The reaction solution was prepared under high pressure to give the compound TPD005313 as a yellow solid (4 mg, 3.6% yield). LCMS (ESI) m/z calcd. for C50H51FN7O9 [M+H]+ 912.4; found 912.5. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005315 by the following synthetic route:
Compound 1 (10 g, 42.2 mmol), benzyl 4-hydroxypiperidine-1-carboxylate (10 g, 42.2 mmol) and potassium carbonate (12.5 g, 90.9 mmol) were dissolved in DMF (100 ml) and stirred at 50° C. overnight. After completion of the reaction the reaction solution was added to (200 ml) ice water and extracted with EA (100 ml×2), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified over a column to give compound 2 as a yellow solid (6 g, 36.3% yield).
Compound 2 (6 g, 16.5 mmol) was dissolved in methanol, palladium carbon (1 g) was added, and 20 Psi hydrogen at room temperature overnight, filtered, and spun-dried to give compound 3 as a yellow solid (2.2 g, 55.7% yield).
Compound Corel (100 mg, 0.16 mmol) and compound 3 (62 mg, 0.24 mmol) were dissolved in DMF (2 ml), NaI (48 mg, 0.32 mmol) and potassium carbonate (44.3 mg, 0.32 mmol) were added, and the reaction was carried out at 100° C. overnight. The reaction was completed by pouring the reaction solution into water, EA (10 ml×2) was extracted, the organic phase was washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and the solvent was spun dry to give the crude compound 4 as a yellow solid (110 mg, 88% yield).
Compound 4 (110 mg, 0.14 mmol) was dissolved in EA (2 ml), 4 N HCl/EA (2 ml) was added and the reaction was carried out at room temperature for 2 hours. The reaction was completed by vacuum spin drying the solvent under high pressure to prepare a yellow solid of compound 5 (10 mg, 10% yield).
Compound 5 (10 mg, 0.015 mmol) and compound 5-1 (5 mg, 0.018 mmol) were dissolved in NMP (2 ml) and DIEA (4 mg, 0.03 mmol) was added at room temperature and the reaction solution reacted overnight at 100. The reaction solution was prepared under high pressure to give the compound TPD005315 as a yellow solid (1 mg, 7.3% yield). LCMS (ESI) m/z calcd. for C51H53FN7O10 [M+H]+ 942.4; found 942.5. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005316 by the following
Compound 1 (10 g, 45.4 mmol), tert-butyl (2-bromoethyl) carbamate (12.16 g, 54.5 mmol) and potassium carbonate (12.5 g, 90.9 mmol) were dissolved in DMF (100 ml) and stirred at 50° C. overnight. After completion of the reaction the reaction solution was added to ice water (200 ml) and extracted with EA (100 ml×2), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified over a column to give compound 2 as a yellow solid (6 g, 36.3% yield).
Compound 2 (6 g, 16.5 mmol) was dissolved in methanol, palladium carbon (1 g) was added, and the mixture was left at room temperature overnight under 20 Psi hydrogen. Filtration and spin drying gave compound 3 as a yellow solid (2.2 g, 59.4% yield).
Compound Corel (100 mg, 0.16 mmol) and compound 3 (62 mg, 0.24 mmol) prepared as in Example 1 were dissolved in DMF (2 ml), NaI (48 mg, 0.32 mmol) and potassium carbonate (44.3 mg, 0.32 mmol) were added, and the reaction was carried out at 100° C. overnight. The reaction was completed the reaction solution was poured into water, EA (10 ml×2) was added to extract, the organic phase was washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and the solvent was spun dry to give the crude compound 4 as a yellow solid (102 mg, 84.2% yield).
Compound 4 (102 mg, 0.13 mmol) was dissolved in EA (2 ml), 4N HCl/EA (2 ml) was added and the reaction was carried out at room temperature for 2 hours. The reaction was completed vacuum spin drying the solvent as a yellow solid of compound 5 (80 mg, 90% yield).
Compound 5 (80 mg, 0.12 mmol) and compound 4-1 (40.4 mg, 0.14 mmol) were dissolved in NMP (2 ml) and DIEA (31 mg, 0.24 mmol) was added at room temperature and the reaction solution reacted overnight at 100. The reaction solution was prepared under high pressure to give the compound TPD005316 as a yellow solid (2 mg, 1.8% yield). LCMS (ESI) m/z calcd. for C49H50FN8O9 [M+H]+ 913.4; found 913.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005317 by the following synthetic route:
Compound 1 (450 mg, 1.63 mmol), tert-butyl glycinate (256.3 mg, 1.96 mmol) and DIEA (420.6 mg, 3.26 mmol) in DMF (10 ml) were stirred at 100° C. overnight. After completion of the reaction the reaction solution was added to ice water (30 ml) and extracted with EA (20 ml×2), the organic layer was dried with Na2 SO4 and concentrated to give a yellow solid crude compound 2 (610 mg, 101.3% yield).
Compound 2 (300 mg, 0.78 mmol) was dissolved in DCM (5 ml), TFA (5 ml) was added, and the reaction was carried out at room temperature for 2 h. The reaction solution was spun dry to obtain a yellow solid of compound 3 (280 mg, crude).
The aforementioned compounds Corel (100 mg, 0.16 mmol) and N-BOC-piperazine (59.7 mg, 0.32 mmol) were dissolved in DMF (5 ml), NaI (48 mg, 0.32 mmol) and potassium carbonate (44.3 mg, 0.32 mmol) were added, and the reaction was carried out at 80° C. overnight. The reaction was completed by pouring the reaction solution into water, EA (10 ml×2) was extracted, the organic phase was washed with saturated saline (10 mL×2), dried with anhydrous sodium sulfate, spun-dried with solvent DCM:MeOH=10:1, and purified by preparative plate to give compound 4 as a yellow solid (54 mg, 47.3% yield).
Compound 4 (54 mg, 0.75 mmol) was dissolved in (2 ml) DCM, TFA (0.4 ml) was added and the reaction was carried out at room temperature for 1 hour. The reaction was completed by vacuum spin drying the solvent to give compound 5 as a yellow solid (80 mg, 90% yield).
Compound 5 (80 mg, 0.13 mmol) and compound 3 (66 mg, 0.2 mmol) were dissolved in DMF (2 ml), HATU (76 mg, 0.2 mmol) and TEA (26 mg, 0.26 mmol) were added at room temperature and the reaction solution reacted at 100° C. overnight. The reaction solution was prepared under high pressure to give the compound TPD005317 as a yellow solid (17 mg, 14.1% yield). LCMS (ESI) m/z calcd. for C49H48FN8O10 [M+H]+ 927.3; found 927.2. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005401 by the following synthetic route:
To a 250 ml triple-necked vial was added compound 1 (10.0 g, 0.05 mol), 4-aminophenol (7.33 g, 0.07 mol) and N,N-dimethylformamide (100 ml). After dissolution, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate (25.5 g, 0.07 mol) and N,N-diisopropylethylamine (17.3 g, 0.13 mol) were added and stirred at room temperature overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over sodium sulfate and spun dry. The crude product was pulped with dichloromethane, filtered and the filter cake was dried to obtain compound 2 (9.00 g, white solid), yield: 64.3%.
A 250 ml single-necked vial was charged with compound 2 (10.0 g, 0.03 mol), 7-benzyloxy-4-chloro-6-methoxyquinoline 5-amino-2-methoxybenzoate methyl ester (8.70 g, 0.03 mol), cesium carbonate (21.0 g, 0.06 mol), and dimethylsulfoxide (100 ml), and the reaction solution was stirred overnight at 120° C., quenched by adding water. It was extracted with ethyl acetate 3 times, washed with water once, washed with saturated brine once, dried with anhydrous sodium sulfate and spun dry. The crude product was passed through a column with petroleum ether/ethyl acetate (1:1) and dichloromethane/methanol (20:1-10:1) to obtain compound 3 (10 g, brown solid), yield: 48.3%.
A 250 ml hydrogenation flask was charged with compound 3 (8.00 g, 0.013 mol), palladium carbon (3.00 g) and tetrahydrofuran (40 ml) and methanol (40 ml). The air was replaced three times with hydrogen and stirred overnight at 40° C. in a 20 Psi hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth and the filtrate was spin dried to give compound 4 (6.00 g, yellow solid), yield: 88.9%.
A 250 ml three-necked vial was charged with compound 4 (7.00 g, 0.01 mol), pyridine (14.0 g, 0.18 mol), dichloromethane (35 ml), and tetrahydrofuran (35 ml). Trifluoromethanesulfonic anhydride (26.0 g, 0.09 mol) was added dropwise at 0° C. with stirring at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed once with saturated brine, dried with anhydrous sodium sulfate, spun dry and purified over a column (petroleum ether/ethyl acetate=30:1-3:1) to give compound 5 (4.00 g, yellow solid), yield: 44.9%.
A 20 ml reaction vial was charged with compound 5 (100 mg, 0.16 mmol), 4-hydroxymethylpiperidine (40.0 mg, 0.35 mmol), tris(dibenzylideneacetone)dipalladium (30.0 mg, 0.03 mmol), 1,1′-binaphthyl-2,2′-bisdiphenyphosphine (40.0 mg, 0.06 mmol), cesium carbonate (105 mg. 0.32 mmol) and 1,4-dioxane (10 ml), nitrogen displacement and stirring at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by column purification (petroleum ether/ethyl acetate=30:1-2:1) to obtain compound 6 (30.0 mg, yellow solid), yield: 31.8%.
A 100 ml triple vial was charged with compound 6 (380 mg, 0.64 mmol), triethylamine (99.0 mg, 0.97 mmol), dichloromethane (5 ml) and methanesulfonyl chloride (89.0 mg, 0.78 mmol). The reaction solution was stirred at 25° C. overnight, quenched with water, extracted 3 times with dichloromethane, the combined organic phases were washed once with water, dried over anhydrous sodium sulfate and spun dry to give compound core2 (320 mg, yellow solid), yield: 74.3%.
A 100 ml single-necked vial was charged with compound core2 (200 mg, 0.30 mmol), tert-butyl 2-(piperidin-1-yl)acetate (723 mg, 0.36 mmol), potassium carbonate (83.0 mg, 0.60 mmol), sodium iodide (90.0 mg, 0.60 mmol), N,N-dimethylformamide (20 ml). The reaction solution was stirred at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun-dried to give compound 7 (200 mg, yellow solid), yield: 86.5%.
To a 100 ml single mouth flask was added compound 7 (400 mg, 0.52 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry and pulped with methyl tert-butyl ether to give the crude compound 8 (400 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 8 (500 mg, 0.70 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (492 mg, 1.05 mmol), and triethylamine (213 mg. 2.11 mmol), dissolved with DCM (5 ml), 1-hydroxybenzotriazole (114 mg, 0.25 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (161 mg, 0.85 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005401 (35 mg, yellow solid), yield: 4.7%. LCMS (ESI) m/z calcd. for C61H72FN10O8S [M+H]+ 1122.5; found 1122.6. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005402B by the following synthetic route:
A 100 ml single-necked vial was charged with compound core2 (200 mg, 0.30 mmol) prepared according to the steps in Example 6, tert-butyl piperidine-4-carboxylate (67.0 mg, 0.36 mmol), potassium carbonate (83.4 mg, 0.60 mmol), sodium iodide (90.0 mg, 0.60 mmol), and N,N-dimethylformamide (20 ml). The reaction solution was stirred at 80° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun-dried to give compound 9 (100 mg, yellow solid), yield: 44.0%.
In a 100 ml single-necked vial, compound 9 (100 mg, 0.13 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml) were added, the reaction solution was stirred at room temperature overnight, spun dry, and pulped with methyl tert-butyl ether to obtain the crude compound 10 (80 mg, yellow solid), yield: 92.3%.
A 100 ml triple-necked vial was charged with compound 10 (95 mg, 0.15 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (102 mg, 0.22 mmol) and triethylamine (44.2 mg. 0.44 mmol), dissolved in dichloromethane (10 ml), 1-hydroxybenzotriazole (23.6 mg, 0.18 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (33.4 mg, 0.18 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried with anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD00542B (19 mg, yellow solid), yield: 11.7%. LCMS (ESI) m/z calcd. for C61H71FN9O8S [M+H]+ 1107.5; found 1107.5. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005406 by the following synthetic route:
A tetrahydrofuran solution (150 ml) of compound 7 (10 g, 42.6 mmol), tert-butyl acrylate (8.17 g. 63.8 mol) and potassium tert-butanolate (12.5 g, 112 mmol) was taken and stirred at room temperature overnight, the reaction solution was quenched with 1N HCl (100 ml) and extracted with ethyl acetate (50 ml×2). The combined organic phases were washed with water (50 ml) and dried over a column with anhydrous sodium sulfate to give compound 8 (5 g, 32.5%).
To a methanolic solution (50 ml) of compound 8 (5 g, 13.8 mmol) was added palladium carbon (1 g) and stirred overnight at room temperature in a hydrogen atmosphere (30 psi). The reaction solution was filtered and spun dry to give compound 9 (1.5 g, 47.6%).
A 100 ml single-necked vial was charged with compound core 2 (100 mg, 0.15 mmol), tert-butyl 3-(piperidin-4-acyloxy)propionate (68.7 mg, 0.30 mmol), potassium carbonate (83.0 mg, 0.60 mmol), sodium iodide (90.0 mg, 0.60 mmol) and N,N-dimethylformamide (10 ml). The reaction solution was stirred at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun-dried to give compound 10 (80 mg, yellow solid), yield: 66.7%.
To a 100 ml single necked vial was added compound 7 (80 mg, 0.11 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry and pulped with methyl tert-butyl ether to give the crude compound 11 (50 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 11 (50 mg, 0.07 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (49 mg, 0.11 mmol) and triethylamine (21.3 mg. 0.2 mmol), dissolved with DCM (2 ml), 1-hydroxybenzotriazole (11.4 mg, 0.03 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (16.1 mg, 0.09 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005406 (10 mg, yellow solid), yield: 12.8%. LCMS (ESI) m/z calcd. for C63H75FN9O9S [M+H]+ 1151.5; found 1151.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005407 by the following
A DMF solution (100 ml) of compound 12 (10 g, 42.6 mmol) was taken and NaH (3.8 g, 112 mmol) was added at 0° C. and reacted at room temperature for 1 h. DMF solution (30 ml) of tert-butyl bromoacetate (12.4 g, 63.8 mol) was added to the above solution at room temperature and stirred at room temperature overnight. The reaction solution was poured into ammonium chloride solution (500 ml) quenched and extracted with ethyl acetate (500 ml×2). The combined organic phases were washed with water (500 ml) and dried over a column with anhydrous sodium sulfate to give compound 8 (5 g, 33%).
To a methanolic solution (50 ml) of compound 13 (5 g, 13.8 mmol), palladium carbon (1 g) was added and stirred overnight at room temperature in a hydrogen atmosphere (30 psi). The reaction solution was filtered and spun dry to give compound 14 (3 g, 80%).
A 100 ml single-necked vial was charged with compound core 1 (100 mg, 0.15 mmol), tert-butyl 2-(piperidin-4-acyloxy)acetate (64.5 mg, 0.30 mmol), potassium carbonate (83.0 mg, 0.60 mmol), sodium iodide (90.0 mg, 0.60 mmol) and N,N-dimethylformamide (10 ml). The reaction solution was stirred at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun-dried to give compound 15 (60 mg, yellow solid), yield: 50.8%.
To a 100 ml single mouth flask was added compound 7 (60 mg, 0.08 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry and pulped with methyl tert-butyl ether to give the crude compound 11 (40 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 16 (50 mg, 0.06 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (49 mg, 0.11 mmol) and triethylamine (21.3 mg. 0.2 mmol), dissolved with DCM (2 ml), 1-hydroxybenzotriazole (11.4 mg, 0.03 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (16.1 mg, 0.09 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005407 (10 mg, yellow solid), yield: 15.9%. LCMS (ESI) m/z calcd. for C62H73FN9O9S [M+H]+ 1137.5; found 1137.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005409 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), tert-butyl piperidine-4-carboxylate (131.6 mg, 0.71 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol), and cesium carbonate (210.4 mg, 0.65 mmol), were dissolved in 1,4-dioxane (5 ml) and reacted at 100° C. overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 6 as a yellow solid (50 mg, 23.6% yield).
Compound 6 (50 mg, 0.08 mmol) was dissolved in TFA (1 ml) and reacted overnight at room temperature. The reaction was completed vacuum spin drying the solvent of compound 7 yellow solid (50 mg, 109.4% yield).
Compound 7 (50 mg, 0.08 mmol), core1 (58.5 mg, 0.13 mmol), HOBT (13.5 mg, 0.10 mmol), EDCI (19.2 mg, 0.10 mmol), and triethylamine (25.4 mg, 0.25 mmol), were dissolved in DMF (2 ml), and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by pre-HPLC to give the compound TPD005409 as a yellow solid (10.5 mg, yield 12.5%). LCMS (ESI) m/z calcd. for C55H60FN8OS [M+H]+ 1011.4; found 1011.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005410 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), tert-butyl 4-piperidineacetate oxalate (141.5 mg, 0.71 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol), and cesium carbonate (210.4 mg, 0.65 mmol), were dissolved in 1,4-dioxane (5 ml) and reacted at 100° C. overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 8 as a yellow solid (50 mg, yield 23.2%).
Compound 8 (50 mg, 0.07 mmol) was dissolved in TFA (1 ml) and the reaction was carried out at room temperature overnight, the reaction was completed by vacuum spin drying the solvent of compound 9 yellow solid (50 mg).
Compound 9 (50 mg, 0.08 mmol), core1 (52.7 mg, 0.12 mmol), HOBT (13.2 mg, 0.10 mmol), EDCI (18.8 mg, 0.10 mmol), and triethylamine (24.8 mg, 0.24 mmol), were dissolved in DMF (2 ml), and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by pre-HPLC to give the compound TPD005410 as a yellow solid (6.5 mg, yield 7.7%). LCMS (ESI) m/z calcd. for C56H62FN8O8S [M+H]+ 1024.4; found 1024.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005411 by the following synthetic route:
A 20 ml reaction vial was charged with compound 5 (100 mg, 0.16 mmol), tert-butyl 2-(piperidin-4-acyloxy)acetate (75.3 mg, 0.35 mmol), tris(dibenzylideneacetone)dipalladium (30.0 mg, 0.03 mmol), 1,1′-binaphthyl-2,2′-bisdiphenylphosphine (40.0 mg, 0.06 mmol), cesium carbonate (105 mg, 0.32 mmol) and 1,4-dioxane (10 ml), nitrogen displacement, and stirring at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by column purification (petroleum ether/ethyl acetate=30:1-2:1) to give compound 21 (70.0 mg, yellow solid), yield: 63.6%.
To a 100 ml single necked vial was added compound 21 (70 mg, 0.10 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry and pulped with methyl tert-butyl ether to give the crude compound 22 (30 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 22 (30 mg, 0.05 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (44.2 mg, 0.1 mmol), and triethylamine (20.2 mg. 0.2 mmol), dissolved with DCM (2 ml), 1-hydroxybenzotriazole (27.2 mg, 0.2 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (38.4 mg, 0.2 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005411 (10 mg, yellow solid), yield: 20%. LCMS (ESI) m/z calcd. for C56H62FN8O9S [M+H]+ 1040.4; found 1040.4. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005412 by the following synthetic route:
A 20 ml reaction vial was charged with compound 5 (100 mg, 0.16 mmol), tert-butyl 3-(piperidin-4-acyloxy)propionate (80 mg, 0.35 mmol), tris(dibenzylideneacetone)dipalladium (30.0 mg, 0.03 mmol), 1,1′-binaphthyl-2,2′-bis(diphenylphosphine) (40.0 mg, 0.06 mmol) Cesium carbonate (105 mg, 0.32 mmol) and 1,4-dioxane (10 ml), nitrogen displacement and stirring at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by column purification (petroleum ether/ethyl acetate=30:1-2:1) to give compound 23 (80.0 mg, yellow solid), yield: 70.8%.
To a 100 ml single-necked vial was added compound 23 (80 mg, 0.11 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry, and pulped with methyl tert-butyl ether to give the crude compound 22 (30 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 24 (30 mg, 0.05 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (44.2 mg, 0.1 mmol), and triethylamine (20.2 mg. 0.2 mmol), dissolved with DCM (2 ml), 1-hydroxybenzotriazole (27.2 mg, 0.2 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (38.4 mg, 0.2 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005412 (10 mg, yellow solid), yield: 20%. LCMS (ESI) m/z calcd. for C57H64FN8O9S [M+H]+ 1054.4; found 1054.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005415 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), tert-butyl 3-aminopropionate (103.1 mg, 0.71 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol) and cesium carbonate (210.4 mg, 0.65 mmol) were dissolved in 1,4-dioxane (5 ml) and reacted at 100° C. overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 8 as a yellow solid (50 mg, yield 25.2%).
Compound 10 (50 mg, 0.07 mmol) was dissolved in TFA (1 ml) and reacted overnight at room temperature. The reaction was completed vacuum spin drying the solvent of compound 11 yellow solid (50 mg, yield 110.1%).
Compound 11 (50 mg, 0.09 mmol), core1 (57.8 mg, 0.13 mmol), HOBT (14.5 mg, 0.11 mmol), EDCI (20.6 mg, 0.11 mmol), and triethylamine (27.2 mg, 0.27 mmol), were dissolved in DMF (2 ml), and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by pre-HPLC to give the compound TPD005415 as a yellow solid (11 mg, yield 12.7%). LCMS (ESI) m/z calcd. for C52H56FN8O8S [M+H]+ 970.4; found 970.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005417 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), tert-butylglycinate (93.2 mg, 0.71 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol) and cesium carbonate (210.4 mg, 0.65 mmol) were dissolved in 1,4-dioxane (5 ml) in 100° C. and reacted overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 12 as a yellow solid (50 mg, yield 25.8%).
Compound 12 (50 mg, 0.08 mmol) was dissolved in TFA (1 ml) and reacted overnight at room temperature. The reaction was completed vacuum spin drying the solvent of compound 13 yellow solid (50 mg, yield 110.3%).
Compound 13 (50 mg, 0.09 mmol), core1 (59.3 mg, 0.14 mmol), HOBT (14.9 mg, 0.11 mmol), EDCI (21.1 mg, 0.11 mmol) and triethylamine (27.9 mg, 0.28 mmol) were dissolved in DMF (2 ml), and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by pre-HPLC to give the compound TPD005417 as a yellow solid (10.1 mg, yield 11.5%). LCMS (ESI) m/z calcd. for C51H54FN8O8S [M+H]+ 956.4; found 956.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005420 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), 4-hydroxypiperidine (49.0 mg, 0.48 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol) and cesium carbonate (210.4 mg, 0.65 mmol) were dissolved in 1,4-dioxane (5 ml) in 100° C. and reacted overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 14 as a yellow solid (50 mg, 27.1% yield).
Compound 14 (250 mg, 0.44 mmol) was dissolved in DCM (10 ml), Des Martin (371.6 mg, 0.88 mmol) was added in batches and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with DCM (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 15 as a yellow solid (150 mg, yield 60.2%).
Compound 15 (150 mg, 0.26 mmol) and tert-butyl piperidine-4-carboxylate (146.6 mg, 0.79 mmol) were dissolved in DCM (5 ml) and tetraisopropyl titanate (1 ml), stirred at room temperature for 3 h. Sodium borohydride acetate (167.9 mg, 0.79 mmol) was added, and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with DCM (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 16 as a yellow solid (40 mg, 20.5% yield).
Compound 12 (70 mg, 0.09 mmol) was dissolved in TFA (1 ml) and reacted overnight at room temperature. The reaction was completed vacuum spin drying the solvent of compound 13 yellow solid (70 mg, 108.2% yield).
Compound 17 (70 mg, 0.10 mmol), core1 (71.8 mg, 0.15 mmol), HOBT (16.7 mg, 0.12 mmol), EDCI (23.6 mg, 0.12 mmol) and triethylamine (31.2 mg, 0.31 mmol) were dissolved in DMF (2 ml), and the reaction was carried out at room temperature overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by pre-HPLC to give the compound TPD005420 as a yellow solid (10.3 mg, 9.1% yield). LCMS (ESI) m/z calcd. for C60H69FN9O8S [M+H]+ 1094.5; found 1094.3. The compound structure is confirmed.
This embodiment was used to prepare the synthetic compound TPD005421 by the following synthetic route:
Compound 5 (200 mg, 0.32 mmol), 4-hydroxypiperidine (81.8 mg, 0.71 mmol), BINAP (80.4 mg, 0.13 mmol), Pd(dba)2 (55.7 mg, 0.10 mmol) and cesium carbonate (210.4 mg, 0.65 mmol) were dissolved in 1,4-dioxane (5 ml) in 100° C. and reacted overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 14 as a yellow solid (50 mg, yield 26.5%).
Compound 18 (500 mg, 0.86 mmol) and triethylamine (129.8 mg, 1.28 mmol) were dissolved in DCM (5 ml), MsCl (117.6 mg, 1.03 mmol) was added dropwise at 0° C. and the reaction was carried out at room temperature for 2 hours. After completion of the reaction the reaction solution was washed with water (2 ml) and the organic phase was dried with anhydrous sodium sulfate and concentrated to give compound 19 as a yellow solid (500 mg, yield 88.2%).
Compound 19 (300 mg, 0.45 mmol), tert-butyl piperidine-4-carboxylate (125.8 mg, 0.68 mmol), sodium iodide (135.8 mg, 0.91 mmol) and potassium carbonate (187.7 mg, 1.36 mmol) were dissolved in DMF (5 ml) and stirred at 80° C. overnight. After completion of the reaction the reaction solution was added to (30 ml) of water and extracted with EA (10 ml×3), the organic layer was dried and concentrated with anhydrous sodium sulfate and purified by preparative plate to give compound 20 as a yellow solid (200 mg, 58.8% yield).
Compound 20 (200 mg, 0.27 mmol) was dissolved in TFA (2 ml) and reacted overnight at room temperature. The reaction was completed vacuum spin drying the solvent of compound 21 yellow solid (150 mg, yield 69.7%).
A 100 ml single-necked vial was charged with compound core 1 (300 mg, 0.45 mmol), tert-butyl azetidine-3-carboxylate (94.2 mg, 0.6 mmol), potassium carbonate (83.0 mg, 0.60 mmol), sodium iodide (90.0 mg, 0.60 mmol), N,N-dimethylformamide (10 ml). The reaction solution was stirred at 100° C. overnight. Quenched with water, extracted with ethyl acetate 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun-dried to give compound 7 (120 mg, yellow solid), yield: 36.5%.
To a 40 ml single-necked vial was added compound 35 (120 mg, 0.17 mmol), dichloromethane (10 ml) and trifluoroacetic acid (2 ml), the reaction solution was stirred at room temperature overnight, spun dry, and pulped with methyl tert-butyl ether to give the crude compound 36 (100 mg, yellow solid).
A 100 ml triple-necked vial was charged with compound 8 (100 mg, 0.15 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (124 mg, 0.3 mmol) and triethylamine (50.5 mg. 0.5 mmol), dissolved with DCM (5 ml), 1-hydroxybenzotriazole (114 mg, 0.25 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (161 mg, 0.85 mmol) were added and stirred at room temperature overnight. Quenched with water, extracted with dichloromethane 3 times, the combined organic phases were washed with water 3 times, saturated brine once, dried over anhydrous sodium sulfate and spun dry. The crude product was purified by high pressure preparation to give compound TPD005423 (10 mg, yellow solid), yield: 6.2%. LCMS (ESI) m/z calcd. for C59H67FN9O8S [M+H]+ 1079.5; found 1079.4. The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound int-A (600 mg, 0.9685 mmol), 4-Boc-aminopiperidine (387.94 mg, 1.937 mmol), cesium carbonate (631.11 mg, 1.937 mmol), BINAP (241.22 mg, 0.3874 mmol), Pd2 (dba)3 (177.37 mg, 0.1937 mmol) and 1,4-dioxane (30 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to obtain the compound TPD5424-1 (440 mg, yellow solid, purity 84.226%), yield: 57.13%. LCMS (ESI) m/z calcd. for C37H40FN5O6 [M+H]+ 670.3; found 670.2. 1H, NMR (400 MHz, CDCl3): δ=9.34 (s, 1H), 8.81 (s, 1H), 8.46 (d, J=5.3 Hz, 1H), 7.62 (d, J=8.9 Hz, 2H), 7.52-7.45 (m, 4H), 7.16 (d, J=8.9 Hz, 2H), 7.05 (t, J=8.6 Hz, 2H), 6.43 (d, J=5.2 Hz, 1H), 4.52 (s, 1H), 4.02 (s, 3H), 3.64 (d, J=12.1 Hz, 2H), 2.83 (t, J=11.0 Hz, 2H), 2.11 (d, J=10.9 Hz, 2H). 1.74-1.71 (m, 2H), 1.68-1.65 (m, 4H), 1.47 (s, 9H).
A 50 ml three-necked vial was charged with compound TPD5424-1 (440 mg, 0.657 mmol), DCM (8 ml) and TFA (4 ml). The reaction solution reacted at 20° C. for 4 hours. After completion of the reaction the reaction solution was directly spun dry to give the crude compound TPD5424-2 (440 mg, yellow solid, purity 91.595%), yield: 89.73%. LCMS (ESI) m/z calcd. for C32H32FN5O4 [M+H]+ 570.2; found 570.2. The compound structure is confirmed.
To a 50 ml triple-necked vial was sequentially added compound TPD5424-2 (300 mg, 0.563 mmol), monomethyl 1,1-cyclopropyl dicarboxylate (91 mg, 0.632 mmol), DMF (5 ml), HATU (300 mg, 0.790 mmol) and triethylamine (266 mg, 2.633 mmol). The reaction mixture reacted at 20° C. for 16 hours. After completion of the reaction the reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 ml) and the aqueous phase was extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5424-3 (250 mg, yellow solid, purity 97.596%), yield: 66.58%. LCMS (ESI) m/z calcd. for C37H36FN4O7 [M+H]+ 696.3; found 696.1. 1H NMR (400 MHz, CDCl3): δ=9.49 (s, 1H), 8.88 (d, J=7.8 Hz, 1H), 8.76 (s, 1H), 8.43 (d, J=5.6 Hz, 1H), 7.69-7.63 (m, 2H), 7.53 (s, 1H), 7.49-7.46 (m, 3H). 7.20-7.15 (m, 2H), 7.09-7.01 (m, 2H), 6.48 (d, J=5.6 Hz, 1H), 4.05 (s, 3H), 3.70-3.62 (m, 5H), 2.96 (t, J=10.4 Hz, 2H), 2.13-2.11 (m, 2H), 1.88-1.64 (m, 8H). 1.59-1.56 (m, 2H). The compound structure is confirmed.
To a 50 ml three-necked vial was added sequentially the compound TPD5424-3 (100 mg, 0.1437 mmol), THF (2 ml), H2O (1 ml) and lithium hydroxide monohydrate (12.06 mg, 0.2874 mmol). the reaction was carried out at 20° C. for 3 h. The reaction solution was adjusted to pH 8 with dilute hydrochloric acid, then the mixture was extracted three times with ethyl acetate (5 ml), anhydrous sodium sulfate. The reaction solution was adjusted to pH 8 with dilute hydrochloric acid, then the mixture was extracted three times with ethyl acetate (5 ml), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spun-dried to give the crude compound, TPD5424-5 (50 mg, yellow solid, purity 97.614%), yield: 49.83%. LCMS (ESI) m/z calcd. for C37H36FN5O7 [M+H]+ 682.26; found 682.2. The compound structure is confirmed.
A 50 ml triple-necked vial was sequentially charged with compound TPD5424-4 (50 mg, 0.0733 mmol), compound TPD5424-5 (34.72 mg, 0.0806 mmol), DMF (4 ml), HATU (41.81 mg, 0.1099 mmol) and triethylamine (22.25 mg, 0.2199 mmol). The reaction mixture reacted at 20° C. for 16 hours. After completion of the reaction the reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 ml) and the aqueous phase was extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005424 (26.6 mg, off-white solid, purity 97.557%), yield: 32.33%. LCMS (ESI) m/z calcd. for C59H64 FN9O9S [M+H]+ 1094.45; found 1094.7. 1H NMR (400 MHz, DMSO-d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 9.10 (d, J=8.6 Hz, 1H), 8.96 (s, 1H), 8.68 (t, J=5.9 Hz, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.74 (dd, J=18.3 8.3 Hz, 3H), 7.67-7.59 (m, 2H), 7.46 (s, 1H), 7.44-7.36 (m, 4H), 7.34 (s, 1H), 7.21 (d, J=9.0 Hz, 2H), 7.15 (dd, J=12.3, 5.5 Hz, 2H), 6.40 (d, J=5.2 Hz, 1H). 5.15 (d, J=3.5 Hz, 1H), 4.58-4.17 (m, 5H), 3.99-3.76 (m, 4H), 3.72-3.47 (m, 4H), 2.86-2.66 (m, 2H), 2.43 (s, 3H), 2.04 (d, J=8.5 Hz, 1H), 1.96-1.65 (m, 5H). 1.47 (s, 4H), 1.29 (s, 4H), 0.95 (s 9H). The compound structure is confirmed.
Compound TPD5488-1 (200 mg, 0.3421 mmol), triethylamine (103.85 mg, 1.0263 mmol) and dichloromethane (10 ml) were added sequentially to a 50 ml triple-necked vial. Cooled down under nitrogen protection to 0° C. The nmethylsulfonyl chloride (58.78 mg, 0.51315 mmol) was added dropwise. After dropwise addition, the cold bath was removed and the reaction was naturally warmed to 20° C. for 1 hour. The reaction solution was decanted into water (20 ml) and extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude compound TPD5430-1 (220 mg, yellow solid, purity 83.913%), yield: 81.44%. LCMS (ESI) m/z calcd. for C34H35FN4O7S [M+H]+ 663.22; found 663.1. The compound structure is confirmed.
To a 25 ml triple-necked vial was sequentially added compound TPD5430-2 (110 mg, 0.166 mmol), tert-butyl 3-acridinecarboxylate hydrochloride (38.58 mg, 0.1992 mmol), potassium carbonate (45.89 mg, 0.332 mmol), sodium iodide (49.8 mg, 0.332 mmol) and N,N-dimethylformamide (5 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. The reaction solution was poured into water (20 ml). It was extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=15/1 to give compound TPD005430-2 (50 mg, yellow solid, purity 96.731%), yield: 40.24%. LCMS (ESI) m/z calcd. for C41H46FN5O [M+H]+ 724.34; found 724.4. 1H NMR (400 MHz, CDCl3): δ=9.19 (s, 1H), 8.88 (s, 1H), 8.39 (d, J=5.2 Hz, 1H), 7.60 (d, J=8.8 Hz, 2H), 7.48-7.46 (m, 2H), 7.41 (s, 1H), 7.15 (t, J=8.8 Hz, 3H), 7.05 (t, J=6.4 Hz, 2H), 6.33 (d, J=5.2 Hz, 1H), 3.96 (s, 3H), 3.66-3.57 (m, 2H), 3.56-3.48 (m, 3H), 3.28-3.18 (m, 4H), 2.52-2.48 (m, 2H), 2.29-2.21 (m, 1H), 2.16-2.10 (m, 1H), 1.73-1.68 (m, 5H), 1.54-1.49 (m, 2H), 1.46 (s, 9H). The compound structure is confirmed.
A 50 ml triple vial was charged with compound TPD5488-2 (100 mg, 0.1382 mmol), dichloromethane (6 ml) and trifluoroacetic acid (3 ml). the reaction was carried out at 20° C. for 16 h. The reaction was carried out at 20° C. for 2 h. The reaction was carried out at 20° C. for 2 h. The reaction was completed with the addition of compound TPD5430-3 (90 mg, yellow solid). Concentration to dryness afforded the crude compound TPD5430-3 (90 mg, yellow solid, purity 79.272%), yield: 66.06%. LCMS (ESI) m/z calcd. for C37H38FN5O6 [M+H]+ 668.28; found 668.3. 1H NMR (400 MHz, DMSO_d6): δ=14.78 (s 1H), 10.32 (s, 1H), 10.00 (s, 1H), 8.57 (d, J=6.8 Hz, 1H), 7.84 (d, J=9.2 Hz, 2H), 7.66-7.62 (m, 2H), 7.54 (s, 1H), 7.33 (d, J=9.2 Hz, 2H), 7.16 (t, J=9.2 Hz, 2H), 6.90 (s, 1H), 6.71 (d, J=6.8 Hz, 1H), 4.29-4.07 (m, 7H), 4.00 (s, 3H), 3.38-3.17 (m, 4H), 2.27-2.19 (m, 1H), 2.16-2.10 (m, 1H), 1.70-1.57 (m, 3H), 1.49 (d J=8.4 Hz, 4H). The compound structure is confirmed.
A 25 ml triple-necked vial was sequentially charged with compound TPD5430-3 (45 mg, 0.0674 mmol), N,N-dimethylformamide (3 ml), (2S,4R)-4-hydroxy-N-(4-(4-(4-methylthiazol-5-yl)phenylmethyl)pyrrolidine-2-carboxamide hydrochloride (32.09 mg, 0.1011 mmol), HOBt (13.66 mg, 0.1011 mmol), EDCI (19.38 mg, 0.1011 mmol) and triethylamine (40.84 mg, 0.4044 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. The reaction solution was decanted into water (10 ml). It was extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=4/1 to give compound TPD005430 (6.65 mg, yellow solid, purity 97.127%), yield: 4.97%. LCMS (ESI) m/z calcd. for C53H55FN8O7S [M+H]+ 967.39; found 967.7. 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.08 (s, 1H), 8.98 (s, 1H), 8.80 (br. s., 0.35H), 8.46 (br. s., 0.76H), 8.35 (br. s., 1H), 7.75 (d, J=6.4 Hz, 2H), 7.65 (br. s., 2H), 7.47-7.43 (m, 2H), 7.38 (s, 3H), 7.21-7.14 (m, 4H), 6.94 (s, 1H), 6.27 (br. s., 1H), 5.12 (s, 1H), 4.42-4.25 (m, 4H), 3.90 (s, 3H), 3.56-3.44 (m, 6H), 3.23 (s, 1H), 3.15 (s, 3H), 2.51 (s, 3H), 2.20 (s, 1H), 2.06-1.96 (m, 3H), 1.90-1.85 (m, 1H), 1.55-1.35 (m, 7H), 1.24 (s, 2H). The compound structure is confirmed.
A 25 ml single-necked vial was charged sequentially with tert-butyl 3-oxo-azetidine-1-carboxylate (70 mg, 0.4065 mmol), compound TPD5431-1 (189.85 mg, 0.4065 mmol), sodium triacetoxyborohydride 258.46 mg, 1.2195 mmol), ethylene dichloride (6 ml) and glacial acetic acid (73.23 mg, 1.2195 mmol), the reaction was carried out at 50° C. for 16 hours. After the reaction was completed, the reaction solution and poured into 10 ml of saturated aqueous ammonium chloride solution, extracted three times with DCM (10 ml), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the residue was purified by means of a preparative plate, the unfolding agent was DCM/MeOH=10/1, to give the compound TPD5431-2 (210 mg, white solid, purity 89.921%), yield: 79.31%. LCMS (ESI) m/z calcd. for C30H43N5O5S [M+H]+ 586.3; found 586.1. 1H NMR (400 MHz, DMSO_d6): δ=8.99 (d, J=6.2 Hz, 1H), 8.59 (t, J=6.0 Hz, 1H). 7.51-7.31 (m, 4H), 5.07 (d, J=2.8 Hz, 1H), 4.50-4.32 (m, 3H), 4.26-4.20 (m, 1H), 4.12-3.69 (m, 3H), 3.81-3.50 (m, 4H), 3.39 (br. s., 1H), 3.00 (br. s., 1H). 2.45 (d, J=3.2 Hz, 3H), 2.13-1.79 (m, 2H), 1.36 (d, J=3.2 Hz, 9H), 0.92 (s, 9H). The compound structure is confirmed.
A 25 ml single-necked vial was charged with compound TPD5431-2 (180 mg, 0.3073 mmol) and hydrogen chloridemethanol solution (4N, 6 mL). The reaction was carried out at 25° C. for 30 minutes. The reaction was completed and concentrated under reduced pressure to give compound TPD5431-3 (185 mg, yellow solid, purity 85.380%), yield: 98.44%. LCMS (ESI) m/z calcd. for C25H35N5O3S [M+H]+ 486.3; found 486.1. The compound structure is confirmed.
A 25 ml single-necked vial was charged with compound TPD5488-1 (100 mg, 0.171 mmol), DCM (6 ml), and triethylamine (51.91 mg, 0.513 mmol), and methanesulfonyl chloride (29.38 mg, 0.2565 mmol) was added dropwise after the temperature was lowered to 0° C. The reaction was carried out at 0° C. for 1 h. The reaction was carried out at 0° C. for 1 h. The reaction was carried out at 0° C. for 2 h. The temperature of the reaction was reduced to 0° C. for 1 hour. The reaction was carried out at 0° C. for 1 hour. After the reaction was completed it was poured into water (10 ml) and extracted three times with DCM (3 ml). The combined organic phases were washed with water, brine, dried over saturated sodium sulfate filtered and concentrated to dryness to give the crude compound TPD5431 (50 mg, yellow oil, purity 40.853%), yield: 25.26%. LCMS (ESI) m/z calcd. for C34H35FN4O7S [M+H]+ 663.2; found 229.2 and 663.1. The compound structure is confirmed.
To a 25 mL single-necked vial was sequentially added compound TPD5431-4 (70 mg, 0.1056 mmol), compound TPD5431-3 (102.57 mg, 0.2112 mmol), potassium carbonate (58.38 mg, 0.4224 mmol), sodium iodide (31.68 mg, 0.2112 mmol) and DMF (5 ml). The reaction was carried out at 80° C. for 16 hours. After the reaction was completed it was cooled to room temperature, decanted into water (10 ml) and extracted three times with EA (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% ammonia); gradient: 30-90/8 min to afford the compound TPD005431 (11.2 mg, off-white solid, purity 95.035%) in a yield of 9.56%. LCMS (ESI) m/z calcd. for C58H66FN9O7S [M/2+H]+526.7; found 526.9. 1H NMR (400 MHz, DMSO_d6): δ=10.17 (s, 1H), 10.06 (s, 1H), 8.97 (d, J=6.0 Hz, 1H), 8.59 (t, J=5.9 Hz, 1H). 8.34 (d, J=5.1 Hz, 1H), 7.74 (d, J=8.8 Hz, 2H), 7.66-7.62 (m, 2H), 7.48-7.32 (m, 5H), 7.23-7.09 (m, 4H), 6.93 (s, 1H), 6.26 (d, J=5.2 Hz, 1H), 5.14 (d, J=10.2 Hz, 1H), 4.50-4.29 (m, 3H), 4.25-4.20 (m, 1H), 3.90 (s, 3H), 3.64 (d, J=11.1 Hz, 1H), 3.58-3.41 (m, 6H), 3.22-3.10 (m, 3H), 3.00 (br. s., 1H), 2.62-2.56 (m 1H), 2.44 (s, 3H), 2.39-2.32 (m, 2H), 2.20-2.14 (m, 1H), 2.10-1.97 (m, 3H), 1.93-1.85 (m, 1H), 1.55-1.43 (m, 4H), 1.42-1.33 (m, 2H), 1.23 (s, 1H), 0.96-0.79 (m, 9H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound int-A (1400 mg, 2.2579 mmol), 4-hydroxypiperidine (502.84 mg, 4.97134 mmol), Pd2 (dba)3 (413.85 mg, 0.45194 mmol), BINAP (562.82 mg, 0.9038 mmol), cesium carbonate (1472.51 mg, 4.5194 mmol) and dioxane (50 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, water was added to the reaction solution and extracted three times with EtOAc (100 ml). The combined organic layers were washed with brine (200 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give a residue. The residue was purified by silica gel column chromatography with eluent and ratio of DCM/MeOH=100/1˜20/1 to give the product compound TPD5432-1 (595 mg, yellow solid, purity 85.651%), yield: 37.67%. LCMS (ESI) m/z calcd. for C32H31FN4O5 [M+H]+ 571.23. found 571.2. The compound structure is confirmed.
Oxalyl chloride (143.48 mg, 1.1304 mmol) and DCM (2 ml) were added to a 50 ml three-necked vial under nitrogen protection. Cooling down to −78° C. a solution of DMSO (176.34 mg, 2.2608 mmol) in DCM (1 ml) was added dropwise. The resulting mixture was kept at −78° C. for 0.5 hours. Then a DCM (2 ml) solution of compound TPD5432-1 (430 mg, 0.7536 mmol) was added dropwise at −78° C. The resulting mixture was kept at −78° C. for 1 hour. Then TEA (228.77 mg, 2.2608 mmol) was added dropwise at −78° C. The resulting mixture was naturally warmed up for the reaction to 20° C. and kept for 0.5 hours. After completion of the reaction, the reaction was quenched by addition of saturated aqueous sodium bicarbonate. The mixture was extracted with DCM (20 ml×3). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give a residue. Purification of the residue by preparative TLC (DCM/MeOH=10/1) afforded the product compound TPD5432-2 (290 mg, yellow solid, purity 92.290%), yield: 62.46%. LCMS (ESI) m/z calcd. for C32H29FN4O5 [M+H]+ 569.21; found 569.0. 1H NMR (400 MHz, CDCl3): δ=9.35 (s, 1H), 8.66 (s, 1H), 8.49 (d, J=5.2 Hz, 1H), 7.58-7.46 (m, 5H), 7.19-7.17 (m, 2H), 7.08-7.04 (m, 2H), 6.46 (d, J=5.2 Hz, 1H). 4.07 (s, 3H), 3.57-3.54 (m, 4H), 2.72-2.69 (m, 4H), 1.75-1.65 (m, 4H). The compound structure is confirmed.
A 50 ml single-necked vial was charged with DCE (10 ml), compound TPD5432-2 (150 mg, 0.2638 mmol), tert-butyl 3-acridinecarboxylate hydrochloride (255.45 mg, 1.319 mmol), sodium triacetoxyborohydride (167.73 mg, 0.7913 mmol), tetraisopropyl titanate (0.5 ml). The reaction solution was heated up to 40° C. under nitrogen protection and then reacted for 16 hours. After the reaction was completed, water was added to the mixture and the mixture was extracted three times with EtOAc (10 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give the residue. Purification of the residue by preparative TLC (DCM/MeOH=10/1) afforded the product compound TPD5432-3 (100 mg, yellow solid, purity 96.741%), yield: 51.67%. LCMS (ESI) m/z calcd. for C40H44 FN5O6 [M+H]+ 710.33; found 710.5. The compound structure is confirmed.
A 50 ml single-necked vial was charged with compound TPD5432-3 (50 mg, 0.0704 mmol), DCM (2 ml), trifluoroacetic acid (2 ml). The resulting mixture was stirred at 20° C. for 2 hours. The reaction was completed and the mixture was spun dry to obtain the crude product compound TPD5432-4 (46 mg, white solid, purity 92.176%), yield: 92.19%. LCMS (ESI) m/z calcd. for C36H36 FN5O6 [M+H]+ 654.26; found 654.3. The compound structure is confirmed.
Compound TPD5432-4 (46 mg, 0.0704 mmol), HATU (40.15 mg, 0.1056 mmol), (2S,4R)-1—((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-formamide hydrochloride (40.3 mg, 0.0704 mmol) and TEA (35.62 mg, 0.352 mmol). The reaction was carried out under nitrogen protection at 15° C. for 16 hours. After completion of the reaction, the reaction solution was quenched with water. It was extracted three times with ethyl acetate (10 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give the residue. The crude product was purified by preparative HPLC (column: sunfire, 5 m, 19-150 mm; detector: 254 nm; mobile phase: ACN/H2O (0.1% FA in H2O); gradient: 10-50/8 min; retention time: 7.0 min) to give the product compound TPD005432 (23.5 mg, yellow solid, purity 97.684%), yield: 22.03%. LCMS (ESI) m/z calcd. for C58H64FN9O8S [M+H]+ 1066.46; found 533.8 (half peak); 356.2 (one-third peak)1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 10.06 (s, 1H), 8.98 (s, 1H), 8.59 (t, J=6.0 Hz, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.18 (s, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.47-7.37 (m, 5H), 7.31 (s, 1H), 7.23-7.13 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 4.57 (d, J=9.4 Hz, 1H), 4.48-4.40 (m, 2H), 4.37 (s, 1H), 4.22 (dd, J=15.7, 5.3 Hz, 1H). 3.94 (s, 3H), 3.67-3.64 (m, 2H), 3.44-3.37 (m, 2H), 3.22 (dd, J=28.7, 6.4 Hz, 3H), 2.74 (t, J=10.0 Hz, 2H), 2.45 (s, 3H), 2.30 (br. s., 1H), 2.15-1.75 (m, 5H). 1.48 (s, 4H), 1.36 (d, J=9.0 Hz, 2H), 1.23 (s, 2H), 0.95-0.93 (m, 9H). The compound structure is confirmed.
To a 250 ml triple-necked vial were sequentially added compound int-A (1.4 g, 2.2597 mmol), ethyl 4-piperidine carboxylate (710.49 mg, 4.5194 mmol), cesium carbonate (1.472 g, 4.5194 mmol), BINAP (562.82 mg, 0.9038 mmol), Pd2 (dba)3 (413.85 mg, 0.4519) and 1,4-dioxane (70 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5436-1 (660 mg, brown solid, purity 72.545%), yield: 33.81%. LCMS (ESI) m/z calcd. for C35H35FN4O6 [M+H]+ 627.3; found 627.4. The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5436-1 (600 mg, 0.9574 mmol), lithium hydroxide monohydrate (80.35 mg, 1.9148 mmol) and methanol/water=2:1 (10 ml). The reaction was carried out at 20° C. for 4 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5436-2 (700 mg, brown solid), yield: 86.17%. LCMS (ESI) m/z calcd. for C33H31FN4O6 [M+H]+ 599.2; found 599.1. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compounds TPD5436-2 (200 mg, 0.3341 mmol), ethyl 4-piperidinecarboxylate (63.03 mg, 0.4009 mmol), HATU (254.07 mg, 0.6682 mmol), triethylamine (101.43 mg, 0.6682 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD5436-3 (200 mg, brown solid, purity 80.484%), yield: 65.31%. LCMS (ESI) m/z calcd. for C41H44FN5O7 [M+H]+ 738.3; found 738.2; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.47 (s, 1H), 7.31 (s, 1H), 7.21 (d, J=9.0 Hz, 2H), 7.18-7.13 (m, 2H), 6.40 (d, J=5.2 Hz, 1H), 4.11-4.05 (m, 2H), 3.95 (s, 3H), 3.62 (d, J=11.1 Hz, 2H), 3.24-3.06 (m, 2H), 2.92-2.83 (m, 3H), 2.79-2.68 (m, 4H), 2.65-2.59 (m, 1H), 1.89-1.70 (m, 6H), 1.47 (s, 4H), 1.21-1.17 (m, 3H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5436-3 (200 mg, 0.2711 mmol), lithium hydroxide monohydrate (22.75 mg, 0.5422 mmol) and methanol/water=2:1 (3 ml). The reaction was carried out at 20° C. for 4 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5436-4 (250 mg, brown solid), yield: 85.58%. LCMS (ESI) m/z calcd. for C39H40FN5O7 [M+H]+ 710.3; found 710.2. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compounds TPD5436-4 (250 mg, 0.3522 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (197.38 mg, 0.4226 mmol), HATU (267.83 mg, 0.7044 mmol), triethylamine (71.28 mg, 0.7044 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to afford compound TPD005436 (31.2 mg, light yellow solid, purity 96.544%), yield: 7.47%. LCMS (ESI) m/z calcd. for C61H68FN9O9S [M+H]+ 1122.5; found 1122.3; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 10.06 (s, 1H), 8.99 (s, 1H), 8.58 ((br. s., 1H), 8.44 (d, J=5.2 Hz, 1H), 7.91 (d, J=8.6 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H). 7.66-7.63 (m, 2H), 7.47 (s, 1H), 7.44-7.38 (m, 4H), 7.31 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.18-7.13 (m, 2H), 6.40 (d, J=5.2 Hz, 1H), 5.14 (d, J=3.3 Hz, 1H). 4.54 (d, J=9.7 Hz, 1H), 4.45-4.31 (m, 3H), 4.35 (s, 1H), 4.25-4.20 (m, 1H), 4.03 (br. s., 1H), 3.95 (s, 3H), 3.68-3.61 (m, 4H), 3.07-3.02 (m, 1H), 2.83-2.67 (m, 4H), 2.45 (s, 3H), 2.08-1.87 (m, 3H), 1.81-1.62 (m, 6H), 1.47 (s, 5H), 1.23 (s, 1H), 0.95-0.92 (m, 9H). The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with the compounds TPD5436-2 (300 mg, 0.5012 mmol), glycine ethyl ester hydrochloride (83.95 mg, 0.6014 mmol), HATU (381.14 mg, 1.0024 mmol), triethylamine (101.43 mg, 1.0024 mmol), and DMF (5 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD5452-1 (190 mg, brown solid, purity 90.489%), yield: 50.18%. LCMS (ESI) m/z calcd. for C37H38FN5O7 [M+H]+ 684.3; found 684.3; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.07 (s, 1H), 8.45 (d, J=5.2 Hz, 1H), 8.32 (t, J=5.9 Hz, 1H), 7.76 (d, J=8.9 Hz, 1H), 7.67-7.63 (m, 2H), 7.48 (s, 1H), 7.34 (s, 1H), 7.23-7.21 (m, 2H), 7.18-7.14 (m, 2H), 6.40 (d, J=5.2 Hz, 1H), 4.13-4.08 (m, 2H), 3.96 (s, 1H), 3.83 (d, J=5.9 Hz, 1H), 3.62 (d, J=11.5 Hz, 1H), 2.74-2.68 (m, 3H), 1.84-1.79 (m, 4H), 1.48 (s, 4H), 1.21 (t, J=7.1 Hz, 1H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5436-3 (190 mg, 0.2779 mmol), lithium hydroxide monohydrate (23.32 mg, 0.5558 mmol) and methanol/water=2:1 (3 ml). The reaction was carried out at 20° C. for 4 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5436-4 (200 mg, brown solid), yield: 76.90%. LCMS (ESI) m/z calcd. for C35H34FN5O7 [M+H]+ 656.2; found 656.2. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound TPD5452-2 (100 mg, 0.1525 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (85.46 mg, 0.183 mmol), HATU (115.97 mg, 0.305 mmol), triethylamine (30.86 mg, 0.305 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=6/1 to afford compound TPD005452 (28.2 mg, light yellow solid, purity 95.282%), yield: 16.52%; LCMS (ESI) m/z calcd. for C57H62FN9O9S [M+H]+ 1068.4; found 534.7; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 10.06 (s, 1H), 8.94 (s, 1H), 8.67 (t, J=6.4 Hz, 1H), 8.44 (d, J=5.2 Hz, 1H), 8.23 (t, J=5.6 Hz, 1H), 7.76 (d, J=8.9 Hz, 1H), 7.70-7.63 (m, 3H), 7.46 (s, 1H), 7.44-7.37 (m, 4H), 7.33 (s, 1H), 7.23-7.21 (m, 2H), 7.18-7.13 (m, 2H), 6.40 (d, J=5.2 Hz, 1H), 5.17 (d, J=3.5 Hz, 1H), 4.54 (d, J=9.5 Hz, 1H), 4.48-4.46 (m, 1H), 4.44-4.41 (m, 1H), 4.36 (br. s., 1H), 4.25-4.19 (m, 1H), 3.93 (s, 3H), 3.78 (d, J=6.0 Hz, 1H), 3.69-3.63 (m, 4H), 2.67 (br. s., 2H), 2.43 (s, 3H), 2.01-1.84 (m, 6H), 1.47 (s, 4H), 1.23 (s, 1H), 0.94-0.92 (m, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5436-2 (300 mg, 0.5012 mmol), ethyl 5-aminopentanoate hydrochloride (109.26 mg, 0.6014 mmol), HATU (381.14 mg, 1.0024 mmol), triethylamine (101.43 mg, 1.0024 mmol), and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to afford compound TPD5453-1 (110 mg, brown solid, purity 82.845%), yield: 25.06%. LCMS (ESI) m/z calcd. for C40H44FN5O7 [M+H]+ 726.32; found 726.2; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 10.06 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.84 (t, J=5.5 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.66-7.62 (m, 2H), 7.47 (s, 1H), 7.33 (s, 1H), 7.23-7.20 (m, 2H), 7.18-7.13 (m, 2H), 6.40 (d, J=5.1 Hz, 1H), 4.08-4.03 (m, 2H), 3.95 (s, 3H), 3.62 (d, J=11.1 Hz, 2H), 3.09-3.04 (m, 2H), 2.89 (s, 4H), 2.70-2.63 (m, 2H), 2.30 (s, 1H), 1.79 (br. s., 4H), 1.47 (s, 4H), 1.43-1.38 (m, 2H), 1.18 (t, J=7.1 Hz, 3H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5453-1 (110 mg, 0.1516 mmol), lithium hydroxide monohydrate (12.72 mg, 0.3032 mmol) and methanol/water=2/1 (3 ml). The reaction was carried out at 25° C. for 4 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5453-2 (110 mg, brown solid), yield: 90.50%. LCMS (ESI) m/z calcd. for C38H40FN5O7 [M+H]+ 698.3; found 698.2. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound TPD5453-2 (100 mg, 0.1433 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (80.31 mg, 0.17196 mmol), HATU (108.97 g, 0.2866 mmol), triethylamine (29 mg, 0.2866 mmol) and DMF (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (6 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=8/1 to give compound TPD005453 (21.8 mg, light yellow solid, purity 97.007%), yield: 13.26%. LCMS (ESI) m/z calcd. for C60H68FN9O9S [M+H]+ 1110.5; found 555.8 (half peak); 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.07 (s, 1H), 8.98 (s, 1H), 8.59 (t, J=6.4 Hz, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.89 (d, J=9.4 Hz, 1H), 7.83 (t, J=5.4 Hz 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.47 (s, 1H), 7.43-7.37 (m, 4H), 7.32 (s, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.18-7.13 (m, 2H), 6.40 (d, J=5.1 Hz, 1H), 5.16 (d, J=3.4 Hz, 1H), 4.55 (d, J=9.4 Hz, 1H), 4.47-4.41 (m, 2H), 4.36 (br. s., 1H), 4.24-4.19 (m, 1H), 3.95 (s, 3H), 3.66-3.60 (m, 4H), 3.06-3.04 (m, 2H). 2.67-2.66 (m, 2H), 2.44 (s, 3H), 2.31-2.24 (m, 2H), 2.16-2.10 (m, 1H), 2.08-1.99 (m, 2H), 1.94-1.87 (m, 1H), 1.79 (br. s., 4H), 1.47 (s, 4H), 1.42-1.35 (m. 2H), 1.23 (s, 1H), 0.94 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound int-A (200 mg, 0.3228 mmol), tert-butyl piperazine-1-carboxylate (132.27 mg, 0.7101 mmol), cesium carbonate (210.35 mg, 0.6456 mmol), dioxane (10 ml), Pd2 (dba)3 (59.12 mg, 0.0645 mmol) and 1,1′-binaphthyl-2,2′-bisdiphenylphosphine (80.4 mg, 0.1291 mmol). The reaction was protected by nitrogen at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜50/1 to obtain the compound TPD5457-1 (63.33 mg, yellow solid, purity 88.841%), yield: 53.16%. LCMS (ESI) m/z calcd. for C36H38FN5O6 [M+H]+ 656.3; found 656.3; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 8.45 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.67-7.62 (m, 2H). 7.50 (s, 1H), 7.34 (s, 1H), 7.24-7.12 (m, 4H), 6.41 (d, J=5.2 Hz, 1H), 3.96 (s, 3H), 3.53 (br. s., 4H), 3.18-3.01 (m, 4H), 1.49-1.37 (m, 13H). It showed that the structure of the compound is correct.
A 25 ml single mouth vial was charged with compound TPD5457-1 (280 mg, 0.4270 mmol), DCM (2 ml) and TFA (2 ml). the reaction was carried out for 30 min at 25° C. The reaction solution was concentrated to dryness to give crude compound TPD5457-2 (440 mg, white solid) in 94.24% yield. The reaction solution was concentrated to dryness to give the crude compound TPD5457-2 (440 mg, white solid, purity 82.094%) in 94.24% yield. LCMS (ESI) m/z calcd. for C31H30FN5O4 [M+H]+ 556.2; found 556.1. The compound structure is confirmed.
A 100 ml single-necked vial was charged with compound TPD5457-2 (300 mg, 0.54 mmol), ethyl 4-bromobutyrate (210.66 mg, 1.08 mmol), potassium carbonate (223.9 mg, 1.62 mmol), potassium iodide (8.96 mg, 0.054 mmol), and acetonitrile (30 mL). the reaction was carried out for 16 h at 20° C. The reaction solution was extracted with EA (30 ml) for 3 times and extracted with EA (30 ml) for 3 times. The reaction solution was poured into water (50 ml) and extracted 3 times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and the residue was purified by preparative plate with unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD5457-3 (100 mg, yellow solid, purity 98.017%), yield: 27.11%. LCMS (ESI) m/z calcd. for C37H40FN5O6 [M+H]+ 670.3; found 670.1; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.69-7.58 (m, 2H). 7.48 (s, 1H), 7.31 (s, 1H), 7.24-7.09 (m, 4H), 6.40 (d, J=5.2 Hz, 1H), 4.07 (q, J=7.1 Hz, 2H), 3.95 (s, 3H), 3.14 (br. s., 4H), 2.56 (br. s., 4H), 2.38-2.32 (m 4H), 1.82-1.65 (m, 2H), 1.48 (s, 4H), 1.19 (t, J=7.1 Hz, 3H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5457-3 (100 mg, 0.1493 mmol), EtOH (0.5 ml), H2O (0.5 ml), and lithium hydroxide monohydrate (9.4 mg, 0.2239 mmol). the reaction was carried out at 25° C. for 2 h. The reaction solution was concentrated to dryness to give crude TPD5457-4 (100 mg, white solid). The reaction solution was concentrated to dryness to give the crude compound TPD5457-4 (100 mg, white solid, purity 91.427%), yield: 94.44%. LCMS (ESI) m/z calcd. for C35H36FN5O6 [M+H]+ 642.3; found 642.1. The compound structure is confirmed.
Compound TPD5457-4 (110 mg, 0.1714 mmol), DMF (6 ml), TEA (34.69 mg, 0.3428 mmol), HATU (130.34 mg, 0.3428 mmol) and (2S,4R)-1-((S)-2-(12-azacyclonyl)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (96.06 mg, 0.2056 mmol). The reaction was carried out at 25° C. for 16 hours. The reaction solution was poured into water (10 ml) and extracted three times with EA (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plates with the unfolding agent ratio of DCM/MeOH=6/1 to give compound TPD005457 (30.1 mg, off-white solid, purity 96.484%), yield: 16.04%. LCMS (ESI) m/z calcd. for C57H64FN9O8S [M+H]+ 1054.5; found 1054.7; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 8.98 (s, 1H), 8.58 (t, J=5.9 Hz, 1H), 8.44 (d, J=5.2 Hz, 1H). 7.92 (br. s., 1H), 7.76 (d, J=8.9 Hz, 2H), 7.88-7.83 (m, 2H), 7.48 (s, 1H), 7.44-7.37 (q, J=8.3 Hz, 4H), 7.32 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz 2H), 6.40 (d, J=5.1 Hz, 1H), 5.15 (d, J=3.2 Hz, 1H), 4.57 (d, J=9.4 Hz, 1H), 4.47-4.12 (m, 2H), 4.36 (br. s., 1H), 4.25-4.19 (m, 1H), 3.98-3.92 (m, 3H), 3.71-3.61 (m, 2H), 3.15 (br. s., 4H), 2.56 (br. s., 2H), 2.44 (s, 4H), 2.39-2.27 (m, 3H), 2.22 (d, J=7.1 Hz, 1H), 2.07-1.99 (m, 1H), 1.94-1.87 (m, 1H), 1.72 (br. s. 2H), 1.47 (s, 4H), 1.23 (s, 1H), 0.98-0.93 (m, 9H). The compound structure is confirmed.
A 100 ml triple-necked vial was sequentially charged with compound int-A (600 mg, 0.9685 mmol), piperidine-4-methanol (245.4 mg, 2.1307 mmol), cesium carbonate (631.11 mg, 1.937 mmol), BINAP (241.22 mg, 0.3874 mmol), Pd2 (dba)3 (177.37 mg, 0.1937) and 1,4-dioxane (30 ml). The reaction was carried out at 100° C. for 16 h under nitrogen protection. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5488-1 (360 mg, yellow solid, purity 63.777%), yield: 40.55%. LCMS (ESI) m/z calcd. for C33H33FN4O5 [M+H]+ 585.2; found 585.1; 1H NMR (400 MHz, CDCl3): δ=9.30 (s, 1H), 8.80 (s, 1H), 8.46 (d, J=5.2 Hz, 1H), 7.82 (d, J=8.8 Hz, 2H), 7.51-7.46 (m, 4H), 7.17 (d, J=8.8 Hz, 2H), 7.07-7.03 (m, 2H), 6.43 (d, J=5.2 Hz, 1H), 4.03 (s, 3H), 3.76-3.73 (m, 2H), 3.60 (d, J=6.4 Hz, 2H), 2.74-2.68 (m, 2H), 1.92-1.89 (m, 2H), 1.75-1.53 (m, 8H). It showed that the structure of the compound is correct.
A 50 ml triple-necked vial was charged with oxalyl chloride (86.85 mg, 0.6842 mmol) and DCM (5 ml). The reaction solution was cooled down to −78° C. under nitrogen protection and then DMSO (106.91 mg, 1.3684 mmol) was added dropwise. After half an hour of reaction at −78° C. a solution of DCM (5 ml) of compound TPD5488-2 (200 mg, 0.3421 mmol) was added dropwise. TEA (276.94 mg, 2.7368 mmol) was added dropwise after 1 h of reaction at −78° C. The reaction was carried out in a cold bath. After dropwise addition, the cold bath was withdrawn and the reaction was naturally warmed up for 1 hour. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 ml) and extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude compound TPD5488-2 (80 mg, yellow solid, purity 53.406%), yield: 21.43%. LCMS (ESI) m/z calcd. for C33H31FN4O5 [M+H]+ 583.2; found 583.0. The compound structure is confirmed.
A 250 ml three-necked flask was charged with oxalyl chloride (5.51 g, 43.4 mmol) and DCM (80 ml). The reaction solution was cooled down to −78° C. under nitrogen protection and then DMSO (6.784 g, 86.8 mmol) was added dropwise. After half an hour of reaction at −78° C. a solution of DCM (20 ml) of compound tert-butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (5, 21.7 mmol) was added dropwise. The reaction was carried out at −78° C. for 1 hour followed by dropwise addition of TEA (17.47 g, 173.6 mmol). After dropwise addition, the cold bath was removed and the reaction was naturally warmed up for 1 hour. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (100 ml), the organic phase was separated and the aqueous phase was extracted twice with DCM (80 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude compound TPD5488-4 (4 g, light yellow oil, purity 90%), yield: 72.81%. LCMS (ESI) m/z calcd. for C11H20N2O3 [M+H]+ 229.2; found 229.2 and 247.2; 1H NMR (400 MHz, DMSO_d6): δ=9.58 (s, 1H), 3.28 (br. s., 1H), 3.21 (s, 3H), 2.41-2.39 (m, 6H), 1.40 (s, 9H). The compound structure is confirmed.
To a 250 ml triple-necked vial was sequentially added compound TPD5488-4 (1.76 g, 7.71 mmol), DCE (50 ml), 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2 g, 7.71 mmol) and sodium triacetoxyborohydride (4.08 g, 19.2 mmol). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. The reaction solution was decanted into saturated aqueous sodium bicarbonate solution (50 ml). The organic phase was separated and the aqueous phase was extracted twice with DCM (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=60/1˜40/1 to afford the compound TPD5488-5 (1.78 g, white solid, purity 92.874%), yield: 42.21%. LCMS (ESI) m/z calcd. for C24H33N5O5 [M+H]+ 472.3; found 472.1; 1H NMR (400 MHz, CDCl3): δ=8.20 (s, 1H), 7.39 (t, J=7.7 Hz, 1H), 7.30 (d, J=7.5 Hz, 1H), 6.80 (d, J=7.9 Hz, 1H), 5.26 (dd, J=13.3, 5.1 Hz, 1H), 4.33 (d, J=15.4 Hz. 1H), 4.25 (br. s., 1H), 4.16 (d, J=15.3 Hz, 1H), 3.46 (s, 4H), 3.29 (dd, J=10.4, 5.1 Hz, 2H), 2.96-2.85 (m, 2H), 2.71 (t, J=5.4 Hz, 2H), 2.48-2.32 (m, 5H), 2.27-2.17 (m, 1H), 1.48 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5488-5 (500 mg, 1.0581 mmol) and hydrogen chloride 1,4-dioxane solution (10 ml, 4 N). The reaction was carried out at 20° C. for 4 h. After completion of the reaction, it was concentrated to dryness to obtain the crude compound TPD5488-6 (480 mg, white solid), yield: 98.09%. LCMS (ESI) m/z calcd. for C19H25N5O3 [M+H]+ 372.2; found 372.0. The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound TPD5488-2 (80 mg, 0.1373 mmol), DCE (4 ml), compound TPD5488-6 (56.1 mg, 0.151 mmol) and sodium triacetoxyborohydride (87.3 mg, 0.4119 mmol). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD005488 (15.6 mg, light yellow solid, purity 99.304%), yield: 12.02%. LCMS (ESI) m/z calcd. for C52H56FN9O7 [M+H]+ 938.4; found 938.4; 1H NMR (400 MHz, CDCl3): δ=9.38 (s, 1H), 8.80 (s, 1H), 8.46 (d, J=5.3 Hz, 1H), 8.23 (br. s., 1H), 7.62 (d, J=8.9 Hz, 2H), 7.54-7.44 (m, 4H), 7.37 (t, J=7.7 Hz, 1H). 7.29-7.26 (m, 1H), 7.16 (d, J=8.9 Hz, 2H), 7.05 (t, J=8.6 Hz, 2H), 6.76 (d, J=7.9 Hz, 1H), 6.43 (d, J=5.3 Hz, 1H), 5.21 (dd, J=13.2, 5.0 Hz, 1H), 4.39-4.30 (m 2H), 4.02 (s, 3H), 3.71 (d, J=11.5 Hz, 2H), 3.35 (br. s., 2H), 2.96-2.57 (m, 12H), 2.45-2.18 (m, 5H), 1.90 (d, J=12.7 Hz, 3H), 1.80-1.61 (m, 6H), 1.59-1.44 (m 3H). The compound structure is confirmed.
Compound TPD5432-2 (550 mg, 0.9673 mmol), 1-tert-butoxycarbonylpiperazine (900.8 mg, 4.8365 mmol), sodium triacetoxyborohydride (615.03 mg, 2.9019 mmol), tetraisopropyl titanate (1 ml), and DCE (10 ml) were added sequentially to a 50 ml triple-necked vial. The reaction mixture was stirred under nitrogen protection and at 40° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, water was added to the reaction solution and extracted three times with DCM (20 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give the residue. Purification of the residue by preparative TLC (DCM/MeOH=10/1) afforded the product compound TPD5494-1 (800 mg, yellow solid, purity 80.329%), yield: 89.92%. LCMS (ESI) m/z calcd. for C41H47FN6O6 [M+H]+ 739.4; found 739.5; 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.68 (s, 1H), 8.40 (d, J=5.2 Hz, 1H), 7.55 (d, J=8.9 Hz, 2H), 7.49-7.33 (m, 4H), 7.10 (d, J=8.9 Hz, 2H), 7.04-6.90 (m, 2H), 6.36 (d, J=5.2 Hz, 1H), 3.95 (s, 3H), 3.73-3.65 (m, 3H), 3.39-3.38 (m, 5H), 2.74 (br. s., 8H), 2.51 (br. s., 4H), 1.61-1.60 (m, 6H), 1.40 (s, 9H). The compound structure is confirmed.
A 25 ml triple-necked vial was charged with compound TPD5494-1 (500 mg, 0.6767 mmol), DCM (2.5 ml), trifluoroacetic acid (2.5 ml). The obtained mixture reacted at 20° C. for 3 hours. After completion of the reaction, the yellow solid product compound TPD5494-2 (400 mg, yellow solid, purity 90.161%) was obtained by direct spin drying, yield: 83.43%. LCMS (ESI) m/z calcd. for C36H39FN6O4 [M+H]+ 639.3; found 639.3. The compound structure is confirmed.
Compound TPD54945-2 (400 mg, 0.6262 mmol), HATU (357.15 mg, 0.9393 mmol), monoethyl adipate (109.08 mg, 0.6262 mmol) and TEA (316.83 mg, 3.131 mmol) were added sequentially in a 25 ml single mouth vial. The reaction solution reacted for 16 hours at 20° C. under nitrogen protection. After the reaction was completed, water was added to the mixture and the mixture was extracted three times with DCM (5 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give the residue. Purification of the residue by preparative TLC (DCM/MeOH=10/1) afforded the yellow solid product compound TPD5494-3 (50 mg, yellow solid, purity 95.357%), yield: 9.58%. LCMS (ESI) m/z calcd. for C44H51FN6O7 [M+H]+ 795.4; found 795.5. The compound structure is confirmed.
To a 25 ml single-necked vial was added compound TPD5494-3 (50 mg, 0.0629 mmol), THF/MeOH/H2O=1/1/1 (2 ml), LiOH·H2O (5.28 mg, 0.1258 mmol). The resulting mixture was stirred at 20° C. for 3 hours. The reaction was completed, and the reaction solution was spun dry to give the crude product compound TPD5494-4 (48 mg, white solid, purity 96.029%), yield: 95.55%. LCMS (ESI) m/z calcd. for C42H47FN6O7 [M+H]+ 767.3; found 767.5. The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added DMF (5 ml), compound TPD5494-4 (48 mg, 0.0626 mmol), HATU (35.7 mg, 0.0939 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-2-carboxamide hydrochloride (35.84 mg, 0.0626 mmol) and TEA (31.67 mg, 0.313 mmol). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was quenched with water. It was extracted three times with ethyl acetate (10 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to give the residue. Purification of the residue by preparative TLC (DCM/MeOH=10/1) afforded the yellow solid product compound TPD005494 (13.5 mg, yellow solid, purity 95.005%), yield: 17.41%. LCMS (ESI) m/z calcd. for C64H75FN10O9S [M+H]+ 1079.5; found 1179.6; 1H NMR (400 MHz, DMSO_d6): δ 10.18 (s, 1H), 10.05 (s, 1H), 8.98 (s, 1H), 8.57 (t, J=6.1 Hz, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.87 (d, J=9.3 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.0 Hz, 2H), 7.51-7.34 (m, 5H), 7.32 (s, 1H), 7.25-7.10 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 5.13 (d, J=3.4 Hz, 1H), 4.55 (d, J=9.3 Hz 1H), 4.44 (dd, J=15.5, 7.0 Hz, 2H), 4.35 (s, 1H), 4.21 (dd, J=15.9, 5.2 Hz, 1H), 3.94 (s, 3H), 3.66 (s, 4H), 3.44 (s, 4H), 2.64 (d, J=10.7 Hz, 2H), 2.45 (d, J=5.5 Hz, 5H), 2.29 (s, 3H), 2.19-1.97 (m, 3H), 1.94-1.81 (m, 3H), 1.63 (d, J=10.2 Hz, 2H), 1.47 (s, 9H), 1.23 (s, 1H), 0.94 (s, 9H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added the compound TPD5495-1 (2 g, 12.33 mmol), tert-butyl 2-hydroxyacetate (1.63 g, 12.33 mmol), sodium methanol (1.33 g, 24.66 mmol) and tetrahydrofuran (50 ml). The reaction was carried out under nitrogen protection at 20° C. for 48 hours. After completion of the reaction, the reaction solution was quenched with saturated ammonium chloride (100 ml). The mixture was extracted three times with ethyl acetate (100 ml). The combined organic phases were washed with saturated saline (100 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was spun dry to give a residue. The residue was purified by silica gel column with eluent ratio of PE/EtOAc=50/1˜15/1 to afford the compound TPD5495-2 (800 mg, colorless oil, purity 88.706%), yield: 19.55%. LCMS (ESI) m/z calcd. for C16H22O5 [M+H]+ 295.2; found 295.1 and 317.1 (plus Na); 1H NMR (400 MHz, CDCl3): δ=7.41-7.27 (m, 3H), 5.15 (s, 2H), 3.97 (s, 1H), 3.84 (t, J=6.4 Hz, 1H), 2.70 (t, J=6.4 Hz, 1H), 1.47 (s, 5H). The compound structure is confirmed.
To a 50 ml single-necked vial was added compound TPD5495-2 (200 mg, 0.68 mmol) and EA (2 ml). The addition of Pd/C (43 mg, 0.41 mmol) was continued and the reaction mixture was stirred at 20° C. for 2 h under H2 environment. The mixture was filtered and concentrated to dryness to obtain the crude compound TPD5495-2 (100 mg, yellow solid, 90% purity), yield: 64.86%. LCMS (ESI) m/z calcd. for C9H16O5 [M−H]− 203.1; found 203.2. The compound structure is confirmed.
A 50 ml single-necked vial was sequentially charged with compound TPD5494-2 (172 mg, 0.27 mmol), compound TPD5494-2 (55 mg, 0.27 mmol), HATU (154 mg, 0.40 mmol), triethylamine (136 mg, 1.35 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and extracted three times with EA (6 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=5/1 to give compound TPD005495-4 (70 mg, light yellow solid, purity 90.63%), yield: 61.20%. LCMS (ESI) m/z calcd. for C45H53FN6O8 [M+H]+ 825.3; found 825.4 and 826.4; 1H NMR (400 MHz, DMSO_d6): δ=10.17 (s, 1H), 10.05 (s, 1H), 8.44 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.65-7.62 (m, 2H), 7.46 (s, 1H), 7.32 (s, 1H). 7.22-7.20 (m, 2H), 7.17-7.13 (m, 2H), 6.39 (d, J=5.2 Hz, 1H), 3.96 (s, 5H), 3.67-3.63 (d, J=11.3 Hz, 4H), 3.45 (m, 5H), 2.59-2.54 (t, J=11.2 Hz, 8H), 1.88 (m 2H), 1.64-1.62 (m, 2H), 1.47 (m, 4H), 1.47 (s, 4H), 1.42 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound TPD5495-4 (100 mg, 0.12 mmol), DCM (3 ml) and trifluoroacetic acid (1 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5495-5 (90 mg, brown solid, purity 82.471%), yield: 79.62%. LCMS (ESI) m/z calcd. for C41H46FN6O8 [M+H]+ 769.3; found 385.2 and 769.3. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound TPD5495-5 (90 mg, 0.12 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (74 mg, 0.13 mmol), HATU (67 mg, 0.18 mmol), triethylamine (59 mg, 0.59 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 ml) and extracted three times with EA (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=8/1 to afford compound TPD005495 (15.5 mg, light yellow solid, purity 97.458%), yield: 10.93%. LCMS (ESI) m/z calcd. for C63H73FN10O10S [M+H]+ 1181.5; found 1181.3; 1H NMR (400 MHz, DMSO_d6): δ=10.16 (s, 1H), 10.05 (s, 1H), 8.99 (s, 1H), 8.59 (br. s., 1H), 8.44 (d, J=4.9 Hz, 1H), 7.74 (d, J=9.0 Hz, 2H), 7.46 (d, J=8.4 Hz, 3H). 7.40-7.38 (m, 5H), 7.22 (s, 1H), 7.17-7.13 (m, 4H), 6.40 (d, J=4.7 Hz, 1H), 5.14 (s, 1H), 4.58 (d, J=9.2 Hz, 1H), 4.39-4.35 (m, 3H), 4.27-4.25 (m, 2H), 3.94 (s, 5H), 3.73-3.64 (m, 6H), 3.46 (s, 4H), 2.66-2.63 (m, 4H), 2.49 (s, 3H), 2.07 (m, 2H), 2.02-1.82 (m, 4H), 1.59 (m, 3H), 1.47 (s, 4H), 1.23 (s, 1H), 0.93 (s, 9H). The compound structure is confirmed.
A 25 ml single-necked vial was charged sequentially with compound TPD5432-2 (350 mg, 0.6156 mmol), tert-butyl piperidine-4-carboxylate hydrochloride (651.67 mg, 3.078 mmol), sodium triacetoxyborohydride (391.41, 1.8468 mmol), tetraisopropyl titanate (1.5 ml) and 1,2-dichloroethane (5 ml). The reaction was carried out under nitrogen protection at 40° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (10 ml) and extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5496-1 (500 mg, brown solid, purity 75.139%), yield: 82.72%. LCMS (ESI) m/z calcd. for C42H48FN5O6 [M+H]+ 738.4; found 738.4; 1H NMR (400 MHz, DMSO_d6): δ=10.17 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.75 (d, J=8.9 Hz, 2H), 7.66-7.61 (m, 2H), 7.46 (s, 1H), 7.31 (s, 1H). 7.23-7.19 (m, 2H), 7.18-7.12 (m, 2H), 6.39 (d, J=5.2 Hz, 1H), 3.95 (s, 3H), 3.65 (d, J=11.3 Hz, 2H), 2.92-2.85 (m, 4H), 2.64 (t, J=11.2 Hz, 2H), 2.46-2.43 (m 2H), 1.84-1.76 (m, 4H), 1.69-1.63 (m, 4H), 1.47 (s, 4H), 1.39 (s, 10H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5496-1 (450 mg, 0.6099 mmol), DCM (2 ml) and trifluoroacetic acid (2 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5496-2 (450 mg, brown solid), yield: 81.41%. LCMS (ESI) m/z calcd. for C38H40FN5O6 [M+H]+682.3; found 682.2. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compounds TPD5496-2 (450 mg, 0.6601 mmol), H-Y-ABU-OTBU hydrochloride (155.01 mg, 0.7921 mmol), HATU (501.98 mg, 1.3202 mmol), triethylamine (133.59 mg, 1.3202 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5496-3 (250 mg, brown solid, purity 76.554%) yield: 35.24%. LCMS (ESI) m/z calcd. for C46H55FN6O7 [M+H]+ 823.4; found 823.4. The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound TPD5496-3 (250 mg, 0.3038 mmol), DCM (3 ml) and trifluoroacetic acid (2 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5496-4 (200 mg, brown solid), yield: 71.49%. LCMS (ESI) m/z calcd. for C42H47FN6O=[M+H]+ 767.4; found 767.3. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compounds TPD5496-4 (200 mg, 0.2608 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (146.16 mg, 0.3129 mmol), HATU (198.33 mg, 0.5216 mmol), triethylamine (52.78 mg, 0.5216 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=5/1 to give compound TPD005496 (10.4 mg, light yellow solid, purity 95.075%), yield: 3.22%. LCMS (ESI) m/z calcd. for C64H75FN10O9S [M+H]+ 1179.5; found 590.4 (half peak); 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.05 (s, 1H), 8.99 (s, 1H), 8.57 (br. s., 1H), 8.45 (d, J=4.9 Hz, 1H), 7.92 (d, J=9.0 Hz, 2H), 7.76 (d, J=8.4 Hz, 2H). 7.66-7.63 (m, 2H), 7.50-7.36 (m, 6H), 7.22-7.13 (m, 4H), 6.41 (d, J=4.7 Hz, 1H), 5.14 (s, 1H), 4.55 (d, J=9.2 Hz, 1H), 4.45-4.36 (m, 3H), 4.25-4.19 (m, 1H). 3.96 (s, 3H), 3.73-3.57 (m, 5H), 3.05 (s, 3H), 2.72-2.67 (m, 2H), 2.45 (s, 3H), 2.33-2.16 (m, 4H), 2.07-1.91 (m, 9H), 1.62 (s, 2H), 1.48 (s, 4H), 1.23 (br. s. 3H), 0.95 (s, 9H). The compound structure is confirmed.
A 25 ml triple-necked vial was sequentially charged with compound int-A (300 mg, 0.4842 mmol), tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (274.03 mg, 0.7263 mmol), sodium carbonate (102.65 mg, 0.9684 mmol), Pd(dppf)Cl2 (70.86 mg, 0.09684) and DMSO/H2O=5/1 (6 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5505-1 (350 mg, brown solid, purity 87.238%), yield: 87.48%. LCMS (ESI) m/z calcd. for C40H41FN6O6 [M+H]+ 721.3; found 721.2; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.06 (s, 1H), 8.52 (d, J=5.1 Hz, 1H), 8.44 (s, 1H), 8.27 (s, 1H), 8.16 (s, 1H), 7.78 (d, J=8.9 Hz, 2H), 7.67-7.62 (i, 3H), 7.26 (d, J=9.0 Hz, 2H), 7.18-7.14 (m, 2H), 6.48 (d, J=5.1 Hz, 1H), 4.48-4.40 (m, 2H), 4.10-4.05 (m, 6H), 2.07-2.04 (m, 2H), 1.92-1.82 (in, 2H). 1.48 (s, 4H), 1.43 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5505-1 (300 mg, 0.4162 mmol), DCM (4 ml) and trifluoroacetic acid (2 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5505-2 (320 mg, brown solid), yield: 77.73%. LCMS (ESI) m/z calcd. for C38H33FNO4 [M+H]+ 621.3; found 621.4. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound TPD5505-2 (320 mg, 0.5156 mmol), monoethyl adipate (107.77 mg, 0.61872 mmol), HATU (392.09 mg, 1.0312 mmol), triethylamine (104.35 mg, 1.0312 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to give compound TPD5505-3 (340 mg, brown solid, purity 94.211%), yield: 79.97%. LCMS (ESI) m/z calcd. for C43H45FN6O7 [M+H]+ 777.3; found 777.4; 1H NMR (400 MHz, DMSO_d6): δ=10.23 (s, 1H), 10.04 (s, 1H), 8.60 (d, J=5.5 Hz, 1H), 8.46 (s, 1H), 8.27 (s, 1H), 8.16 (s, 1H), 7.80 (d, J=9.0 Hz, 2H), 7.67-7.63 (m, 3H), 7.30-7.28 (m, 2H), 7.19-7.14 (m, 2H), 6.57 (d, J=5.5 Hz, 1H), 4.54-4.50 (m, 2H), 4.08-4.03 (m, 6H), 3.17 (s, 2H), 2.38 (t, J=6.9 Hz, 2H), 2.32 (t J=7.0 Hz, 2H), 2.12-2.06 (m, 2H), 1.99-1.93 (m, 1H), 1.86-1.80 (m, 1H), 1.58-1.54 (m, 4H), 1.48 (s, 4H), 1.18 (t, J=7.1 Hz, 3H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5505-3 (300 mg, 0.3862 mmol), lithium hydroxide monohydrate (32.41 mg, 0.7724 mmol) and methanol/water=2/1 (3 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5505-4 (300 mg, brown solid), yield: 85.24%. LCMS (ESI) m/z calcd. for C41H41FN6O7 [M+H]+ 749.3; found 749.2. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound TPD5505-4 (300 mg, 0.4006 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (224.51 mg, 0.48072 mmol), HATU (304.64 mg, 0.8012 mmol), triethylamine (81.07 mg, 0.8012 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to give compound TPD005505 (13.3 mg, white solid, purity 98.177%), yield: 2.80%. LCMS (ESI) m/z calcd. for C63H69FN10O9S [M+H]+ 1161.5; found 1161.6; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.05 (s, 1H), 8.98 (s, 1H), 8.59-8.52 (m, 2H), 8.43 (s, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 7.88 (d, J=9.4 Hz, 1H), 7.78 (d, J=8.9 Hz, 2H), 7.67-7.60 (m, 3H), 7.43-7.37 (m, 5H), 7.26 (d, J=9.0 Hz, 2H), 7.18-7.13 (m, 2H), 6.49 (d, J=5.3 Hz, 1H), 5.13 (s, 1H), 4.57-4.51 (m 3H), 4.46-4.41 (m, 2H), 4.35 (s, 1H), 4.24-4.19 (m, 1H), 4.04 (s, 4H), 3.65 (br. s., 2H), 3.23-3.17 (m, 1H), 2.70 (d, J=25.1 Hz, 1H), 2.44 (s, 4H), 2.39-2.35 (m, 2H), 2.33-2.28 (m, 1H), 2.18-1.99 (m, 4H), 1.95-1.75 (m, 3H), 1.56-1.45 (m, 8H), 0.94 (s, 9H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound TPD5706-1 (5 g, 21.3 mmol), tert-butyl acrylate (4.10 g, 31.95 mmol), potassium tert-butoxide (0.48 g, 4.26 mmol) and 1,4-dioxane (50 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and poured into saturated aqueous ammonium chloride solution (100 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude compound TPD5706-2 (1.4 g, yellow solid, purity 20.909%), yield: 3.76%. LCMS (ESI) m/z calcd. for C20H29NO5 [M+H]+ 364.2; found 308.1. The compound structure is confirmed.
Compound TPD5706-2 (1.4 g, 3.9 mmol), THF (15 ml), and Pd/C (0.14 g, 10% Pd, 50% wet) were added sequentially in a 50 ml single-necked vial. The reaction was carried out for 4 h at 25° C. under hydrogen protection. After completion of the reaction, the reaction solution was filtered and concentrated to dryness to afford the crude compound TPD5706-3 (900 mg, brown oil, purity 37.009%), yield: 33.33%; LCMS (ESI) m/z calcd. for C12H23NO3 [M+H]+ 230.2; found 230.1; 1H NMR (400 MHz, DMSO_d6): δ=3.58 (t, J=6.0 Hz, 2H), 3.29-3.25 (m, 1H), 3.13 (s, 2H), 2.87 (d, J=12.0 Hz, 2H), 2.44-2.41 (m, 1H), 2.38 (t, J=6.4 Hz, 2H), 1.78-1.74 (m, 2H), 1.40 (s, 9H), 1.26-1.17 (m, 2H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compounds TPD5706-3 (800 mg, 3.4886 mmol), int-A (2.161 g, 3.4886 mmol), cesium carbonate (2.27331 g, 6.9772 mmol), BINAP (868.9 mg, 1.3954 mmol), Pd2 (dba)3 (638.92 mg, 0.6977 mmol) and 1,4-dioxane (40 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=150/1˜30/1 to afford the compound TPD5706-4 (470 mg, brown solid, purity 72.189%), yield: 13.92%. LCMS (ESI) m/z calcd. for C39H43FN4O7 [M+H]+ 699.3; found 350.2 and 699.2; 1H NMR (400 MHz, DMSO_d6): δ 10.19 (s, 1H), 10.07 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.66-7.63 (m, 2H). 7.47 (s, 1H), 7.23-7.13 (m, 5H), 6.39 (d, J=5.2 Hz, 1H), 3.95 (s, 3H), 3.66 (t, J=6.0 Hz, 2H), 3.50 (s, 1H), 3.39 (s, 2H), 2.89 (t, J=9.2 Hz, 2H), 2.44 (t, J=6.0 Hz, 2H), 1.96 (s, 2H), 1.65-1.63 (m, 2H), 1.47 (s, 4H), 1.42 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5706-4 (400 mg, 0.5724 mmol), DCM (2 ml) and trifluoroacetic acid (2 ml). The reaction was carried out at 25° C. for 4 h. After completion of the reaction, it was concentrated to dryness to obtain the crude compound TPD5706-5 (380 mg, brown solid), yield: 57.95%. LCMS (ESI) m/z calcd. for C35H35FN4O7 [M+H]+ 643.3; found 643.4. The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5706-5 (100 mg, 0.1556 mmol), DCM (10 ml) and 1-chloro-N,N,2-trimethylpropenylamine (41.58 mg, 0.3112 mmol), and the mixture was stirred for 0.5 hr at 25° C. The mixture was then mixed with pyridine (36.92 mg, 0.4668 mmol) and 5-amino-2-(2,6-dioxopiperidin-3-yl). Then a DCM mixture of pyridine (36.92 mg, 0.4668 mmol) and 5-amino-2-(2,6-dioxopiperidin-3-yl)isodihydroindole-1,3-dione (85.04 mg, 0.3112 mmol) was added. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. The reaction solution was decanted into water (10 ml). The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-55/8 min to afford the compound TPD005706 (11.6 mg, yellow solid, purity 98.769%) in the following yield: 7.97%. LCMS (ESI) m/z calcd. for C48H44FN7O10 [M+H]+ 898.3; found 449.6 and 898.3; 1H NMR (400 MHz, DMSO_d6): δ=11.13 (s, 1H), 10.67 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.42 (d, J=4.9 Hz. 1H), 8.28 (s, 1H), 8.24 (s, 0.5H), 7.93 (d, J=7.6 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.66-7.63 (m, 2H), 7.45 (s, 1H), 7.25-7.13 (m, 5H). 6.38 (d, J=4.8 Hz, 1H), 5.15-5.10 (m, 1H), 3.93 (s, 3H), 3.82-3.80 (m, 2H), 3.55 (br. s., 2H), 2.89-2.84 (m, 3H), 2.69-2.66 (m, 2H), 2.62-2.57 (m, 2H), 2.01-1.98 (m, 3H), 1.65-1.63 (m, 2H), 1.47 (s, 3H), 1.23 (s, 2H). The compound structure is confirmed.
To a 500 ml autoclave was sequentially added compound TPD5714-1 (5 g, 28.6 mmol), palladium acetate (1.28 g, 5.72 mmol), dppf (3.17 g, 5.72 mmol)methanol (50 ml), triethylamine (10 ml) and DMF (150 ml). Carbonmonoxidegas at 0.4 MPa was flushed in and the reaction was carried out at 80° C. for 16 hours. After the reaction was completed, the reaction solution was filtered and the filtrate was spin-dried to obtain the residue. The residue was purified by silica gel column with the eluent ratio of DCM/MeOH=80/1˜60/1 to obtain the compound TPD5714-2 (2.4 g, brown solid, purity 70%), yield: 38.11%. LCMS (ESI) m/z calcd. for C5H6N4O2 [M+H]+ 155.1; found 155.1; 1H NMR (400 MHz, CDCl3): δ=8.83 (s, 1H), 6.02 (br. s, 2H), 4.06 (s, 3H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added the compounds TPD5714-2 (1.8 g, 7.82 mmol), ACN (30 ml), tert-butyl nitrite (4.84 g, 46.91 mmol) and dimethyl disulfide (7.36 g, 78.19 mmol). The reaction was carried out under nitrogen protection at 60° C. for 16 hours. After the reaction was completed, the reaction solution was concentrated to dryness. The residue was purified by silica gel column with eluent ratio of PE/EA=10/1˜5/1 to afford the compound TPD5714-2 (800 mg, light yellow oil, purity 83.445%), yield: 32.68%. LCMS (ESI) m/z calcd. for C9H16O5[M−H]+186.0; found 186.0; 1H NMR (400 MHz, CDCl3): δ=8.95 (s, 1H), 4.08 (s, 3H), 2.83 (s, 3H). The compound structure is confirmed.
Compound TPD5494-3 (2 g, 10.80 mmol), piperidinemethanol (1.49 g, 12.94 mmol) and ACN (40 ml) were added sequentially in a 50 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 5 hours. After the reaction was completed, the reaction solution was poured into water (150 ml) and extracted three times with EA (80 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of PE/EA=5/1˜1/1 to give compound TPD005714-4 (310 mg, light yellow solid, purity 79.48%), yield: 9.26%. LCMS (ESI) m/z calcd. for C11H16N4O3 [M+H]+ 253.1; found 253.2; 1H NMR (400 MHz, CDCl3): δ=8.69 (s, 1H), 4.00 (s, 3H), 3.56 (d, J=5.9 Hz, 2H), 3.06 (br. s, 2H), 1.91-1.85 (m, 3H), 1.49-1.41 (m, 1H), 1.35-1.23 (m, 3H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5714-4 (140 mg, 0.56 mmol), LiOH (47 mg, 1.11 mmol) and THF (3 ml), MeOH (3 ml), H2O (3 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5714-5 (81 mg, yellow solid, purity 88.152%), yield: 54.00%. LCMS (ESI) m/z calcd. for C10H14N4O3 [M+H]+ 239.1; found 239.1. The compound structure is confirmed.
Compound TPD5714-5 (170 mg, 0.71 mmol), int-B (347 mg, 0.71 mmol), HATU (407 mg, 1.07 mmol), TEA (369 mg, 2.85 mmol), and DMF (3 mL) were added sequentially to a 50 ml single-necked vial. The reaction was carried out at 25° C. for 16 h. After completion of the reaction, the reaction solution was poured into water (10 ml) and extracted three times with EA (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. High pressure preparation afforded the compound TPD5714-6 (85 mg, brown solid, purity 88.183%), yield: 14.87%. LCMS (ESI) m/z calcd. for C37H35FN8O6 [M+H]+ 707.3; found 707.4. The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5714-6 (70 mg, 0.10 mmol), Dess-Martin oxidizer (63 mg, 0.15 mmol) and DCM (3 ml). The reaction was carried out at 25° C. for 0.5 h. After completion of the reaction, the reaction solution was poured into saturated sodium bicarbonate solution (10 ml) and extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude compound TPD5714-7 (35 mg, brown solid, purity 65.428%) was obtained, yield: 32.83%. LCMS (ESI) m/z calcd. for C37H34FN8O6 [M+H]+ 705.3; found 705.1. The compound structure is confirmed.
Compound TPD5714-7 (35 mg, 0.05 mmol), 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (18.8 mg, 0.05 mmol), sodium triacetoxyborohydride (21 mg, 0.09 mmol), and DCE (2 ml) were added sequentially to 25 ml of single-necked vials. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was decanted into sodium bicarbonate (10 ml) and extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=15/1 to give compound TPD005714 (3.0 mg, light yellow solid, purity 88.487%), yield: 5.23%. LCMS (ESI) m/z calcd. for Cs4H51FN12O9 [M+H]+ 1031.39; found 1031.60; 1H NMR (400 MHz, CDCl3:CD3 OD=10:1): δ=9.39 (s, 1H), 8.97 (s, 1H), 8.70 (d, J=6.0 Hz, 1H), 7.76-7.73 (m, 4H), 7.56-7.49 (m, 3H), 7.23-7.19 (m, 2H), 7.15-7.11 (m 1H), 7.08-7.02 (m, 2H), 6.76-6.72 (m, 1H), 5.36-5.34 (m, 1H), 4.19 (s, 3H), 3.70-3.62 (m, 2H), 3.39-3.35 (m, 4H), 3.17-3.14 (m, 2H), 2.83-2.80 (m, 4H). 2.64-2.61 (m, 1H), 2.16-2.08 (m, 3H), 2.05-1.99 (m, 3H), 1.74-1.69 (m, 4H), 1.63-1.56 (m, 4H). The compound structure is confirmed.
To a 50 ml triple-necked vial was sequentially added compound TPD5735-6 (500 mg, 1.3199 mmol), 4-formyl-N-CBZ piperidine (359.04 mg, 1.45189 mmol) and 1,2-dichloroethane (8 ml). Then sodium triacetoxyborohydride (839.22 mg, 3.9597 mmol) was added at room temperature. The reaction was then carried out at 25° C. for 16 hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 ml) and extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio of DCM/MeOH=20/1 to give the crude compound TPD5734-6 (400 mg, yellow solid, 60% purity), yield: 31.7%. LCMS (ESI) m/z calcd. for C31H35N5O6 [M+H]+ 574.26; found 574.5. The compound structure is confirmed.
Compound TPD5734-6 (800 mg, 1.3946 mmol), palladium hydroxide (300 mg) and ethyl acetate (20 ml) were added sequentially to a 50 ml triple-necked vial. The reaction was then carried out at 30° C. under 20 psi hydrogen for 3 hours. The reaction solution was filtered, the filter cake was washed with ethyl acetate, and the filtrate was concentrated to dryness to obtain the crude compound TPD5734-7 (500 mg, yellow solid, purity 60%), yield: 48.95%. LCMS (ESI) m/z calcd. for C23H29N5O4 [M+H]+ 440.22; found 440.1. The compound structure is confirmed.
A 25 ml triple-necked vial was charged with int B (100 mg, 0.2056 mmol), triethylamine (83.22 mg, 0.8224 mmol) and dichloromethane (3 ml), tetrahydrofuran (3 ml). Then a solution of acryloyl chloride (37.22 mg, 0.4112 mmol) in dichloromethane (0.5 ml) was added dropwise at 0° C. The reaction was then carried out at 0° C. for 2 hours. The reaction solution was decanted into water (10 ml). It was extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=15/1 to give compound TPD5734-8 (50 mg, yellow solid, purity 99.242%), yield: 44.65%. LCMS (ESI) m/z calcd. for C30H25FN4O5 [M+H]+ 541.18; found 542.2; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.06 (s, 1H), 9.72 (s, 1H), 8.88 (s, 1H), 8.52 (s, 1H), 7.78 (d, J=8.8 Hz, 2H), 7.66-7.63 (m, 2H), 7.61 (s, 1H), 7.25 (d, J=8.8 Hz, 2H), 7.16 (t, J=8.8 Hz, 2H), 6.90-6.83 (m, 1H), 6.47 (d, J=5.2 Hz, 1H), 6.36 (d, J=16.8 Hz, 1H), 5.81 (d, J=11.2 Hz, 1H), 4.05 (s, 3H), 1.48 (s, 4H). The compound structure is confirmed.
To a 25 ml triple-necked vial was sequentially added compound TPD5734-8 (45 mg, 0.0832 mmol), acetonitrile (3 ml), compound TPD5734-7 (36.57 mg, 0.0832 mmol) and DBU (38 mg, 0.2496 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. The reaction solution was decanted into water (10 ml). It was extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005734 (10.2 mg, yellow solid, purity 97.581%), yield: 12.2%. LCMS (ESI) m/z calcd. for C53H54FN9O9 [M+H]+ 980.40; found 980.3; 1H NMR (400 MHz, DMSO_d6): δ=11.09 (s, 1H), 10.82 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.92 (s, 1H), 8.49 (d, J=4.8 Hz, 1H), 7.77 (d, J=8.4 Hz, 2H), 7.69-7.63 (m, 3H)), 7.59 (s, 1H), 7.35 (s, 1H), 7.27-7.23 (m, 3H), 7.16 (t, J=8.8 Hz, 2H), 6.45 (d, J=4.8 Hz, 1H), 5.10-5.04 (m, 1H), 4.05 (s, 3H), 3.45 (s, 3H), 3.05 (br. s., 2H). 2.93-2.83 (m, 2H), 2.64 (br. s., 8H), 2.25 (br. s., 2H), 2.01 (br. s., 3H), 1.80 (d, J=10.4 Hz, 2H), 1.63 (br. s., 1H), 1.47 (s, 4H), 1.31-1.24 (m, 4H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound TPD5735-4 (4 g, 0.0145 mol), 1-tert-butoxycarbonylpiperazine (3.26 g, 0.0174 mol), triethylamine (2.93 g, 0.029 mol) and dimethylsulfoxide (50 ml). The reaction was then carried out at 90° C. for 16 hours. The reaction solution was poured into water (100 ml), filtered, the filter cake was washed with a small amount of water, and the filter cake was purified by silica gel chromatography column with eluent and ratio of PE/EtOAc=3/1˜1/1 to obtain the compound TPD5735-5 (2.5 g, yellow solid, purity 98.803%), yield: 38.62%. LCMS(ESI) m/z calcd. for C22H26N4O6 [M+H]+ 443.19; found 343.0, 387.0; 1H NMR (400 MHz, DMSO_d6): δ=11.09 (s, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.35 (s, 1H), 7.25 (d, J=8.8 Hz, 1H), 5.10-5.06 (m, 1H), 3.47 (s, 8H), 2.93-2.84 (m, 1H), 2.61-2.50 (m, 2H), 2.03-1.99 (m, 1H), 1.43 (s, 9H). The compound structure is confirmed.
To a 100 ml triple-necked flask was sequentially added compound TPD5735-5 (2.5 g, 0.0056 mol), ethyl acetate (10 ml) and ethyl acetate hydrochloric acid gas (50 ml). The reaction was then carried out at 25° C. for 16 hours. The reaction solution was filtered and the filter cake was dried to obtain the crude compound TPD5735-6 (1.9 g, yellow solid, purity 75.193%), yield: 67.86%. LCMS (ESI) m/z calcd. for C17H18N4O4 [M+H]+ 343.13; found 343.0; 1H NMR (400 MHz, DMSO_d6): δ=11.10 (s, 1H), 9.28 (s, 2H), 7.75 (d, J=8.4 Hz, 1H), 7.45 (s, 1H), 7.33 (d, J=8.8 Hz, 1H), 5.12-5.07 (m, 1H), 3.57 (s, 4H), 3.21 (s, 4H), 2.93-2.84 (m, 1H), 2.67-2.53 (m, 2H), 2.04-1.99 (m, 1H). The compound structure is confirmed.
Compound int B (100 mg, 0.2056 mmol), triethylamine (83.22 mg, 0.8224 mmol), tetrahydrofuran (3 mL), and dichloromethane (3 mL) were added sequentially to a 25 mL triple-necked vial. A solution of chloroacetyl chloride (34.85 mg, 0.3084 mmol) in dichloromethane (0.5 mL) was then added dropwise at 0° C. The reaction was then carried out at 0° C. for 2 hours. The reaction solution was poured into water (10 ml) and extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=15/1 to afford the compound TPD5735-9 (70 mg, yellow solid, purity 98.963%), yield: 59.85%. LCMS (ESI) m/z calcd. for C29H24ClFN4O5 [M+H]+ 563.14; found 563.0; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.05 (s, 1H), 9.84 (s, 1H), 8.79 (s, 1H), 8.52 (d, J=5.2 Hz, 1H), 7.77 (d, J=8.8 Hz, 2H), 7.66-7.63 (m, 3H), 7.25 (d, J=8.8 Hz, 2H), 7.15 (t, J=8.8 Hz, 2H), 6.48 (d, J=4.8 Hz, 1H), 4.53 (s, 2H), 4.06 (s, 3H), 1.48 (s, 4H). The compound structure is confirmed.
To a 25 ml triple-necked vial was sequentially added compound TPD5735-9 (65 mg, 0.1155 mmol), compound TPD5734-7 (50.76 mg, 0.1155 mmol), potassium carbonate (31.88 mg, 0.231 mmol), potassium iodide (1.92 mg, 0.01155 mmol) and acetonitrile (3 ml). The reaction was then carried out at 25° C. for 16 hours. The reaction solution was filtered and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=15/1 to give compound TPD005735 (8.85 mg, yellow solid, purity 95.813%), yield: 7.62%. LCMS (ESI) m/z calcd. for C52H52FN9O9 [M+H]+ 966.39, 967.39; found 966.3, 967.3; 1H NMR (400 MHz, DMSO_d6): δ 11.09 (s, 1H), 10.27 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.86 (s, 1H), 8.51 (d, J=5.2 Hz, 1H), 7.78 (d, J=8.8 Hz, 2H)), 7.69-7.63 (m, 4H), 7.35 (s, 1H), 7.28-7.24 (m, 3H), 7.16 (t, J=9.2 Hz, 2H), 6.47 (d, J=5.2 Hz, 1H), 5.10-5.05 (dd, J=5.2, 5.6 Hz, 1H), 4.09 (s, 3H), 3.45 (s 4H), 3.21 (s, 2H), 2.92-2.85 (m, 3H), 2.67-2.55 (m, 5H), 2.30-2.25 (m, 4H), 2.03-2.00 (m, 1H), 1.82 (d, J=10.4 Hz, 2H), 1.62 (br. s., 1H), 1.48 (s, 4H), 1.30-1.22 (m, 3H). The compound structure is confirmed.
To a 100 ml single-necked vial was added the compound TPD5736-1 (5 g, 0.027 mmol), benzyl 4-formylpiperidine-1-carboxylate (6.68 g, 0.027 mmol), sodium triacetoxyborohydride (11.44 g, 0.054 mmol), and ethylene dichloride (100 ml) in this order. the reaction was carried out at 15° C. for 16 h. After completion of the reaction, the system was poured into water (50 ml) and extracted with dichloromethane (50 ml) for three times. After completion of the reaction the system was poured into water (50 ml) and extracted three times with dichloromethane (50 ml). The combined organic phases were washed with water (50 ml), saturated saline (50 ml), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column with eluent ratio of PE/EA=10/1˜3/1 to obtain the compound TPD5736-2 (6.2 g, colorless oil, purity 93.260), yield: 51.48%. LCMS (ESI) m/z calcd. for C24H36N2O4 [M+H]+ 417.3; found 417.2; 1H NMR (400 MHz, CDCl3): δ=7.41-7.28 (m, 5H), 5.12 (s, 2H), 4.16 (s, 2H), 2.78 (d, J=11.3 Hz, 4H), 2.24-2.07 (m, 3H), 1.94 (t, J=10.9 Hz, 2H), 1.82 (d, J=10.6 Hz, 2H). 1.75-1.63 (m, 4H), 1.60 (s, 1H), 1.43 (s, 9H), 1.08 (d, J=11.7 Hz, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial was sequentially added compound TPD5736-2 (2.0 g, 4.8 mmol), THF (40 ml) and palladium charcoal (0.4 g, 10% content, 50% water content). The system reacted under 1 atm hydrogen at 15° C. for 16 hours. After completion of the reaction filtration was carried out with diatomaceous earth and washed with THF. The filtrate was concentrated under reduced pressure to give the compound TPD5736-3 (1.2 g, white solid, purity 55.619%), yield: 50.00%. LCMS (ESI) m/z calcd. for C16H30N2O2 [M+H]+ 283.2; found 283.1; 1H NMR (400 MHz, CDCl3): δ=3.06 (d, J=12.1 Hz, 2H), 2.80 (d, J=11.5 Hz, 2H), 2.58 (td, J=12.1, 2.0 Hz, 2H), 2.19-2.14 (m, 1H), 2.12 (d, J=7.1 Hz, 2H), 1.92 (t, J=11.3 Hz, 2H), 1.82 (d, J=10.0 Hz, 2H), 1.74-1.88 (m, 4H), 1.82-1.57 (m, 3.8 Hz, 1H), 1.43 (s, 8H), 1.12-1.02 (m, 2H). The compound structure is confirmed.
Compound TPD5736-3 (562.36 mg, 1.991 mmol) was added to a 40 ml single-necked vial, 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindole-1,3-dione (500 mg, 1.810 mmol), DIEA (701.85 mg, 5.431 mmol), and DMSO (5 ml). 100° C. reacted for 16 hours. After the reaction was completed it was cooled to room temperature and then poured into cold water (20 ml) and filtered the filter cake was purified by silica gel column with eluent ratio of PE/EA=5/1˜2/1 to obtain the compound TPD5736-4 (750 mg, yellow solid, purity 91.595%), yield: 70.46%. LCMS (ESI) m/z calcd. for C29H38N4O6 [M+H]+ 539.3; found 229.2 and 539.1; 1H NMR (400 MHz, CDCl3): δ=8.25 (s, 1H), 7.66 (d, J=8.6 Hz, 1H), 7.28 (d, J=2.2 Hz, 1H), 7.05-7.02 (m, 1H), 4.96-4.92 (m, 1H), 4.02-3.86 (m, 2H), 2.98-2.92 (m, 2H), 2.88-2.73 (m, 4H), 2.26-2.06 (m, 4H), 1.97 (t, J=10.8 Hz, 2H), 1.86 (t, J=13.1 Hz, 4H), 1.77-1.53 (m, 4H), 1.44 (s, 9H), 1.29-1.21 (m, 2H). The compound structure is confirmed.
A 50 ml single-necked vial was charged with compound TPD5736-4 (500 mg, 0.928 mmol) and HCl/EA (4N, 20 ml). the reaction was carried out at 20° C. for 16 h and then warmed up to 60° C. for another 8 h. After the reaction, the system was concentrated to dryness under reduced pressure to give compound TPD5736-5 (450 mg, yellow solid), yield: 79.45%. After completion of the reaction the system was concentrated to dryness under reduced pressure to give the compound TPD5736-5 (450 mg, yellow solid, purity 85.053%), yield: 79.45%. LCMS (ESI) m/z calcd. for C25H30N4O6 [M+H]+ 483.2; found 483.0. The compound structure is confirmed.
Compound TPD5736-5 (100 mg, 0.2072 mmol), int-C (103.71 mg, 0.2072 mmol), HATU (157.57 mg, 0.4144 mmol), triethylamine (41.93 mg, 0.4144 mmol), and DMF (3 ml) were added sequentially to a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (3 ml) and extracted three times with EA (1 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005736 (25 mg, yellow solid, purity 97.359%), yield: 12.16%. LCMS (ESI) m/z calcd. for C53H53FN8O9 [M+H]+ 965.4; found 965.3; 1H NMR (400 MHz, DMSO_d6): δ=11.09 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.51 (d, J=5.2 Hz, 1H), 8.40 (s, 1H), 7.79-7.64 (m, 6H), 7.54 (s, 1H), 7.31-7.13 (m, 6H), 6.52 (d, J=4.8 Hz, 1H), 5.09-5.04 (m, 1H), 4.40 (s, 2H), 4.05 (d, J=12.4 Hz, 2H), 3.98 (s, 3H), 3.00-2.88 (m, 5H), 2.14 (s, 2H), 1.99 (s, 2H), 1.90 (s, 2H), 1.79 (d, J=12.8 Hz, 4H), 1.79 (d, J=12.8 Hz, 4H), 1.68 (br. s., 2H), 1.47 (s, 4H), 1.23-1.16 (m, 5H). The compound structure is confirmed.
To a 50 ml triple-necked vial was sequentially added compound int-A (500 mg, 0.807 mmol), zinc cyanide (189.52 mg, 1.614 mmol), tetrakis(triphenylphosphine)palladium (93.25 mg, 0.0807 mmol) and N,N-dimethylformamide (10 ml). The reaction was then carried out at 110° C. for 3 hours. The reaction solution was poured into water (30 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent and ratio of PE/EtOAc=3/1˜1/1 to give compound int-C-1 (250 mg, yellow solid, purity 98.176%), yield: 61.26%. LCMS (ESI) m/z calcd. for C28H21FN4O4 [M+H]+ 497.15; found 497.6; 1H NMR (400 MHz, DMSO_d6): δ=10.23 (s, 1H), 10.49 (s, 1H), 8.66 (d, J=4.8 Hz, 1H), 8.57 (s, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.77 (s, 1H), 7.66-7.63 (m, 2H), 7.28 (d, J=8.8 Hz=8.8 Hz, 2H), 7.16 (t, J=8.8 Hz, 2H), 6.66 (d, J=5.2 Hz, 1H), 4.08 (s, 3H), 1.48 (s, 4H). The compound structure is confirmed.
Compound int-C-1 (250 mg, 0.5035 mmol), ethanol (5 ml) and ammonia (3 ml) were sequentially added to a 50 ml single-necked vial. Then Raney nickel (100 mg) was added. The reaction was then carried out under a hydrogen balloon at 20° C. for 4 hours. The reaction solution was filtered and the filtrate was concentrated to dryness to give compound int-C (150 mg, white solid, purity 99.425%), yield: 59.19%. LCMS (ESI) m/z calcd. for C28H25FN4O4 [M+H]+ 501.19; found 501.4; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (br. s., 1H), 10.05 (br. s., 1H), 8.51 (d, J=5.2 Hz, 1H), 7.80 (s, 1H), 7.77 (d, J=8.8 Hz, 2H), 7.66-7.63 (m, 2H), 7.50 (s, 1H), 7.23 (d, J=8.8 Hz, 2H). 7.15 (t, J=8.8 Hz, 2H), 6.50 (d, J=5.2 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 2H), 1.47 (s, 4H). The compound structure is confirmed.
To a 50 ml triple-necked vial was sequentially added compound 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindole-1,3-dione (200 mg, 0.7241 mmol), tert-butyl 2-(piperidin-1-yl)acetate, DIEA (280.75 mg, 2.1723 mmol) and DMSO solution (10 ml). The reaction was carried out under nitrogen protection at 90° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (20 ml) to precipitate a yellow solid, filtered and dissolved the yellow solid with dichloromethane, dried over anhydrous sodium sulfate and spun dry to give the compound TPD5745-13 (280 mg, yellow solid, purity 60.632%), yield: 79.50%. LCMS (ESI) m/z calcd. for C23H28N4O6 [M+H]+ 457.5; found 457.1. The compound structure is confirmed.
To a 50 ml triple-necked vial was sequentially added compound TPD5745-13 (200 mg, 0.4381 mmol), 3N hydrogen chloride 1,4-dioxane solution (10 ml). The reaction was carried out under nitrogen protection at 15° C. for 4 hours. After completion of the reaction, the reaction solution was concentrated to dryness to give the compound TPD5745-14 (150 mg, yellow solid, purity 92.973%), yield: 79.50%. LCMS (ESI) m/z calcd. for C19H20N4O6 [M+H]+ 401.14; found 401.0. The compound structure is confirmed.
A 100 ml single mouth flask was charged with sulfoxide chloride (20 ml) and compound TPD5745-9 (1 g, 0.0036 mol) was added. The reaction solution was warmed up to 70° C. for 3 hours under nitrogen protection. The reaction solution was concentrated to dryness to obtain the crude compound TPD5745-10 (0.9 g, light yellow oil, purity 93.903%), yield: 72.81%. LCMS (ESI) m/z calcd. for C15H18ClNO3 [M-Cl+OCH3+H]+292.35; found 292.0. The compound structure is confirmed.
A 250 ml three-necked vial was charged with int-B (450 mg, 0.925 mmol) and TEA (280.80 mg, 2.7750 mmol) in DCM (55 ml). The reaction solution was cooled down to 0° C. under nitrogen protection followed by dropwise addition of compound TPD-5745-11 (410.37 mg, 1.3875 mmol) dissolved in DCM (5 ml). After dropwise addition the reaction reacted at 15° C. for one and a half hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (30 ml) and extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5745-11 (360 mg, white solid, purity 87.842%) in the yield: 45.84%. LCMS (ESI) m/z calcd. for C42H40FN5O7 [M+H]+ 746.29; found 764.4. The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5745-11 (300 mg, 0.4023 mmol) and Pd(OH)2/C (100 mg, 10%) in ethyl acetate (5 ml). The reaction was carried out at 15° C. for 6 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5745-12 (70 mg, white solid, purity 30.526%), yield: 8.68%. LCMS (ESI) m/z calcd. for C19H25N5O3 [M+H]+ 612.67; found 612.1. The compound structure is confirmed.
Compound TPD5745-12 (70 mg, 0.1144 mmol), HATU (65.25 mg, 0.1716 mmol), compound TPD5745-14 (45.8 mg, 0.1144 mmol), and TEA (57.88 mg, 0.572 mmol) were added sequentially in a 50 ml single mouth vial. The reaction was carried out for 16 h at 15° C. under nitrogen protection. After the reaction was completed, the reaction solution was quenched with water. It was extracted three times with ethyl acetate (10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and evaporated to give a residue. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005745 (23.5 mg, yellow solid, purity 97.684%), yield: 22.03%. LCMS (ESI) m/z calcd. for C53H52FN9O10 [M+H]+ 994.05; found 994.4; 1H NMR (400 MHz, DMSO_d6): δ=11.08 (s, 1H), 10.19-10.09 (m, 1H), 10.05 (br. s., 1H), 9.41 (br. s., 1H), 8.78 (s, 1H), 8.49-8.47 (m, 1H), 7.68 (br. s., 2H), 7.64 (br. s., 3H), 7.35 (s, 1H), 7.23 (s, 1H), 7.16 (br. s., 3H), 7.13 (br. s., 2H), 6.47-6.44 (m, 1H), 5.10-5.05 (m, 1H), 4.36-4.33 (m, 1H), 4.02-4.01 (m, 3H), 3.44 (s, 3H), 3.12-3.04 (m, 4H), 2.56 (br. s., 5H), 2.08 (br. s., 4H), 1.75 (m, 2H), 1.48 (s, 3H), 1.23-0.93 (m, 7H). The compound structure is confirmed.
To a 250 ml single-necked vial were sequentially added 1-Boc-piperazine (5.0 g, 26.7 mmol), 1-Cbz-4-piperidinecarboxaldehyde (6.6 g, 26.7 mmol), acetic acid (4.81 g, 80.1 mmol) and sodium triacetoxyborohydride (11.32 g, 53.4 mmol). The reaction was carried out at 20° C. for 16 hours. After the reaction was completed the reaction solution was poured into saturated aqueous sodium bicarbonate solution (200 ml). It was extracted three times with DCM (100 ml). The combined organic phases were washed with saturated saline (100 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was spun dry to give a residue. The residue was purified by silica gel column with eluent and ratio of DCM/MeOH=60/1˜40/1 to obtain compound TPD5746-10 (5.1 g, colorless oil, purity 85.387%), yield: 38.95%. LCMS (ESI) m/z calcd. for C23H35N3O4 [M+H]+ 418.2; found 418.0; 1H NMR (400 MHz, CDCl3): δ=7.36-7.30 (m, 5H), 5.12 (s, 2H), 4.17 (br. s. 2H), 3.40 (t, J=4.8 Hz, 4H), 2.77 (br. s. 2H), 2.33 (t, J=4.8 Hz, 4H), 2.17-2.15 (m, 2H), 1.76-1.61 (m, 3H), 1.46 (s, 9H), 1.13-1.05 (m, 2H). The compound structure is confirmed.
A 100 ml single-necked vial was charged with compound TPD5746-10 (1 g, 2.4 mmol), EA (20 ml) and palladium carbon hydroxide (0.1 g, 10% palladium, 50% water). The reaction was carried out at 25° C. by passing 20 psi hydrogen for 2 hours. Filtration and concentration to dryness afforded compound TPD5746-11 (320 mg, gray solid, purity 43.468%), yield: 20.83%. LCMS (ESI) m/z calcd. for C15H29N3O2 [M+H]+ 284.2; found 284.1; 1H NMR (400 MHz, DMSO_d6): δ=3.28 ((t, J=5.2 Hz, 5H), 2.87 (d, J=12.0 Hz, 2H), 2.45-2.35 (m, 2H), 2.28-2.21 (m, 4H), 2.07 (d, J=7.0 Hz, 2H), 1.64-1.49 (m, 3H), 0.97-0.87 (m, 2H). The compound structure is confirmed.
A 25 mL single-necked vial was charged with compound TPD5746-11 (320 mg, 1.1251 mmol), DIEA (436.22 mg, 3.3753 mmol), DMSO (2 ml), and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindole-1,3-dione (310.78 mg, 1.1251 mmol). 100° C. for 16 hours. Cooled to room temperature, the reaction solution was poured into water (20 ml) and stirred for 10 min. Filtration, the filter cake was washed with water and purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜50/1 to obtain compound TPD5746-12 (510 mg, yellow solid, purity 97.998%), yield: 82.17%. LCMS (ESI) m/z calcd. for C28H37N5O6 [M+H]+ 540.3; found 540.1 and 484.1.
A 25 mL single-necked vial was charged with compound TPD5746-12 (260 mg, 0.4809 mmol) and hydrogen chloride dioxane solution (3 N, 10 ml). the reaction was carried out at 25° C. for 2 h. The reaction solution was concentrated to dryness to give compound TPD5746-13 (248 mg, yellow solid, purity: 95.127%). The reaction solution was concentrated to dryness to give compound TPD5746-13 (248 mg, yellow solid, purity 95.127%), yield: 95.74%. LCMS (ESI) m/z calcd. for C23H29N5O4 [M+H]+ 440.22; found 440.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5746-13 (43 mg, 0.0839 mmol), compound TPD5735-9 (47.23 mg, 0.0839 mmol), potassium carbonate (34.79 mg, 0.0839 mmol), acetonitrile (6 ml) and potassium iodide (1.39 mg, 0.00839 mmol). the reaction was carried out at 25° C. for 16 hours. The reaction solution was poured into water (10 ml), EA (10 ml) was extracted 3 times, the combined organic phases were washed with water, brine, dried with anhydrous sodium sulfate filtered and concentrated and purified by preparative plates, the ratio of unfolding agent was DCM/MeOH=10/1 to give compound TPD005746 (31.4 mg, yellow solid, purity 97.503%) in 37.78% yield. LCMS (ESI) m/z calcd. for C52H52FN9O9 [M+H]+ 966.4; found 966.4; 1H NMR (400 MHz, DMSO_d6): δ=11.10 (s, 1H), 10.22 (d, J=4.9 Hz, 2H), 10.07 (s, 1H), 8.88 (s, 1H), 8.52 (t, J=5.3 Hz, 1H), 7.79 (d, J=8.9 Hz, 2H), 7.69-7.62 (m, 4H), 7.33 (s, 1H), 7.26 (d, J=9.0 Hz, 3H), 4.12 (t, J=8.9 Hz, 2H), 6.49 (d, J=5.1 Hz, 1H), 5.11-5.06 (m 1H), 4.11-4.05 (d, J=19.4 Hz, 5H), 3.25 (s, 2H), 3.07-2.83 (m, 4H), 2.73-2.59 (m, 6H), 2.23 (d, J=6.1 Hz, 2H), 2.10-1.92 (m, 2H), 1.83 (d, J=12.6 Hz, 2H). 1.49 (s, 4H), 1.26-1.16 (m, 5H). The compound structure is confirmed.
To a 100 ml triple-necked vial was sequentially added the compound tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (2 g, 7.2 mmol), 3-bromopiperidine-2,6-dione (2.76 g, 14.4 mmol), DIEA (1.86 g, 14.4 mmol) and DMF (20 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (30 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5752m-2 (1.1 g, green solid, purity 95.775%) in the yield: 37.50%. LCMS (ESI) m/z calcd. for C20H28N4O4 [M+H]+ 389.2; found 389.1; 1H NMR (400 MHz, DMSO_d6): δ=10.77 (s, 1H), 6.78-6.76 (m, 2H), 6.62-6.60 (m, 2H), 5.45 (d, J=7.4 Hz, 1H), 4.24-4.18 (m, 1H), 3.43 (br. s., 4H), 2.87-2.85 (m, 4H), 2.77-2.68 (m, 1H), 2.60-2.54 (m, 1H), 2.12-2.08 (m, 1H), 1.88-1.78 (m, 1H), 1.41 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5752m-2 (250 mg, 0.6419 mmol), EA (1 ml) and HCl/EA (2 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5752m-3 (250 mg, blue solid), yield: 75.14%. LCMS (ESI) m/z caled. for C15H20N4O2 [M+H]+ 289.2; found 289.2. The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound int-B (862.6 mg, 1.7731 mmol), TEA (717.68 mg, 7.0924 mmol) and DCM (10 ml). After cooling down to 0° C. compound TPD5752m-4 (500 mg. 3.5462 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was lowered to 0° C. and decanted into water (30 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the crude compound TPD5752m-5 (480 mg, brown oil, purity 76.441%), yield: 17.51%. LCMS (ESI) m/z calcd. for C31H28ClFN4O5 [M+H]+ 591.2; found 591.2; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.07 (s, 1H), 9.54 (s, 1H), 8.78 (s, 1H), 8.50 (d, J=5.2 Hz, 1H), 7.78 (d, J=8.9 Hz, 2H), 7.67-7.62 (m, 3H), 7.26-7.24 (m, 2H), 7.19-7.13 (m, 3H), 4.03 (s, 3H), 3.73 (t, J=6.6 Hz, 2H), 2.70 (t, J=7.3 Hz, 2H), 2.10-2.06 (m, 2H), 1.48 (s, 4H). The compound structure is confirmed.
A 50 ml triple-necked vial was sequentially charged with compounds TPD5752m-5 (500 mg, 0.846 mmol), 4-hydroxymethylpiperidine (194.75 mg, 1.692 mmol), K2CO3 (233.85 mg, 1.692 mmol), KI (28.09 mg, 0.1692 mmol) and acetonitrile (10 ml). The reaction was carried out under nitrogen protection at 60° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and poured into water (10 ml) and extracted three times with EA (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5752m-6 (240 mg, brown solid, purity 97.264%) in the yield: 41.19%. LCMS (ESI) m/z calcd. for C37H40FN5O6 [M+H]+ 670.3; found 670.3; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.06 (s, 1H), 9.39 (s, 1H), 8.79 (s, 1H), 8.49 (d, J=5.2 Hz, 1H), 7.77 (d J=8.9 Hz, 1H), 7.66-7.63 (m, 3H), 7.27-7.23 (m, 2H), 7.18-7.13 (m, 3H), 4.44 (s, 1H), 4.03 (s, 2H), 3.22-3.17 (m, 4H), 2.97 (br. s., 2H), 2.55-2.54 (m, 2H). 1.82 (br. s., 2H), 1.67-1.64 (m, 3H), 1.48 (s, 3H), 1.36-1.34 (m, 2H), 1.17 (br. s., 2H). The compound structure is confirmed.
Compound oxalyl chloride (90.96 mg, 0.7166 mmol) and DCM (2 ml) were sequentially added to a 50 ml triple-necked vial, cooled down to −78° C. A DCM (1 ml) solution of DMSO (111.79 mg, 1.4332 mmol) was added and −78° C. stirred for 0.5 hours. Then a DCM (2 ml) solution of compound TPD5752m-6 (240 mg, 0.3583 mmol) was added and −78° C. stirred for 0.5 hours. Finally, TEA (290.05 mg, 2.8664 mmol) was added and the reaction was slowly restored to room temperature and protected by nitrogen for 1 hour. After completion of the reaction, the reaction solution was poured into saturated NaHCO3 solution (6 ml) and extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give the crude compound TPD5752m-7 (240 mg, brown solid, purity 31.626%), yield: 31.73%. LCMS (ESI) m/z calcd. for C37H38FN5O6 [M+H]+ 668.3; found 668.3. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5752m-7 (160 mg, 0.2396 mmol), compound TPD5752m-3 (82.91 mg, 0.2875 mmol), sodium triacetoxyborohydride (101.56 mg, 0.4792 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. The reaction solution was decanted into water (5 ml) and appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% NH H32 O); gradient: 40-80/8 min to afford compound TPD005752m (2.47 mg, gray solid, purity 87.474%), yield: 0.96%. LCMS(ESI) m/z calcd. for C52H58FN9O7 [M+H]+ 940.4; found 314.3 and 470.8 (one-third peaks and half peaks); 1H NMR (400 MHz, CD3 OD): δ=8.93 (s, 1H), 8.43 (d, J=5.4 Hz, 1H), 7.73-7.70 (m, 3H), 7.58-7.54 (m, 2H), 7.23-7.21 (m, 2H), 7.09-7.05 (m, 2H), 6.89-6.87 (m, 2H), 6.73-6.71 (m, 2H), 6.56 (d, J=5.3 Hz, 1H), 4.21-4.17 (m, 1H), 4.09 (s, 3H), 3.13-3.11 (m, 2H), 3.02 (br. s., 3H), 2.79-2.71 (m, 2H), 2.61-2.56 (m, 7H), 2.34-2.30 (m, 1H), 2.20 (t, J=6.7 Hz, 1H), 2.03-1.97 (m, 3H), 1.92-1.88 (m, 1H), 1.85-1.81 (m, 2H), 1.64 (br. s., 3H), 1.30 (s, 9H). The compound structure is confirmed.
A 50 ml single-necked vial was charged with int-H (280 mg, 0.87 mmol), benzyl 4-formylpiperidine-1-carboxylate (323 mg, 1.31 mmol), DCE (10 ml), sodium triacetoxyborohydride (554 mg, 2.61 mmol) and glacial acetic acid (157 mg, 2.61 mmol). The reaction was carried out at 25° C. for 16 hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with dichloromethane (5 ml). The organic phases were combined and washed sequentially with water (10 ml) and saturated saline (10 ml). Afterwards, it was dried with anhydrous sodium sulfate, filtered and concentrated to dryness to obtain the crude product, which was purified by silica gel column with eluent ratio of PE/EA=5/1˜0/1 to obtain the compound TPD5760-1 (160 mg, white solid, purity 92.58%), yield: 30.76%. LCMS (ESI) m/z calcd. for C29H37FN6O4 [M+H]+ 553.3; found 553.1; 1H NMR (400 MHz, CDCl3): δ=7.90 (s, 1H), 7.37-7.28 (m, 6H), 6.36 (d, J=8.4 Hz, 1H), 5.51-5.47 (m, 1H), 5.13 (s, 2H), 4.18 (br. s., 2H). 2.96 (d, J=4.2 Hz, 4H), 2.91-2.85 (m, 3H), 2.84-2.72 (m, 4H), 2.56 (s, 4H), 2.40-2.29 (m, 1H), 2.23 (d, J=7.0 Hz, 2H), 1.83-1.63 (m, 4H), 1.11 (d, J=10.8 Hz 2H). The compound structure is confirmed.
A 250 ml single mouth flask was charged with compound TPD5760-1 (160 mg, 0.35 mmol), EA (4 ml) and palladium carbon hydroxide (40 mg, 10%). The reaction was carried out at 25° C. bypassing 1 atm hydrogen for 1 hour. Filtration and concentration to dryness gave the crude compound TPD5760-2 (120 mg, brown oil, purity 39.10%), yield: 41.56%. LCMS (ESI) m/z calcd. for C21H31FN6O2 [M+H]+ 419.3; found 419.1. The compound structure is confirmed.
A 25 mL single-necked vial was charged with compound TPD5760-2 (55 mg, 0.13 mmol), compound TPD5735-9 (73.98 mg, 0.13 mmol), acetonitrile (5 ml), potassium carbonate (36.32 mg, 0.26 mmol), and potassium iodide (2 mg, 0.01 mmol). the reaction was carried out for 16 h at 25° C. The reaction was carried out at 25 C for 16 hours. The reaction solution was poured into water (20 ml) and extracted three times with ethyl acetate (10 ml), the combined organic phases were washed sequentially with water (20 ml) and saturated saline (20 ml), and then dried with anhydrous sodium sulfate. After filtration, purification was carried out by preparative plates with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD005760 (12.6 mg, off-white solid, purity 99.553%) in a yield of 8.56%. LCMS (ESI) m/z calcd. for C50H54F2N10O7 [M+H]+ 945.4; found 945.4; 1H NMR (400 MHz, DMSO_d6): δ=10.81 (s, 1H), 10.23 (d, J=24.7 Hz, 2H), 10.06 (s, 1H), 8.86 (s, 1H), 8.51 (d, J=5.2 Hz, 1H), 7.78 (d, J=8.9 Hz, 2H), 7.68-7.59 (m, 3H), 7.47-7.42 (m, 1H), 7.25 (d, J=9.0 Hz, 2H), 7.19-7.11 (m, 2H), 6.50-6.46 (m, 2H), 5.20 (d, J=7.5 Hz, 1H), 4.09 (s, 3H), 3.20 (s, 2H), 2.87 (d, J=14.8 Hz, 6H). 2.80 (s, 3H), 2.56 (s, 2H), 2.45-2.32 (m, 2H), 2.26 (t, J=9.1 Hz, 4H), 1.86-1.79 (m, 3H), 1.59 (br. s., 1H), 1.48 (s, 4H), 1.24 (d, J=10.4 Hz, 4H). The compound structure is confirmed.
To a 250 ml triple-necked vial was sequentially added compound int-A (1.0 g, 1.61 mmol), tert-butylglycinate (423 mg, 3.23 mmol), cesium carbonate (1051 mg, 3.23 mmol), XantPhos (187 mg, 0.323 mmol), Pd2 (dba)3 (148 mg, 0.16 mmol) and 1,4-dioxane (50 ml). The reaction was carried out at 100° C. for 16 h under nitrogen protection. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford compound TPD5773-1 (680 mg, yellow solid, purity 98.573%), yield: 69.14%. LCMS (ESI) m/z calcd. for C33H33FN4O6 [M+H]+ 601.2; found 601.2; 1H NMR (400 MHz, DMSO_d6): δ=10.16 (s, 1H), 10.06 (s, 1H), 8.34 (d, J=5.2 Hz, 1H), 7.74 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.39 (s 1H), 7.20-7.13 (m, 4H), 6.73 (s, 1H), 6.28 (d, J=5.2 Hz, 1H), 6.08 (t, J=6.1 Hz, 1H), 3.99-3.97 (m, 5H), 1.47 (s, 4H), 1.44 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5773-1 (250 mg, 0.416 mmol), DCM (2 ml) and trifluoroacetic acid (1 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5773-2 (250 mg, yellow solid), yield: 89.33%. LCMS (ESI) m/z calcd. for C29H25FN4O6 [M+H]+ 545.2; found 545.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5773-2 (100 mg, 0.184 mmol), compound TPD5760-2 (77 mg, 0.184 mmol), HATU (105 mg, 0.275 mmol), triethylamine (37 mg, 0.367 mmol) and DMF (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and extracted three times with EA (2 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005773 (26.4 mg, off-white solid, purity 96.12%), yield: 14.65%. LCMS (ESI) m/z calcd. for C50H54F2N10O7 [M+H]+ 945.4; found 945.4; 1H NMR (400 MHz, DMSO_d6): δ=10.81 (s, 1H), 10.17 (s, 1H), 10.06 (s, 1H), 8.36 (d, J=5.3 Hz, 1H), 7.75 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.48-7.43 (m, 1H), 7.40 (s, 1H), 7.21-7.13 (m, 4H), 6.97 (s, 1H), 6.50 (d, J=8.3 Hz, 1H), 6.27 (d, J=5.3 Hz, 1H), 5.98 (br. s., 1H), 5.20 (d, J=9.6 Hz, 1H), 4.41 (d, J=13.9 Hz, 1H), 4.14-4.07 (m, 2H), 4.00 (br. s., 4H), 3.06 (t, J=12.0 Hz, 1H), 2.89-2.84 (m, 5H), 2.81 (s, 3H), 2.69 (t, J=11.4 Hz, 1H), 2.56 (br. s., 1H), 2.43-2.30 (m, 2H), 2.21 (s, 2H), 2.04-1.73 (m, 5H), 1.47 (s, 4H), 1.23 (s, 2H), 1.14 (d, J=12.6 Hz, 1H), 1.02-0.95 (m, 1H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD5773-1 (400 mg, 0.67 mmol), DCM (2 ml) and trifluoroacetic acid (1 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5773-2 (400 mg, yellow solid), yield: 72.60%. LCMS (ESI) m/z calcd. for C29H25FN4O6 [M+H]+ 545.2; found 545.0. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compounds TPD5773-2 (350 mg, 0.64 mmol), 4-hydroxymethylpiperidine (111 mg, 0.964 mmol), EDCI (185 mg, 0.96 mmol), HOBT (130 mg, 0.96 mmol), triethylamine (195 mg, 1.93 mmol) and DCM (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (6 ml) and extracted three times with DCM (2 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to give compound TPD5774-1 (240 mg, yellow solid, purity 96.292%), yield: 56.02%. LCMS (ESI) m/z calcd. for C35H36FN5O6 [M+H]+ 642.3; found 642.1; 1H NMR (400 MHz, DMSO_d6): δ=10.17 (s, 1H), 10.07 (s, 1H), 8.35 (d, J=5.2 Hz, 1H), 7.74 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.39 (s, 1H), 7.21-7.13 (m, 4H), 6.97 (s, 1H), 6.27 (d, J=5.2 Hz, 1H), 5.96 (t, J=4.2 Hz, 1H), 4.54 (t, J=5.2 Hz, 1H), 4.44-4.36 (m, 1H), 4.13-4.04 (m, 3H), 4.00 (s, 3H), 3.27 (t, J=5.6 Hz 3H), 3.17 (d, J=4.7 Hz, 1H), 3.03 (t, J=11.9 Hz, 1H), 2.65 (t, J=11.7 Hz, 1H), 1.77-1.65 (m, 3H), 1.47 (s, 4H). The compound structure is confirmed.
Compound TPD5774-1 (190 mg, 0.30 mmol) and DCM (5 ml) were added sequentially in a 25 ml triple-necked vial. After cooling down to 0° C., Dace-Martin oxidizer (251 mg, 0.59 mmol) was added. The reaction was carried out for 3 hours at 25° C. under nitrogen protection. After completion of the reaction, saturated NaHCO3 solution (3 ml) and water (5 ml) were added to the reaction solution and extracted three times with DCM (3 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plate with unfolding reagent ratio of DCM/MeOH=10/1 to afford the compound TPD5774-2 (90 mg, white solid, purity 84.277%), yield: 40.05%. LCMS (ESI) m/z calcd. for C35H34FN5O6 [M+H]+ 640.2; found 640.1; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 10.07 (s, 1H), 9.62 (s, 1H), 8.35 (d, J=5.2 Hz, 1H), 7.74 (d, J=8.9 Hz, 2H), 7.66-7.62 (m, 2H), 7.39 (s, 1H), 7.21-7.13 (m, 4H), 6.97 (s, 1H), 6.27 (d, J=5.2 Hz, 1H), 5.95 (t, J=4.3 Hz, 1H), 4.20-4.09 (m, 3H), 4.00 (s, 3H), 3.92 (d, J=13.7 Hz 1H), 3.27-3.16 (m, 2H), 3.01-2.92 (m, 1H), 2.68-2.60 (m, 1H), 1.99-1.87 (m, 2H), 1.60-1.53 (m, 1H), 1.47 (s, 4H). The compound structure is confirmed.
Compound TPD5774-2 (90 mg, 0.14 mmol), Int-1-5 (86 mg, 0.28 mmol), sodium triacetoxyborohydride (60 mg, 0.28 mmol), acetic acid (25 mg, 0.42 mmol), and DCE (3 ml) were added sequentially to a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 30° C. for 16 hours. The reaction solution was poured into water (5 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% NH3H2O); gradient: 10-55/8 min to give compound TPD005774 (1.05 mg, white solid, purity 93.818%), yield: 0.78%. LCMS (ESI) m/z calcd. for C49H52F2N10O7[M+H]+ 931.4; found 931.4; 1H NMR (400 MHz, CD3 OD): δ=8.41 (d, J=6.9 Hz, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.67 (s, 1H), 7.58-7.54 (m, 2H). 7.45-7.38 (m, 1H), 7.31 (d, J=8.9 Hz, 2H), 7.07 (t, J=8.8 Hz, 2H), 6.85 (s, 1H), 6.68 (d, J=6.8 Hz, 1H), 6.46 (d, J=8.4 Hz, 1H), 4.29 (d, J=6.1 Hz, 2H), 4.15 (s 3H), 3.75-3.55 (m, 4H), 3.18-3.13 (m, 4H), 2.85-2.69 (m, 4H), 2.26-2.17 (m, 4H), 2.07-2.02 (m, 3H), 1.65 (s, 3H), 1.29 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound DMF (15 ml), compound TPD55901-1 (1 g, 3.90 mmol), 4-Boc-1-piperazineacetic acid (1.05 g, 4.29 mmol), HATU (2.22 g, 5.85 mmol) and DIEA (1.26 g, 9.75 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, it was poured into saturated aqueous sodium bicarbonate solution (60 ml) and extracted three times with ethyl acetate (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD5901-1 (1 g, white solid, purity 98.733%), yield: 51.28%. LCMS (ESI) m/z calcd. for C24H31N5O6 [M+H]+ 486.2; found 486.1; 1H NMR (400 MHz, CDCl3): δ=9.10 (s, 1H), 8.47 (s, 1H), 7.73 (dd, J=7.7, 4.5 Hz, 2H), 7.50 (t, J=7.8 Hz, 1H), 5.20 (dd, J=13.3, 5.1 Hz, 1H), 4.42 (s, 2H), 3.54-3.47 (m, 4H), 3.21 (s, 2H), 2.93-2.75 (m, 2H), 2.61 (t, J=4.8 Hz, 4H), 2.38 (dd, J=13.1, 5.3 Hz, 1H), 2.28-2.14 (m, 1H), 1.47 (s, 9H). The compound structure is confirmed.
Compound TPD5901-2 (200 mg, 0.41 mmol) and 1,4-dioxane/HCl (4N, 10 ml) were added sequentially in a 20 ml single-necked vial. The reaction was carried out at 25° C. for 4 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5901-3 (180 mg, brown solid), yield: 94.55 00 LCMS (ESI) m/z calcd. for C19H23N5O4 [M+H]+ 386.2; found 386.1. The compound structure is confirmed.
Compound TPD5488-2 (100 mg, 0.17 mmol), compound TPD5901-3 (66 mg, 0.17 mmol), DCE (4 ml) and STAB (73 mg, 0.34 mmol) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005901 (16.7 mg, light yellow solid, purity 95.093%), yield: 19.46%. LCMS (ESI) m/z calcd. for C52H54FN9O8 [M+H]+ 952.4; found 952.7; 1H NMR (400 MHz, CDCl3): δ=9.52 (s, 1H), 9.33 (s, 1H), 8.84 (s, 1H), 8.54 (d, J=2.8 Hz, 1H), 8.27 (s, 1H), 7.82 (d, J=7.3 Hz, 2H), 7.72 (d, J=7.0 Hz, 2H), 7.67-7.52 (m, 5H), 7.25 (d, J=6.7 Hz, 2H), 7.13 (t, J=7.4 Hz, 2H), 6.53 (d, J=2.6 Hz, 1H), 5.30 (d, J=13.3 Hz, 1H), 4.54 (s, 2H), 4.11 (s, 3H), 3.81 (d, J=9.6 Hz, 2H), 3.28 (s, 2H), 3.09-2.88 (m, 2H), 2.79 (br. s., 6H), 2.64 (br. s., 3H), 2.56-2.28 (m, 5H), 2.00 (d, J=12.5 Hz, 3H), 1.89-1.73 (m, 9H), 1.67-1.55 (m, 3H). The compound structure is confirmed.
To a 100 ml single-necked vial was sequentially added compound TPD5907-1 (2.0 g, 0.01 mol), benzyl-1-piperazine carbonate (2.20 g, 0.01 mol), sodium cyanoborohydride (1.26 g, 0.02 mol) and methanol (30 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (30 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5907-2 (3.5 g, colorless oil, purity 56.272%), yield: 49.00%. LCMS (ESI) m/z calcd. for C22H33N3O4 [M+H]+ 404.2; found 404.1. The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compounds TPD5907-2 (1.5 g, 0.0037 mol), Pd(OH)2 (3 g, 10%) and EA (25 ml). The reaction was carried out under hydrogen atmosphere at 50° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, filtered and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=20/1˜4/1 to afford the compound TPD5907-3 (0.75 g, yellow solid), yield: 67.57%. LCMS (ESI) m/z calcd. for C14H27N3O2 [M+H]+ 270.21; found 270.1; 1H NMR (400 MHz, DMSO_d6): δ=3.93 (d, J=11.1 Hz, 1H), 2.80-2.58 (m, 3H), 2.47-2.37 (m, 2H), 2.35-2.25 (m, OH), 1.69 (d, J=12.7 Hz, 1H), 1.38 (s, 4H), 1.29-1.17 (m, 1H).
Compound TPD5907-3 (482 mg, 1.78 mmol), sm-1 (480 mg, 1.49 mmol), PEPPSI IHept-C1 (145 mg, 0.149 mmol) cesium carbonate (968 mg, 2.97 mmol), and 1,4-dioxane (25 ml) were added sequentially to a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (50 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative silica gel column with eluent ratio of DCM/MeOH=50/1˜20/1 to afford compound TPD5907-4 (170 mg, yellow solid, purity 63.146%), yield: 14.68%. LCMS (ESI) m/z calcd. for C27H37N5O5 [M+H]+ 512.28; found 512.1. The compound structure is confirmed.
Compound TPD5907-4 (170 mg, 0.27 mmol) 4N HCl/1,4-dioxane (8 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5907-5 (180 mg, yellow solid). LCMS (ESI) m/z calcd. for C22H29N5O3 [M+H]+ 412.22; found 412.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5907-5 (177 mg, 0.43 mol), compound TPD5488-2 (250 mg, 0.43 mmol), sodium triacetoxyborohydride (1832 mg, 0.86 mmol) and DCE (8 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (20 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted twice with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give compound TPD005907 (10.8 mg, yellow solid, purity 96.778%) in yield: 2.49%. LCMS (ESI) m/z calcd. for C55H60FN9O7 [M+H]+ 978.46; found 489.7, 978.4; 1H NMR (400 MHz, CD3 OD): δ=8.41 (d, J=5.4 Hz, 2H), 7.72 (d, J=8.8 Hz, 2H), 7.63 (s, 1H), 7.56 (dd, J=8.9 4.9 Hz, 2H), 7.48 (t, J=6.9 Hz, 2H), 7.41 (s, 1H), 7.24 (dd, J=19.1, 7.4 Hz, 3H), 7.07 (t, J=8.7 Hz, 2H), 6.53 (d, J=5.4 Hz, 1H), 5.17 (dd, J=13.2, 5.1 Hz, 1H). 4.87 (s, 1H), 4.50 (d, J=17.1 Hz, 2H), 4.03 (s, 3H), 3.74 (d, J=9.6 Hz, 2H), 3.54 (s, 2H), 3.20 (s, 4H), 3.01-2.63 (m, 13H), 2.59-2.45 (m, 1H), 2.25-2.12 (m, 3H), 2.09-2.01 (m, 1H), 2.01-1.83 (m, 4H), 1.70-1.52 (m, 6H). The compound structure is confirmed.
To a 50 ml triple-necked vial were sequentially added compounds TPD5937-1 (400 mg, 1.24 mmol), PdCl2 (PPh)32 (87 mg, 0.12 mmol), CuI (24 mg, 0.12 mmol), DMF (16 ml), propargyl oxytrimethylsilane (238 mg, 1.86 mmol) and DIEA (320 mg, 2.48 mmol). The reaction was carried out under nitrogen protection at 80° C. for 6 hours. After the reaction was completed the reaction solution was poured into water (10 ml) and extracted six times with ethyl acetate (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD5937-2 (250 mg, yellow solid, purity 95.746%), yield: 69.59%. LCMS (ESI) m/z calcd. for C16H14N2O4 [M+H]+ 299.1; found 299.0; 1H NMR (400 MHz, DMSO_d6): δ=11.01 (s, 1H), 7.74 (d, J=6.9 Hz, 1H), 7.69-7.66 (m, 1H), 7.54 (t, J=7.6 Hz, 1H), 5.43 (t. J=5.9 Hz, 1H), 5.17-5.13 (m, 1H), 4.35-4.33 (m, 2H), 3.16 (d, J=5.2 Hz, 2H), 3.13-3.10 (m, 1H), 2.95-2.86 (m, 1H), 2.58 (d, J=17.3 Hz, 1H), 2.46-2.38 (m, 1H), 2.03-1.97 (m, 1H). The compound structure is confirmed.
A 300 ml hydrogenation flask was charged with compound TPD5937-2 (800 mg, 2.68 mmol), Pd/C (800 mg, ethanol washed to remove water) and ethanol (80 ml). The reaction was carried out for 72 hours after passing 20-40 psi hydrogen gas and heating up to 50° C. The reaction was completed by filtration. The reaction was completed by filtration and the filter cake was washed with a large amount of ethanol. The filtrate was concentrated to dryness to afford the compound TPD5937-3 (220 mg, white solid, purity 54.175%), yield: 14.70%; LCMS (ESI) m/z calcd. for C16H18N2O4 [M+H]+ 303.1; found 303.0; 1H NMR (400 MHz, DMSO_d6): δ=11.01 (s, 1H), 7.58-7.55 (m, 1H), 7.48-7.44 (m, 2H), 5.17-5.12 (m, 1H), 4.49-4.29 (m, 2H), 3.46-3.40 (m, 3H), 2.76-2.53 (m, 4H), 2.46-2.30 (m, 2H), 1.83-1.68 (m, 2H). The compound structure is confirmed.
A 100 ml triple-necked vial was charged with compound TPD5937-3 (290 mg, 0.96 mmol) and DCM (25 ml). The reaction solution was cooled down to 0° C. under nitrogen protection and then DMP (814 mg, 1.92 mmol) was added and the reaction was carried out at 30° C. for 3 hours. After the reaction was completed the reaction solution was poured into saturated aqueous sodium bicarbonate and sodium thiosulfate (50 ml), the organic phase was separated and the aqueous phase was extracted three times with DCM (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the crude compound TPD5937-4 (52 mg, white solid, purity 87.420%), yield: 15.78%. LCMS (ESI) m/z calcd. for C16H16N2O4 [M+H]+ 301.1; found 301.0; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 9.73 (s, 1H), 7.59-7.56 (m, 1H), 7.52-7.40 (m, 2H), 5.16-5.12 (m, 1H), 4.54-4.31 (m, 2H), 2.98-2.86 (m, 4H), 2.61 (d, J=17.5 Hz, 1H), 2.47-2.37 (m, 2H), 2.07-1.95 (m, 1H). The compound structure is confirmed.
To a 25 ml triple-necked vial were sequentially added compound TPD5488-2 (170 mg, 0.29 mmol), DCE (3.4 ml), tert-butyl piperazine-1-carboxylate (60 mg, 0.32 mmol), glacial acetic acid (53 mg, 0.88 mmol) and sodium triacetoxyborohydride (124 mg, 0.58 mmol). The reaction was carried out under nitrogen protection at 30° C. for 2 hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml). The organic phase was separated and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD5937-5 (120 mg, yellow solid, purity 97.751%), yield: 53.32%. LCMS (ESI) m/z calcd. for C42H49FN6O6 [M+H]+ 753.4; found 753.3; 1H NMR (400 MHz, DMSO_d6): δ=10.21-10.02 (m, 2H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.66-7.62 (m, 2H), 7.46 (s, 1H), 7.31 (s, 1H), 7.25-7.11 (m, 4H). 6.39 (d, J=5.2 Hz, 1H), 3.94 (s, 3H), 3.59 (d, J=11.2 Hz, 2H), 3.33 (d, J=3.2 Hz, 6H), 2.64 (t, J=11.1 Hz, 2H), 2.31 (br. s., 4H), 2.21 (d, J=6.8 Hz, 2H), 1.83 (d J=11.9 Hz, 2H), 1.71 (br. s., 1H), 1.47 (s, 4H), 1.40 (s, 9H), 1.33-1.21 (m, 2H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD5937-5 (90 mg, 0.12 mmol), dichloromethane (3 ml) and trifluoroacetic acid (1.5 ml). The reaction was carried out at 30° C. for 2 h. After completion of the reaction, the pH was adjusted to 8 with saturated sodium bicarbonate, the organic phase was separated, and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to the dry compound TPD5937-6 (82 mg, yellow solid, purity 93.306%), yield: 98.16%. LCMS (ESI) m/z calcd. for C37H41FN6O4 [M+H]+ 653.3; found 653.4; 1H NMR (400 MHz, DMSO_d MHz, DMSO_d6): δ=10.13 (d, J=48.7 Hz, 2H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.46 (s, 1H), 7.31 (s, 1H), 7.24-7.12 (m, 4H). 6.39 (d, J=5.2 Hz, 1H), 3.94 (s, 3H), 3.58 (d, J=11.5 Hz, 2H), 2.74 (s, 4H), 2.63 (t, J=11.1 Hz, 2H), 2.32 (br. s., 4H), 2.17 (d, J=7.1 Hz, 2H), 1.82 (d, J=12.1 Hz, 2H), 1.70 (br. s., 1H), 1.48 (s, 4H), 1.35-1.24 (m, 3H). The compound structure is confirmed.
To a 25 ml triple-necked vial were sequentially added compound TPD5488-2 (82 mg, 0.13 mmol), DCE (6 ml), compound TPD5937-4 (41 mg, 0.14 mmol), glacial acetic acid (23 mg, 0.38 mmol) and sodium triacetoxyborohydride (53 mg, 0.25 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml). The organic phase was separated and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD005937 (39 mg, yellow solid, purity 99.422%), yield: 32.96%. LCMS (ESI) m/z calcd. for C53H57FN8O7 [M+H]+ 937.4; found 937.4; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.13 (d, J=49.4 Hz, 2H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.58-7.56 (m, 1H), 7.50-7.42 (m, 3H), 7.31 (s, 1H), 7.25-7.10 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 5.17-5.13 (m, 1H), 4.51-4.31 (m, 2H), 3.94 (s, 3H), 3.58 (d, J=11.1 Hz, 2H), 2.98-2.88 (m, 1H), 2.68-2.61 (m, 5H), 2.45-2.25 (m, 8H), 2.18 (d, J=6.3 Hz, 2H), 2.05-2.00 (m, 1H), 1.82-1.75 (m, 4H), 1.47 (s, 4H), 1.30-1.19 (m, 6H). The compound structure is confirmed.
Compound TPD005937 (10 mg, 0.0097 mmol), DCM (2 ml) and TFA (1 ml) were added sequentially in a 25 ml single-necked vial. The system reacted at 30° C. for 10 minutes. Concentration to dryness gave the product compound TPD005937-TFA (7.4 mg, yellow solid, purity 96.678), yield: 52.58%. LCMS (ESI) m/z calcd. for C53H57FN8O7 [M+H]+ 937.4; found 937.4; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 10.33 (s, 1H), 10.02 (s, 1H), 8.73 (d, J=6.6 Hz, 1H), 7.85 (d, J=8.9 Hz, 2H), 7.67-7.61 (m, 4H), 7.52-7.47 (m, 3H), 7.35 (d, J=9.0 Hz, 2H), 7.16 (t, J=8.9 Hz, 2H), 6.74 (d, J=6.6 Hz, 1H), 5.20-5.15 (m, 1H), 4.52-4.31 (m, 2H), 4.05 (s, 3H), 3.79 (d, J=11.0 Hz, 2H), 3.06-2.90 (m, 4H), 2.84 (t, J=11.5 Hz, 2H), 2.73-2.68 (m, 2H), 2.66-2.55 (m, 2H), 2.46-2.24 (m, 3H), 2.22-1.74 (m, 8H), 1.49 (d, J=6.5 Hz, 4H), 1.37 (d, J=11.2 Hz, 2H), 1.23 (s, 4H). The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound DMSO (10 ml), compound TPD55972-1 (500 mg, 1.81 mmol), 4-N-(2-aminoethyl)-1-N-Boc-piperidine (500 mg, 2.17 mmol) and DIEA (468 mg, 3.62 mmol). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, it was poured into saturated aqueous sodium bicarbonate solution (30 ml) and extracted three times with ethyl acetate (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜60/1 to afford the compound TPD5972-2 (420 mg, yellow solid, purity 97.762%), yield: 46.62%. LCMS (ESI) m/z calcd. for C24H31N5O6[M+H]+ 486.2; found 486.1; 1H NMR (400 MHz, CDCl3): δ=11.10 (s, 1H), 7.63-7.56 (m, 1H), 7.10 (d, J=8.6 Hz, 1H), 7.04 (d, J=7.0 Hz, 1H), 6.79 (t, J=4.7 Hz, 1H), 5.07 (dd, J=12.9, 5.3 Hz, 1H), 3.42-3.28 (m, 8H), 2.95-2.80 (m, 1H), 2.65-2.56 (m, 3H), 2.43-2.34 (m, 4H), 2.09-1.96 (m, 1H), 1.37 (s, 9H). The compound structure is confirmed.
To a 20 ml single-necked vial was sequentially added compound TPD5972-2 (420 mg, 0.86 mmol) and HCl(g)/EA (4N, 10 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD5972-3 (360 mg, brown solid), yield: 97.87%. LCMS (ESI) m/z calcd. for C19H23N5O4 [M+H]+ 386.2; found 386.1. The compound structure is confirmed.
Compound TPD5488-2 (100 mg, 0.17 mmol), compound TPD5972-3 (79 mg, 0.21 mmol), DCE (4 ml) and STAB (73 mg, 0.34 mmol) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to afford compound TPD005972 (12.4 mg, yellow solid, purity 95.422%), yield: 7.23%. LCMS (ESI) m/z calcd. for C52H54FN9O8 [M+H]+ 952.4; found 952.7; 1H NMR (400 MHz, CDCl3): δ=11.11 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.68-7.54 (m, 3H), 7.46 (s, 1H), 7.31 (s, 1H). 7.26-7.07 (m, 5H), 7.03 (d, J=7.1 Hz, 1H), 6.77 (t, J=4.6 Hz, 1H), 6.39 (d, J=5.2 Hz, 1H), 5.08 (dd, J=13.0, 5.4 Hz, 1H), 4.22 (t, J=6.6 Hz, 1H), 3.94 (s, 4H). 3.59 (d, J=11.3 Hz, 2H), 3.43-3.35 (m, 3H), 2.95-2.81 (m, 1H), 2.71-2.55 (m, 6H), 2.43-2.35 (m, 4H), 2.21 (d, J=6.9 Hz, 2H), 2.06-1.97 (m, 1H), 1.83 (d, J=11.4 Hz, 2H), 1.76-1.51 (m, 3H), 1.47 (s, 4H), 1.41-1.25 (m, 4H), 0.95-0.84 (m, 2H). The compound structure is confirmed.
Compound 7022-1 (500 mg) underwent amide condensation with intermediate 7022-2 to give compound 7022-3. This product was oxidized to prepare its corresponding aldehyde derivative 7022-4 (100 mg). compounds TPD7022-5 (81 mg), DCE (4 ml) and STAB (80 mg) were added sequentially to a 25 ml single mouth vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD007022 (17 mg, yellow solid, purity 97%). LCMS (ESI) m/z calcd. for C48H49FN11O5 [M+H]+ 877.4; found 877.7. The compound structure is confirmed.
Compound 7026-1 (700 mg) underwent a condensation reaction with bromoacetic acid amide to give compound 7026-2. This product was condensed with 4-hydroxymethylpiperidine under alkaline conditions to give 7026-3, followed by oxidation to prepare its corresponding aldehyde derivative 7026-4 (90 mg). It was added to a 25 ml single-necked vial and compound TPD7022-5 (75 mg), DCE (4 ml) and STAB (77 mg) were added sequentially. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD007022 (15 mg, light yellow solid, purity 97%). LCMS (ESI) m/z calcd. for C48H48FN12O6 [M+H]+ 907.4; found 907.6. The compound structure is confirmed.
Compound 7022-1 (600 mg) was condensed with 9004-1 to give compound 9004-2. Hydrolysis of the product in lithium hydroxide solution gave 9004-3, which was added to a 25 ml vial and compound VH032, DCM (5 ml), HOBt (5 mg), and EDCI were added sequentially. the reaction was carried out for 15 h at 25° C. under nitrogen protection. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to give the compound TPD009004 (19 mg, pale yellow solid, purity 96%). LCMS (ESI) m/z calcd. for C56H64FN12O5S [M+H]+ 1035.5; found 1035.6. The compound structure is confirmed.
Compound 9006-1 (500 mg) reacted with EDCI, HOBt in DCM (10 ml) and amide condensation with 9004-1 to give compound 9006-2. Hydrolysis of the product in lithium hydroxide solution afforded 9006-3, which was added to a 25 ml single-necked bottle and compound VH032, DCM (5 ml), HOBt. The reaction was carried out under nitrogen protection at 25° C. for 17 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to give the compound TPD009006 (13 mg, pale yellow solid, purity 98%). LCMS (ESI) m/z calcd. for C58H67FN13O6S [M+H]+ 1092.5; found 1092.6. The compound structure is confirmed.
Compound 10001-1 (550 mg) reacted with DIAD, PPh3 in DCM (10 ml) and then 10001-2 was added for the condensation reaction to give compound 10001-3. the product was hydrolyzed in lithium hydroxide solution to give 10001-4, the latter was added to 25 ml of a single-necked vial and compound VH032, DCM (5 ml) was added sequentially, HOBt (5 mg) and EDCI. The reaction was carried out under nitrogen protection at 25° C. for 19 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD10001 (11 mg, light yellow solid, purity 98%). LCMS (ESI) m/z calcd. for C53H59N10O6S [M+H]+ 963.4; found 963.6. The compound structure is confirmed.
Compound 10001-4 (90 mg) reacted with compound lenalidomide, HOBt (5 mg) and EDCI in DCM (5 ml). The reaction was stirred under nitrogen protection at 25° C. for 19 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD10001 (17 mg, light yellow solid, purity 96%). LCMS (ESI) m/z calcd. for C44H42N9O6 [M+H]+ 792.3; found 792.5. The compound structure is confirmed.
Compound 10040-4 (70 mg) reacted with DIAD, PPh3 in DCM (7 ml) followed by the addition of N-Boc-protected 4-hydroxypiperidine for the condensation reaction to give the crude product deprotected by TFA/DCM to give 10040-2. The product was added to a 25 ml single-necked vial and compound 10040-3, potassium carbonate, and methanol were added sequentially. The reaction was carried out under nitrogen protection at 25° C. for 12 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to give the compound TPD10040 (9 mg, pale yellow solid, 98% purity). LCMS (ESI) m/z calcd. for C37H38Cl2FN8O5 [M+H]+ 763.2; found 763.3. The compound structure is confirmed.
Compound 10040-2 (90 mg) reacted with STAB, 2,2-dimethoxyacetaldehyde in DCE (7 ml) and then the crude product obtained was stripped of the protecting groups in HCl/THF system to give 10041-1. The product was added to a 25 ml single mouth vial and compound 10041-2, STAB and DCE were added sequentially. nitrogen protected reaction at 25° C. for 16 h. The reaction was carried out under nitrogen protection for 16 h. The reaction was carried out under nitrogen protection at 25° C. for 16 h. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to give the compound TPD10041 (7 mg, pale yellow solid, 98% purity). LCMS (ESI) m/z calcd. for C40H43Cl2FN9O5 [M+H]+ 818.3; found 818.4. The compound structure is confirmed.
Compound 10050-1 (100 mg) reacted with NaH in anhydrous THF (9 ml), followed by the addition of N-Boc-4-bromopiperidine, and the crude compound obtained from the reaction reacted with TFA/DCM to remove the protecting group to give 10050-2. The product was added to a 25 mlmono flask and compound 10040-3, potassium carbonate, and methanol were added sequentially. The reaction was carried out under nitrogen protection at 25° C. for 12 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to give the compound TPD10040 (11 mg, light yellow solid, purity 96%). LCMS (ESI) m/z calcd. for C39H43C1N9O6S [M+H]+ 800.3; found 800.3. The compound structure is confirmed.
Compound 10050-1 (100 mg) was oxidized by Swern reagent to give compound 10051-1, the latter reacted with STAB, 4-hydroxymethylpiperidine in DCE (7 ml) to give 10051-2, which was then oxidized by Swern reagent to give compound 10051-3. The product was added to a 25 ml single-necked vial and compound 10041-2 was added in turn, STAB and DCE. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD 10051 (9 mg, light yellow solid, purity 98%). LCMS (ESI) m/z calcd. for C41H46ClN10O5S [M+H]+ 825.4; found 825.5. The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compounds TPD12001-1 (2 g, 10.8 mmol), NBS (2.88 g, 16.2 mmol), AIBN (0.18 g, 1.08 mmol), and CCl4 (20 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the crude compound TPD12001-2 (3.1 g, yellow oil, purity 85.695%), yield: 92.59%. LCMS (ESI) m/z calcd. for C8H7BrClNO2 [M+H]+ 263.9, 263.9; found 264.1, 266.0; 1H NMR (400 MHz, CDCl3): δ=8.48 (d, J=5.0 Hz, 1H), 7.71 (d, J=5.0 Hz, 1H), 5.05 (s, 2H), 4.02 (s, 3H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compound TPD12001-2 (3 g, 11.3 mmol), 3-amino-2,6-piperidinedione hydrochloride (2.23 g, 13.5 mmol), DIEA (2.92 g, 22.6 mmol) and DMF (30 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (100 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12001-3 (1.72 g, yellow solid, purity 92.354%), yield: 50.44%. LCMS (ESI) m/z calcd. for C12H10ClN3O3 [M+H]+ 280.0; found 280.1; 1H NMR (400 MHz, DMSO_d6): δ=11.06 (s, 1H), 8.63 (d, J=4.9 Hz, 1H), 7.80 (d, J=4.9 Hz, 1H), 5.18 (dd, J=13.3, 5.1 Hz, 1H), 4.60 (d, J=18.2 Hz, 1H), 4.44 (d, J=18.3 Hz, 1H), 2.95-2.87 (m, 1H), 2.63-2.58 (m, 1H), 2.49-2.42 (m, 1H), 2.05-2.01 (m, 1H). The compound structure is confirmed.
Compound TPD12001-3 (500 mg, 1.79 mmol), 4-N-(2-aminoethyl)-1-N-BOC-piperidine (492 mg, 2.15 mmol), DIEA (462 mg, 3.58 mmol), and DMSO (10 ml) were added sequentially in 25 ml triple vials. The reaction was carried out under nitrogen protection at 140° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (50 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12001-4 (130 mg, yellow solid, purity 97.218%), yield: 14.93%. LCMS (ESI) m/z calcd. for C23H32N6O5 [M+H]+ 473.3; found 473.3; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 8.15 (d, J=5.2 Hz, 1H), 6.82 (d, J=5.2 Hz, 1H), 6.67 (t, J=5.4 Hz, 1H), 5.12 (dd, J=13.3, 5.1 Hz, 1H), 4.27 (d, J=18.1 Hz, 1H), 4.27 (d, J=18.1 Hz, 1H), 4.15 (d, J=18.0 Hz, 1H), 3.52 (dd, J=13.1, 6.6 Hz, 2H), 2.96-2.84 (m, 2H), 2.64-2.54 (m, 2H), 2.39-2.29 (m, 8H), 2.08-2.03 (m, 2H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12001-4 (130 mg, 0.27 mmol), 1,4-dioxane (5 ml) and 4N HCl/1,4-dioxane (3 ml). The reaction was carried out at 25° C. for 3 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12001-5 (140 mg, yellow solid), yield: 94.68%. LCMS (ESI) m/z calcd. for C18H24N6O3 [M+H]+ 373.2; found 373.2. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12001-5 (70 mg, 0.19 mmol), compound TPD5488-2 (110 mg, 0.19 mmol), sodium triacetoxyborohydride (80 mg, 0.38 mmol), and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=8/1 to give compound TPD12001 (12.5 mg, light yellow solid, purity 96.23%), yield: 6.81%. LCMS (ESI) m/z calcd. for C51H55FN10O7 [M+H]+ 940.1; found 940.2; 1H NMR (400 MHz, DMSO_d6): δ=11.05 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.16 (d, J=5.1 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.46 (s, 1H), 7.31 (s, 1H), 7.21 (d, J=9.0 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 7.02 (s, 1H), 6.83 (d, J=5.2 Hz, 1H), 6.39 (d, J=5.2 Hz, 1H), 5.13 (dd, J=13.3, 5.1 Hz, 1H). 4.28 (d, J=18.0 Hz, 1H), 4.16 (d, J=17.9 Hz, 1H), 3.94 (s, 4H), 3.60-3.53 (m, 4H), 2.95-2.87 (m, 1H), 2.67-2.59 (m, 4H), 2.33-2.24 (m, 8H), 2.07-2.04 (m, 1H), 1.82 (d, J=11.6 Hz, 2H), 1.70 (s, 1H), 1.47 (s, 4H), 1.32 (d, J=14.9 Hz, 4H), 1.25 (d, J=3.8 Hz, 2H). The compound structure is confirmed.
To a 100 ml triple-necked vial was sequentially added compound TPD12003-1 (3 g, 17.5 mmol), dimethyl sulfate (4.41 g, 35 mmol), K2CO3 (7.26 g, 52.5 mmol) and DMF (40 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (150 ml) and extracted three times with EA (80 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=20/1˜5/1 to afford compound TPD12003-2 (2.5 g, white solid, purity 93.222%), yield: 61.76%. LCMS (ESI) m/z calcd. for C8H8ClNO2 [M+H]+ 186.0; found 186.0; 1H NMR (400 MHz, CDCl3): δ=8.88 (s, 1H), 7.25 (s, 1H), 3.94 (s, 3H), 2.63 (s, 3H). The compound structure is confirmed.
Compound TPD12003-2 (1.5 g, 8.1 mmol), NBS (2.16 g, 12.2 mmol), AIBN (0.13 g, 0.81 mmol) and CCl4 (30 ml) were added sequentially to a 100 ml three-necked vial. The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (60 ml) and extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the crude compound TPD12003-3 (2.4 g, yellow oily substance), yield: 33.33%. LCMS (ESI) m/z calcd. for C8H7BrClNO2 [M+H]+ 263.9, 265.9; found 263.9, 265.9. 265.9. The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compounds TPD12003-3 (2.4 g, 9.1 mmol), 3-amino-2,6-piperidinedione hydrochloride (1.8 g, 10.9 mmol), DIEA (2.35 g, 18.2 mmol) and DMF (30 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (100 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to give compound TPD12003-4 (500 mg, mauve solid, purity 99.318%), yield: 19.78%. LCMS (ESI) m/z calcd. for C12H10ClN3O3 [M+H]+ 280.0; found 280.1; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 8.80 (s, 1H), 7.87 (s, 1H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.57 (d, J=18.8 Hz, 1H), 4.44 (d, J=18.7 Hz, 1H). 2.96-2.86 (m, 1H), 2.63-2.57 (m, 1H), 2.46-2.35 (m, 1H), 2.04-2.00 (m, 1H). The compound structure is confirmed.
Compound TPD12003-4 (200 mg, 0.72 mmol), 4-N-(2-aminoethyl)-1-N-BOC-piperidine (197 mg, 0.86 mmol), DIEA (185 mg, 1.43 mmol), and DMSO (5 ml) were added sequentially in 25 ml triple vials. The reaction was carried out under nitrogen protection at 120° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (20 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to give compound TPD12003-5 (100 mg, brown solid, purity 90.95%), yield: 26.86%. LCMS (ESI) m/z calcd. for C23H32N6O5 [M+H]+ 473.2; found 473.2; 1H NMR (400 MHz, DMSO_d6): δ=10.96 (s, 1H), 8.35 (s, 1H), 7.09 (s, 1H), 6.59 (s, 1H), 5.03 (dd, J=13.3, 5.1 Hz, 1H), 4.32 (d, J=17.4 Hz, 1H), 4.18 (d, J=17.5 Hz, 1H), 3.46-3.42 (m, 2H), 3.31 (br. s, 4H), 2.94-2.85 (m, 1H), 2.60-2.51 (m, 2H), 2.38-2.31 (m, 6H), 1.97-1.92 (m, 1H), 1.39 (s, 9H). The compound structure is confirmed.
Compound TPD12003-5 (100 mg, 0.21 mmol), 1,4-dioxane (1 ml) and 4N HCl/1,4-dioxane (2 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12003-6 (100 mg, brown solid), yield: 95.50%. LCMS (ESI) m/z calcd. for C18H24N6O3 [M+H]+ 373.2; found 373.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12003-6 (100 mg, 0.27 mmol), compound TPD5488-2 (156 mg, 0.27 mmol), sodium triacetoxyborohydride (114 mg, 0.54 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (5 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=6/1 to give compound TPD12003 (16.1 mg, yellow solid, purity 98.655%), yield: 6.29%. LCMS (ESI) m/z calcd. for C51H55FN10O7 [M+H]+ 939.4; found 939.4; 1H NMR (400 MHz, DMSO_d6): δ=10.97 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.36 (s, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.63 (m, 2H), 7.46 (s, 1H). 7.31 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 7.08 (br. s, 1H), 6.60 (s, 1H), 6.39 (d, J=5.2 Hz, 1H), 5.03 (dd, J=13.2, 5.0 Hz, 1H), 4.33 (d, J=17.4 Hz, 1H), 4.18 (d, J=17.4 Hz, 1H), 3.94 (s, 3H), 3.59 (d, J=11.1 Hz, 2H), 3.44 (s, 2H), 2.95-2.85 (m, 1H), 2.67-2.56 (m, 4H), 2.38-2.28 (m, 6H), 2.20 (br. s, 2H), 1.96-1.91 (m, 1H), 1.82 (d, J=11.4 Hz, 2H), 1.70 (br. s, 1H), 1.47 (s, 4H), 1.34-1.23 (m, 6H). The compound structure is confirmed.
A 25 ml single-necked vial was charged sequentially with compound TPD12009-1 (50 mg, 0.18 mmol), tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (62 mg, 0.27 mmol), DMSO (5 ml) and DIEA (69 mg, 0.54 mmol). The reaction was carried out at 140° C. for 16 h under nitrogen protection. The system was cooled to room temperature and poured into ice water (20 ml) and extracted three times with ethyl acetate (10 ml). The organic phases were combined and washed sequentially with water (10 ml) and saturated saline (10 ml). Anhydrous sodium sulfate was dried, filtered and concentrated to dryness. The residue was purified by preparative plate with unfolding agent DCM/MeOH=15/1 to afford the compound TPD12009-2 (10 mg, brown oil, purity 58.279%), yield: 6.88%. LCMS (ESI) m/z calcd. for C23H32N6O5 [M+H]+ 473.2; found 473.1. The compound structure is confirmed.
To a 40 ml single-necked vial was added compound TPD12009-2 (20 mg, 0.042 mmol), DCM (1 ml) and hydrogen chloride dioxane solution (2 ml, 4 M). The system was spun dry after 1 h of reaction at 30° C. DCE (0.5 ml) and sodium triacetoxyborohydride (18 mg, 0.084 mmol) were added. The reaction was carried out under nitrogen protection at 30° C. for 72 hours. The system was poured into saturated aqueous sodium bicarbonate solution (2 ml) and extracted three times with DCM (2 ml). The organic phases were combined and washed sequentially with water (2 ml) and saturated saline (2 ml). Anhydrous sodium sulfate was dried, filtered and concentrated to dryness. The residue was purified by preparative plate with unfolding agent DCM/MeOH=10/1 to give compound TPD12009 (2.4 mg, yellow solid, purity 83.866%), yield: 4.98%. LCMS (ESI) m/z calcd. for C51H55FN10O7 [M+H]+ 939.4; found 939.7; 1H NMR (400 MHz, CD3 OD): δ=8.38 (d, J=5.4 Hz, 1H), 8.24 (s, 1H), 7.71 (d, J=8.9 Hz, 2H), 7.60 (s, 1H), 7.58-7.54 (m, 3H), 7.47-7.42 (m, 1H), 7.39 (s, 1H), 7.22-7.19 (m, 2H), 7.09-7.05 (m, 3H), 6.88 (d, J=1.0 Hz, 1H), 6.50 (d, J=5.4 Hz, 1H), 5.34 (t, J=4.7 Hz, 2H), 5.41-5.27 (m, 1H), 4.02 (s, 3H), 3.69 (d, J=11.2 Hz, 2H), 3.49 (d, J=5.9 Hz, 2H), 3.13-3.10 (m, 1H), 2.78-2.73 (m, 4H), 2.59-2.55 (m, 2H), 2.39 (d, J=7.0 Hz, 2H), 2.21-2.17 (m, 2H), 2.02 (d, J=5.0 Hz, 2H), 1.92 (d, J=8.7 Hz, 2H), 1.80 (br. s, 2H), 1.64 (s, 4H), 1.33 (s, 6H). The compound structure is confirmed.
Compound TPD12013-1 (5.0 g, 33.1 mmol) and DCM (100 ml) were added sequentially in a 250 ml three-necked vial. The system was lowered to 0° C. and m-CPBA (8.57 g, 49.6 mmol) was added. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into saturated NaHCO3 solution (100 ml) and extracted three times with DCM (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to obtain the compound TPD12013-2 (6 g, yellow oil, purity 81.067%), yield: 87.92%. LCMS (ESI) m/z calcd. for C8H9NO3 [M+H]+ 168.1; found 168.1; 1H NMR (400 MHz, CDCl3): δ=8.13 (d, J=6.4 Hz, 1H), 7.24-7.20 (m, 1H), 7.15 (d, J=7.9 Hz, 1H), 4.03 (s, 3H), 2.31 (s, 3H). The compound structure is confirmed.
Compound TPD12013-2 (6.0 g, 35.9 mmol), POCl3 (27.52 g, 179.5 mmol) and DCE (60 ml) were added sequentially in a 100 ml triple-necked vial. The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, concentrated and adjusted to alkaline with saturated NaHCO3 solution and extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=20/1˜5/1 to obtain the compound TPD12013-3 (1.9 g, brown oil, purity 97.863%) in the yield: 27.86%. LCMS(ESI) m/z calcd. for C8H8ClNO2 [M+H]+ 186.0; found 186.0; 1H NMR (400 MHz, CDCl3): δ=7.90 (d, J=1.8 Hz, 1H), 7.23 (d, J=5.2 Hz, 1H), 3.89 (s, 3H), 2.32 (s, 3H). The compound structure is confirmed.
Compound TPD12013-3 (1.7 g, 9.2 mmol), NBS (2.46 g, 13.8 mmol), AIBN (0.15 g, 0.92 mmol) and CCl4 (50 ml) were added sequentially in a 100 ml triple-necked vial. The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (100 ml) and extracted three times with DCM (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=50/1˜30/1 to afford the compound TPD12013-4 (1.7 g, yellow oil, purity 97.915%) in the yield: 68.48%. LCMS (ESI) m/z calcd. for C8H7BrClNO2 [M+H]+ 263.9; found 264.0; 1H NMR (400 MHz, CDCl3): δ=8.54 (d, J=5.2 Hz, 1H), 7.54 (d, J=5.2 Hz, 1H), 5.04 (s, 2H), 4.05 (s, 3H). The compound structure is confirmed.
To a 100 ml triple-necked vial were sequentially added compounds TPD12013-4 (1.7 g, 6.4 mmol), 3-amino-2,6-piperidinedione hydrochloride (1.26 g, 7.68 mmol), DIEA (1.65 g, 12.8 mmol) and DMF (30 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (100 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to give compound TPD12013-5 (1 g, purple solid, purity 90.452%), yield: 50.00%. LCMS (ESI) m/z calcd. for C12H10ClN3O3 [M+H]+ 280.0; found 280.1; 1H NMR (400 MHz, DMSO_d6): δ=11.06 (s, 1H), 8.75 (d, J=5.3 Hz, 1H), 7.81 (d, J=5.3 Hz, 1H), 5.20 (dd, J=13.3, 5.1 Hz, 1H), 4.58 (d, J=17.8 Hz, 1H), 4.42 (d, J=17.8 Hz, 1H), 2.98-2.89 (m, 1H), 2.65-2.60 (m, 1H), 2.55-2.44 (m, 1H), 2.07-2.03 (m, 1H). The compound structure is confirmed.
Compound TPD12013-5 (350 mg, 1.25 mmol), 4-N-(2-aminoethyl)-1-N-BOC-piperidine (344.37 mg, 1.50 mmol), DIEA (323.49 mg, 2.50 mmol), and DMSO (5 ml) were added sequentially in 25 ml triple vials. The reaction was carried out under nitrogen protection at 120° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (10 ml) and extracted three times with EA (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD12013-6 (240 mg, yellow solid, purity 57.007%), yield: 23.09%. LCMS (ESI) m/z calcd. for C23H32N6O5 [M+H]+ 473.2; found 473.3. The compound structure is confirmed.
Compound TPD12013-6 (240 mg, 0.5068 mmol), 1,4-dioxane (2 ml) and 4N HCl/1,4-dioxane (4 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12013-7 (240 mg, yellow solid), yield: 74.88%. LCMS (ESI) m/z calcd. for C18H24N6O3 [M+H]+ 373.2; found 373.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12003-6 (100 mg, 0.27 mmol), compound TPD5488-2 (156 mg, 0.27 mmol), sodium triacetoxyborohydride (114 mg, 0.54 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (5 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=6/1 to afford compound TPD12003 (16.1 mg, yellow solid, purity 98.655%), yield: 6.29%. LCMS (ESI) m/z calcd. for C51H55FN10O7 [M+H]+ 939.4; found 470.2; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.25 (d, J=5.7 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.66-7.62 (m, 2H), 7.46 (s, 1H), 7.31 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.17-7.13 (m, 2H), 6.68 (d, J=5.7 Hz, 1H), 6.57 (t, J=5.2 Hz, 1H), 6.39 (d, J=5.2 Hz, 1H), 5.13 (dd, J=13.3, 5.0 Hz, 1H), 4.25 (d, J=17.1 Hz, 1H), 4.14 (d, J=17.2 Hz, 1H), 3.94 (s, 3H), 3.59 (d, J=10.7 Hz, 4H), 3.32 (d, J=5.9 Hz, 2H), 2.97-2.88 (m, 1H), 2.67-2.61 (m, 4H). 2.42-2.26 (m, 6H), 2.20 (d, J=6.8 Hz, 2H), 2.07-1.99 (m, 1H), 1.82 (d, J=12.1 Hz, 2H), 1.69 (br. s, 1H), 1.47 (s, 4H), 1.36-1.24 (m, 4H). The compound structure is confirmed.
Compound int-N-1 (5.0 g, 21.3 mmol) and THF (100 ml) were added sequentially in a 250 ml triple-necked vial. NaH (1.70 g, 42.6 mmol) was added after the system was lowered to 0° C. The reaction was carried out under nitrogen protection at 0° C. for 1 hour. Then bromoacetonitrile (5.11 g, 42.6 mmol), potassium iodide (0.35 g, 2.13 mmol) and tetrabutylammonium bromide (0.69 g, 2.13 mmol) were added sequentially. The reaction was carried out under nitrogen protection at 25° C. for 15 hours. After the reaction was completed, the reaction solution was poured into saturated NH4 C1 solution (300 ml) and extracted three times with EA (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with the eluent ratio of DCM/MeOH=100/1˜30/1 to obtain the compound int-N-2 (4.7 g, brown solid, purity 92.512%) in the yield: 74.65%. LCMS (ESI) m/z calcd. for C14H15N3O3 [M+H]+ 274.1; found 274.1; 1H NMR (400 MHz, DMSO_d6): δ=7.39-7.32 (m, 5H), 5.11 (s, 2H), 4.46 (s, 2H), 4.08 (s, 2H), 3.69 (s, 2H), 3.47 (t, J=5.5 Hz, 2H). The compound structure is confirmed.
To a 100 ml single-necked flask were added sequentially the compounds int-N-2 (5.0 g, 18.3 mmol), EtOH/NH H32 O=10/1 (50 ml) and Raney nickel (1.07 g, 18.3 mmol). The reaction was carried out under hydrogen (1 atm) atmosphere for 16 hours at 25° C. After the reaction was completed, the reaction solution was filtrated, the filtrate was concentrated to dryness and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=50/1˜30/1 to obtain the compound int-N (2.0 g, brown oily substance, purity 94.845%), yield: 37.16%. LCMS (ESI) m/z calcd. for C14H19N3O3 [M+H]+ 278.1; found 278.2. The compound structure is confirmed.
Compound TPD12001-3 (670 mg, 2.40 mmol), int-N (664 mg, 2.40 mmol), DIEA (619 mg, 4.79 mmol) and DMSO (10 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 145° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (60 ml) and extracted three times with EA (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12068-1 (100 mg, brown solid, purity 93.032%), yield: 7.46%. LCMS (ESI) m/z calcd. for C26H28N6O6 [M+H]+ 521.2; found 521.3; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 8.14 (d, J=5.1 Hz, 1H), 7.38-7.35 (m, 5H), 6.89 (t, J=5.0 Hz, 1H), 6.83 (d, J=5.2 Hz, 1H), 5.09 (s, 2H), 4.24 (d J=17.9 Hz, 1H), 4.13 (d, J=17.9 Hz, 1H), 3.94 (s, 2H), 3.58-3.52 (m, 7H), 3.42-3.39 (m, 2H), 2.95-2.86 (m, 1H), 2.61 (d, J=16.4 Hz, 1H), 2.35-2.24 (m, 1H). 2.07-2.02 (m, 1H). The compound structure is confirmed.
Compound TPD 12068-1 (100 mg, 0.38 mmol), EA (5 ml) and Pd(OH)2/C (54 mg, 10% wet) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under hydrogen (1 atm) atmosphere at 40° C. for 48 hours. After completion of the reaction, the reaction solution was filtered and concentrated to dryness to afford the crude compound TPD12068-2 (30 mg, yellow oil, purity 92.378%), yield: 21.42%. LCMS (ESI) m/z calcd. for C18H22N6O4 [M+H]+ 387.2; found 387.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12068-2 (30 mg, 0.078 mmol), compound TPD5488-2 (54 mg, 0.093 mmol), sodium triacetoxyborohydride (33 mg, 0.16 mmol), acetic acid (14 mg, 0.23 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection for 6 hours at 25° C. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 15-40/6 min to afford the compound TPD12068 (8.62 mg, yellow solid, purity 98.240%) in the following yield: 11.08%. LCMS (ESI) m/z calcd. for C51H53FN10O8 [M+H]+ 953.4; found 477.3; 1H NMR (400 MHz, DMSO_d6): δ=11.06 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.1 Hz, 1H), 8.22 (s, 0.6H). 8.17 (d, J=5.1 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.8, 5.1 Hz, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 7.21 (d, J=8.8 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.87 (d, J=5.8 Hz, 1H), 6.84 (d, J=5.1 Hz, 1H), 6.39 (d, J=5.1 Hz, 1H), 5.12 (dd, J=13.2, 4.8 Hz, 1H), 4.26 (d, J=18.0 Hz, 1H), 4.15 (d, J=18.0 Hz, 1H), 3.94 (s, 3H), 3.58 (d, J=8.0 Hz, 5H), 3.50 (d, J=3.6 Hz, 3H), 2.97 (s, 2H), 2.93-2.87 (m, 1H), 2.67-2.60 (m, 5H), 2.36-2.29 (m, 1H), 2.24 (d, J=6.6 Hz, 2H), 1.80 (d, J=11.9 Hz, 2H), 1.69 (s, 1H), 1.47 (s, 4H), 1.35-1.23 (m, 3H). The compound structure is confirmed.
To a 100 ml triple-necked vial was sequentially added compound TPD12081-1 (2 g, 10.09 mmol), acetonitrile (30 ml), bromoacetonitrile (1.21 g, 10.09 mmol) and potassium carbonate (2.07 g, 15.13 mmol). The reaction was carried out under nitrogen protection at 25° C. for 4 hours. After the reaction was completed, the reaction solution was filtered, the filtrate was concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=50/1˜30/1 to obtain the compound TPD12081-2 (1.6 g, yellow solid, purity 98.554%), yield: 66.00%. LCMS (ESI) m/z calcd. for C12H19N3O2 [M+H]+ 238.2; found 237.8; 1H NMR (400 MHz, CDCl3): δ=4.15-4.09 (m, 2H), 3.60 (s, 2H), 3.38-3.25 (m, 2H), 2.83-2.81 (m, 2H), 2.47-2.44 (m, 1H), 1.61-1.58 (m, 1H), 1.46 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial were sequentially added compound TPD12081-2 (1 g, 4.21 mmol), EtOH (10 ml), ammonia (1 ml) and Raney nickel (0.49 g). The reaction was carried out under 1 atm hydrogen at 25° C. for 16 hours. After the reaction was completed, the filtrate was withdrawn and concentrated to dryness and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜10/1 to obtain the compound TPD12081-3 (0.26 g, brown solid, purity 86.928%), yield: 21.43%. LCMS (ESI) m/z calcd. for C12H23FN3O2 [M+H]+ 242.18; found 242.2; 1H NMR (400 MHz, DMSO_d6): δ=4.00-3.99 (m, 2H), 3.80 (s, 2H), 3.15-3.06 (m, 3H), 2.75-2.65 (m, 3H), 2.34-2.27 (m, 2H), 1.55-1.53 (m, 2H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12081-3 (260 mg, 1.07 mmol), compound TPD12003-4 (330 mg, 1.18 mmol), DIEA (416 mg, 3.22 mmol) and DMSO (3 ml). The reaction was carried out under nitrogen protection at 120° C. for 16 hours. The reaction was completed the reaction solution was reduced to room temperature. The reaction solution was poured into water (10 ml) and the aqueous phase was extracted three times with ethyl acetate (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=15/1 to afford the compound TPD12081-4 (100 mg, brown solid, purity 95.665%), yield: 18.37%. LCMS (ESI) m/z calcd. for C24H32N6O5 [M+H]+ 485.24; found 485.1; 1H NMR (400 MHz, DMSO_d6): δ=10.96 (br. s, 1H), 8.35 (s, 1H), 7.04 (s, 1H), 6.56 (s, 1H), 5.02 (dd, J=13.3, 5.1 Hz, 1H), 4.25 (dd. J=58.9, 17.4 Hz, 2H), 3.95 (d, J=5.9 Hz, 2H), 3.50-3.41 (m, 2H), 3.06-2.76 (m, 7H), 2.68 (t, J=6.7 Hz, 2H), 2.36-2.23 (m, 3H), 2.09-1.84 (m, 2H), 1.58 (d, J=7.8 Hz, 1H), 1.38 (s, 9H).
To a 25 ml single-necked vial was sequentially added compound TPD12081-4 (50 mg, 0.103 mmol) and HCl(g)/1.4-dioxane (40 ml, 4N). the reaction was carried out for half an hour at 25° C. The reaction solution was concentrated to dryness to give the crude compound TPD12081-5 (40 mg, yellow solid), yield: 90%. The reaction solution was concentrated to dryness to give the crude compound TPD12081-5 (40 mg, yellow solid), yield: 90%. LCMS (ESI) m/z calcd. for C24H33N5O5 [M+H]+ 385.2; found 385.1. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD5488-2 (100 mg, 0.18 mmol), DCE (4 ml), compound TPD12081-5 (69 mg, 0.18 mmol) and sodium triacetoxyborohydride (76 mg, 0.36 mmol). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml) and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD12081 (15.3 mg, yellow solid, purity 99.365%), yield: 8.90%. LCMS (ESI) m/z calcd. for C52H55FN10O7 [M+H+ 951.4; found 952.4; 1H NMR (400 MHz, CD3 OD): δ=8.56-8.32 (m, 3H), 7.72 (d, J=8.9 Hz, 2H), 7.62 (s, 1H), 7.59-7.53 (m, 2H), 7.39 (s, 1H), 7.27-7.18 (m, 2H), 7.12-7.03 (m, 2H), 6.65 (s, 1H), 6.52 (d, J=5.4 Hz, 1H), 5.05 (dd, J=13.0, 4.7 Hz, 1H), 4.48-4.30 (m, 3H), 4.02 (s, 3H), 3.71 (br. s, 2H), 3.65-3.56 (m 2H), 3.51-3.32 (m, 4H), 3.16-2.87 (m, 4H), 2.85-2.60 (m, 4H), 2.33 (br. s, 2H), 2.11-1.78 (m, 4H), 1.68-1.46 (m, 6H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12086-7 (50 mg, 0.13 mmol), compound TPD5488-2 (76 mg, 0.13 mmol), sodium triacetoxyborohydride (55 mg, 0.26 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 5-40/8 min to afford compound TPD12082 (9.44 mg, yellow solid, purity 98.466%), yield: 7.07%. LCMS (ESI) m/z calcd. for C52H55FN10O7 [M+H]+ 951.4; found 951.5; 1H NMR (400 MHz, DMSO_d6): δ=10.98 (s, 1H), 10.19 (s, 1H), 10.07 (s, 1H), 8.43 (d, J=5.1 Hz, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.76 (d, J=8.6 Hz, 2H), 7.64 (dd, J=8.4, 5.2 Hz, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 7.25-7.13 (m, 5H), 6.59 (s, 1H), 6.39 (d, J=5.1 Hz, 1H), 5.03 (dd, J=13.1 5.1 Hz, 1H), 4.33 (d, J=17.2 Hz, 1H), 4.19 (d, J=17.3 Hz, 1H), 3.94 (s, 3H), 3.66 (s, 3H), 2.97-2.84 (m, 6H), 2.67-2.57 (m, 4H), 2.36-2.26 (m, 4H), 1.98-1.79 (m, 5H), 1.70 (s, 1H), 1.47 (s, 4H), 1.35-1.23 (m, 4H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound sm-1 (500 mg, 2.51 mmol), 2-bromoacetamide (415 mg, 3.01 mmol), potassium carbonate (1040 mg, 7.53 mmol) and acetonitrile (10 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After the reaction was completed, the reaction solution was poured into water (30 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12083-3a (500 mg, white solid, purity 96.019%) in the yield: 74.65%. LCMS (ESI) m/z calcd. for C12H21N3O3 [M+H]+ 256.2; found 255.7; 1H NMR (400 MHz, DMSO_d6): δ=7.16 (s, 1H), 7.01 (s, 1H), 3.94 (d, J=5.7 Hz, 2H), 3.10-2.99 (m, 4H), 2.80 (dd, J=41.5. 9.0 Hz, 2H), 2.30 (dd, J=13.7, 6.4 Hz, 1H), 1.59 (d, J=8.0 Hz, 1H), 1.38 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12083-3a (200 mg, 0.78 mmol), compound TPD12003-4 (218 mg, 0.78 mmol), palladium acetate (35 mg, 0.156 mmol), Xant-Phos (90 mg, 0.156 mmol), cesium carbonate (381 mg. 1.17 mmol) and 1,4-dioxane (5 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with eluent ratio of DCM/MeOH=15/1 to afford the compound TPD12083-4 (57 mg, brown solid, purity 94.190%), yield: 13.78%. LCMS (ESI) m/z calcd. for C24H30N6O [M+H]+ 499.2; found 499.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12083-4 (57 mg, 0.114 mmol), 1,4-dioxane (2 ml) and 4N HCl/1,4-dioxane (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12083-5 (60 mg, white solid), yield: 89.48%. LCMS (ESI) m/z calcd. for C19H22N6O4 [M+H]+ 399.2; found 399.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12083-5 (60 mg, 0.15 mmol), compound TPD5488-2 (88 mg, 0.15 mmol), sodium triacetoxyborohydride (64 mg, 0.30 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 6 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: T3 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-35/7 min. to obtain the compound TPD12083 (6.86 mg, yellow solid, purity 97.191%) in the following yield: 4.38%. LCMS (ESI) m/z calcd. for C52H53FN10O8 [M+H]+ 965.4; found 965.1; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.31 (s, 1H), 10.01 (s, 1H), 8.74-8.68 (m, 2H), 8.34 (d, J=8.0 Hz, 1H). 7.84 (d, J=8.8 Hz, 2H), 7.65-7.62 (m, 4H), 7.42 (s, 1H), 7.32 (d, J=8.5 Hz, 2H), 7.16 (t, J=8.9 Hz, 3H), 6.69 (br. s, 1H), 5.11 (dd, J=13.0, 4.8 Hz, 1H), 4.55 (d J=18.2 Hz, 1H), 4.41 (d, J=18.4 Hz, 1H), 4.04 (s, 3H), 3.77 (br. s, 4H), 3.65-3.56 (m, 3H), 2.81 (br. s, 3H), 2.67-2.57 (m, 1H), 2.33-2.25 (m, 5H), 2.03-1.91 (m, 6H), 1.62 (s, 1H), 1.48 (d, J=16.1 Hz, 8H). The compound structure is confirmed.
To a 500 ml triple-necked vial were sequentially added compound int-O-1 (10 g, 52.6 mmol), EtOH (200 ml) and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxahexane-4,6-dione (10.77 g, 57.8 mmol). The reaction was refluxed under nitrogen protection for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature and filtered by extraction to afford the compound int-O-2 (16 g, yellow solid, purity 99.855%), yield: 92.02%. LCMS (ESI) m/z calcd. for C13H11BrFNO4 [M+H]+ 344.0; found 286.0; 1H NMR (400 MHz, DMSO_d6). 6=11.23 (d, J=14.0 Hz, 1H), 8.52 (d, J=14.3 Hz, 1H), 8.06 (dd, J=6.0, 2.8 Hz, 1H), 7.67-7.63 (m, 1H), 7.44 (t, J=8.7 Hz, 1H), 1.68 (s, 6H). The compound structure is confirmed.
Compound int-O-2 (8 g, 23.2 mmol) and diphenyl ether (80 ml) were added sequentially in a 250 ml single-necked vial. The reaction was carried out at 240° C. for 15 min under nitrogen protection. The other batch was operated identically. After completion of the reaction, the reaction solution was reduced to room temperature and the compound int-O-3 (7.8 g, brown solid, purity 52.957%) was obtained by filtration, yield: 36.13%. LCMS (ESI) m/z calcd. for C9H5BrFNO [M+H]+ 241.9; found 242.0, 244.0; 1H NMR (400 MHz. DMSO_d6): δ=11.90 (s, 1H), 7.99-7.91 (m, 1H), 7.86 (dd, J=11.3, 6.3 Hz, 1H), 7.67-7.57 (m, 1H), 6.05 (dd, J=13.9, 7.4 Hz, 1H). The compound structure is confirmed.
To a 250 ml triple-necked flask was added sequentially compound int-O-3 (7.8 g, 32.2 mmol), toluene (100 ml) and POCl3 (14.81 g, 96.5 mmol). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, concentrated and adjusted to alkaline with saturated NaHCO3 solution and extracted three times with DCM (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=50/1˜30/1 to obtain the compound int-O-4 (2.5 g, yellow solid, purity 98.538%) in the yield: 29.50%. LCMS (ESI) m/z calcd. for C9H4BrClFN [M+H]+ 259.9; found 259.9, 262.0. The compound structure is confirmed.
Compound int-A-1 (20 g, 89.6 mmol), 4-aminophenol (13.69 g, 125.4 mmol), HATU (47.7 g, 125.4 mmol), DIEA (28.95 g, 224 mmol), and DMF (200 ml) were added sequentially to a 500 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound int-A-2 (24 g, brown oil, purity 82.759%), yield: 70.54%. LCMS (ESI) m/z calcd. for C17H15FN2O3 [M+H]+ 315.1; found 315.1; 1H NMR (400 MHz, DMSO_d6): δ=10.17 (s, 1H), 9.74 (s, 1H), 9.23 (s, 1H), 7.64-7.61 (m, 2H), 7.35 (d, J=8.8 Hz, 2H), 7.16-7.12 (m, 2H), 6.70 (dd, J=7.0, 5.1 Hz, 2H). 1.44 (d, J=1.6 Hz, 4H). The compound structure is confirmed.
The compounds int-O-4 (2 g, 7.7 mmol), int-A-2 (2.90 g, 9.24 mmol), cesium carbonate (5.02 g, 15.4 mmol) and DMSO (30 ml) were added sequentially in a 100 ml triple vial. The reaction was carried out under nitrogen protection at 120° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, decanted into water (100 ml) and extracted three times with EA (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of PE/EtOAc=5/1˜2/1 to give compound int-O (1.0 g, brown oil, purity 96.582%), yield: 23.38%. LCMS (ESI) m/z calcd. for C26H18BrF2N3O3 [M+H]+ 538.1; found 538.1. 540.1; 1H NMR (400 MHz, DMSO_d6): δ=10.22 (s, 1H), 10.05 (s, 1H), 8.70 (d, J=5.1 Hz, 1H), 8.45 (d, J=6.7 Hz, 1H), 8.17 (d, J=9.4 Hz, 1H), 7.79 (d, J=8.9 Hz, 2H). 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.27 (d, J=9.0 Hz, 2H), 7.16 (t, J=8.9 Hz, 2H), 6.65 (d, J=5.0 Hz, 1H), 1.47 (s, 4H). The compound structure is confirmed.
Compound TPD12086-1 (5 g, 25.1 mmol), sodium bicarbonate (4.22 g, 50.2 mmol) and DCM/H2O=1/1 (100 ml) were added sequentially in a 250 ml triple-necked vial. CbzCl (5.14 g, 30.1 mmol) was added after the system was lowered to 0° C. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was poured into water (100 ml) and extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of PE/EtOAc=10/1˜5/1 to afford the compound TPD12086-2 (5.7 g, yellow oil, purity 96%) in the yield: 65.74%. LCMS (ESI) m/z calcd. for C18H24N2O4 [M+H]+ 333.2; found 233.2, 355.2; 1H NMR (400 MHz, CDCl3): δ=7.37-7.32 (m, 5H), 5.17 (s, 2H), 4.13 (d, J=21.6 Hz, 3H), 3.99 (s, 1H), 3.44 (d, J=12.2 Hz, 2H). 2.58 (dd, J=15.0, 6.5 Hz, 1H), 1.61 (s, 1H), 1.41 (s, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial was sequentially added compound TPD12086-2 (5.7 g, 17.1 mmol), DCM (30 ml) and trifluoroacetic acid (60 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12086-3 (6 g, brown oil), yield: 87.13%. LCMS (ESI) m/z calcd. for C13H16N2O2 [M+H]+ 233.1; found 233.1. The compound structure is confirmed.
Compound TPD12086-3 (6 g, 25.8 mmol), bromoacetonitrile (3.71 g, 31.0 mmol), potassium carbonate (10.70 g, 77.4 mmol) and ACN (100 ml) were added sequentially in a 250 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 3 hours. After the reaction was completed, the reaction solution was poured into water (300 ml) and extracted three times with EA (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=200/1˜50/1 to afford the compound TPD12086-4 (5.4 g, brown oil, purity 92.063%) in the yield: 70.93%. LCMS (ESI) m/z calcd. for C15H17N3O2 [M+H]+ 272.1; found 272.2. The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added the compounds TPD12086-4 (4.4 g, 16.2 mmol), EtOH/NH H32 O=10/1 (60 ml) and Raney nickel (2.85 g). The reaction was carried out under hydrogen (1 atm) atmosphere at 40° C. for 16 hours. After completion of the reaction, the reaction solution was filtered and concentrated to dryness, and the residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12086-5 (2 g, brown oil, purity 95.061%), yield: 42.59%. LCMS (ESI) m/z calcd. for C15H21N3O2 [M+H]+ 276.2; found 276.2; 1H NMR (400 MHz, DMSO_d6): δ=7.39-7.34 (m, 5H), 5.11 (s, 2H), 3.62 (d, J=12.1 Hz, 1H), 3.51 (t, J=15.4 Hz, 4H), 3.26 (d, J=11.4 Hz, 3H), 2.40-2.31 (m, 4H), 1.41 (d, J=8.4 Hz, 1H). The compound structure is confirmed.
To a 25 ml triple-necked vial were sequentially added compound TPD12086-5 (500 mg, 1.82 mmol), compound TPD12003-4 (508 mg, 1.82 mmol), DIEA (469 mg, 3.63 mmol) and DMSO (5 ml). The reaction was carried out under nitrogen protection at 120° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (30 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12086-6 (200 mg, brown solid, purity 86.411%), yield: 21.24%. LCMS (ESI) m/z calcd. for C27H30N6O5 [M+H]+ 519.2; found 519.1. The compound structure is confirmed.
Compound TPD12086-6 (80 mg, 0.15 mmol) and trifluoroacetic acid (2 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 75° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12086-7 (100 mg, brown solid), yield: 54.50%. LCMS (ESI) m/z calcd. for C19H24N6O3 [M+H]+ 385.2; found 385.2. The compound structure is confirmed.
A 25 ml single-necked vial was sequentially charged with compound int-O (250 mg, 0.46 mmol), 4-hydroxymethylpiperidine (107 mg, 0.93 mmol), Pd2 (dba)3 (43 mg, 0.046 mmol), BINAP (58 mg, 0.093 mmol), cesium carbonate (303 mg, 0.93 mmol), and 1,4-dioxane (10 ml). The reaction was carried out for 16 h at 100° C. under nitrogen protection. The other five batches were operated identically. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (1200 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12086-8 (310 mg, yellow solid, purity 91.691%), yield: 17.82%. LCMS (ESI) m/z calcd. for C32H30F2N4O4 [M+H]+ 573.2; found 573.2; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.06 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 7.86 (d, J=13.5 Hz, 1H), 7.76 (d, J=9.0 Hz, 2H), 7.66-7.63 (m, 2H), 7.45 (d, J=8.5 Hz, 1H), 7.24-7.20 (m, 2H), 7.18-7.13 (m, 2H), 6.45 (d, J=5.2 Hz, 1H), 4.54 (t, J=5.3 Hz, 1H), 3.58 (d, J=11.4 Hz, 2H), 3.33 (s, 1H), 2.77 (t, J=11.1 Hz, 2H), 1.82 (d, J=10.8 Hz, 2H), 1.58 (br. s, 1H), 1.47 (s, 4H), 1.38-1.23 (m, 3H). The compound structure is confirmed.
Compound oxalyl chloride (137 mg, 1.08 mmol) and DCM (4 ml) were sequentially added to a 50 ml triple-necked vial, cooled down to −78° C. A DCM (2 ml) solution of DMSO (169 mg, 2.17 mmol) was added and −78° C. stirred for 0.5 hours. Then DCM (4 ml) solution of compound TPD12086-8 (310 mg, 0.54 mmol) was added and −78° C. stirred for 0.5 hours. Finally, TEA (438 mg, 4.33 mmol) was added and the reaction was slowly returned to room temperature and protected by nitrogen for 1 hour. After completion of the reaction, the reaction solution was poured into saturated NaHCO3 solution (30 ml) and extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=12/1 to afford the compound TPD12086-9 (180 mg, yellow solid, purity 95.968%), yield: 55.91%. LCMS (ESI) m/z calcd. for C32H28F2N4O4 [M+H]+ 571.2; found 571.3; 1H NMR (400 MHz, DMSO_d6): δ=10.19 (s, 1H), 10.06 (s, 1H), 9.68 (s, 1H), 8.57 (d, J=5.2 Hz, 1H), 7.87 (d, J=13.4 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.46 (d, J=8.5 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.46 (d, J=5.2 Hz, 1H), 3.52-3.49 (m, 2H). 2.94 (t, J=10.3 Hz, 2H), 2.73 (s, 1H), 2.04 (d, J=10.3 Hz, 2H), 1.77-1.68 (m, 2H), 1.47 (s, 4H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12086-7 (50 mg, 0.13 mmol), compound TPD12086-9 (74 mg, 0.13 mmol), sodium triacetoxyborohydride (55 mg, 0.26 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-35/7 min to afford the compound TPD12086 (13.26 mg, yellow solid, purity 98.425%) in the following yield: 10.68%. LCMS (ESI) m/z calcd. for C51H52F2N10O6 [M+H]+ 939.4; found 470.2; 939.1; 1H NMR (400 MHz, DMSO_d6): δ=10.98 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 8.38 (s. 1H), 8.22 (s, 2H), 7.86 (d, J=13.4 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.44 (d, J=8.5 Hz, 1H), 7.23-7.13 (m, 5H), 6.59 (s, 1H), 6.45 (d J=5.2 Hz, 1H), 5.04 (dd, J=13.2, 5.0 Hz, 1H), 4.33 (d, J=17.4 Hz, 1H), 4.19 (d, J=17.5 Hz, 1H), 3.71 (s, 3H), 2.98-2.84 (m, 6H), 2.79-2.74 (m, 4H), 2.41-2.22 (m, 4H), 1.93-1.83 (m, 5H), 1.73 (s, 1H), 1.47 (s, 4H), 1.33-1.23 (m, 4H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound sm-1 (500 mg, 2.51 mmol), 2-bromoacetamide (415 mg, 3.01 mmol), potassium carbonate (1040 mg, 7.53 mmol) and acetonitrile (10 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After the reaction was completed, the reaction solution was poured into water (30 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12088-1 (410 mg, white solid, purity 92.085%) in the yield: 58.70%. LCMS (ESI) m/z calcd. for C12H21N3O3 [M+H]+ 256.2; found 255.8; 1H NMR (400 MHz, DMSO_d6): δ=7.23 (s, 1H), 7.09 (s, 1H), 3.56-3.39 (m, 4H), 3.28-3.20 (m, 2H), 2.86 (s, 2H), 2.43 (dd. J=13.9, 6.4 Hz, 1H), 1.43 (s, 9H), 1.38 (d, J=8.5 Hz, 1H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12088-1 (410 mg, 1.60 mmol), compound TPD12003-4 (537 mg, 1.92 mmol), palladium acetate (72 mg, 0.32 mmol), Xant-Phos (185 mg, 0.32 mmol), cesium carbonate (782 mg. 2.40 mmol) and 1,4-dioxane (10 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12088-2 (200 mg, yellow solid, purity 93.691%), yield: 23.45%. LCMS (ESI) m/z calcd. for C24H30N6O6 [M+H]+ 499.2; found 499.1; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.44 (s, 1H), 8.70 (s, 1H), 8.32 (s, 1H), 5.10 (dd, J=13.3, 5.1 Hz, 1H), 4.55 (d, J=18.5 Hz, 1H), 4.40 (d, J=18.5 Hz, 1H), 3.68-3.49 (m, 4H), 3.34-3.26 (m, 5H), 2.95-2.86 (m, 1H), 2.63-2.54 (m, 2H), 2.43-2.34 (m, 1H), 2.02-1.99 (m, 1H), 1.44 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12088-2 (200 mg, 0.40 mmol), 1,4-dioxane (1 ml) and 4N HCl/1,4-dioxane (4 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12088-3 (200 mg, off-white solid), yield: 102.57%. LCMS (ESI) m/z calcd. for C19H22N6O4 [M+H]+ 399.2; found 399.0. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12088-3 (100 mg, 0.25 mmol), compound TPD12086-9 (143 mg, 0.25 mmol), sodium triacetoxyborohydride (106 mg, 0.50 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-35/7 min to afford the compound TPD12088 (19.5 mg, yellow solid, purity 98.134%) in the following yield: 7.73%. LCMS (ESI) m/z calcd. for C51H50F2N10O7 [M+H]+ 953.4; found 953.2; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.41 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.71 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 8.33 (s, 1H), 8.19 (s, 0.7H), 7.86 (d, J=13.5 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.45 (d, J=8.5 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.45 (d, J=5.2 Hz, 1H), 5.11 (dd, J=13.3, 5.1 Hz, 1H), 4.55 (d, J=18.5 Hz, 1H), 4.41 (d, J=18.5 Hz, 1H), 3.64-3.58 (m, 5H), 2.95-2.78 (m, 8H), 2.67-2.53 (m, 3H), 2.43-2.33 (m, 2H), 2.01-1.99 (m, 1H), 1.87 (dd, J=20.3, 10.1 Hz, 3H), 1.78 (s, 1H), 1.47 (s, 4H), 1.34-1.32 (m, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound TPD12096-1 (2 g, 0.01 mol), bromoacetonitrile (1.44 g, 0.012 mol), K2CO3 (4.15 g, 0.03 mol), and ACN (30 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (90 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the compound TPD12096-2 (2 g, yellow oil, purity 82.6%), yield: 69.00%. LCMS (ESI) m/z calcd. for C12H19N3O2 [M+H]+ 238.2; found 238.2; 1H NMR (400 MHz, DMSO_d MHz, DMSO_d6): δ=4.20 (d, J=9.6 Hz, 1H), 3.82-3.66 (m, 2H), 3.32-3.17 (m, 2H), 3.10 (t, J=10.0 Hz, 1H), 2.81-2.65 (m, 2H), 1.80 (d, J=9.5 Hz, 1H), 1.63 (t, J=9.8 Hz, 1H), 1.39 (d, J=3.5 Hz, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compounds TPD12096-2 (1 g, 4.20 mmol), Raney Ni (0.74 g, 12.6 mol), EtOH (20 ml) and NH H32 O (2 ml). The reaction was carried out for 8 hours at 40° C. under hydrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature, filtered and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12096-3 (0.7 g, yellow oil), yield: 54.76%. LCMS (ESI) m/z calcd. for C12H23N3O2 [M+H]+ 242.2; found 242.2; 1H NMR (400 MHz, DMSO_d MHz, DMSO_d6): δ=4.11 (d, J=9.7 Hz, 1H), 3.04 (dd, J=13.8, 10.1 Hz, 2H), 2.79 (d, J=9.2 Hz, 1H), 2.55-2.52 (m, 1H), 2.50-2.43 (m, 4H), 2.39 (d, J=9.5 Hz, 1H). 1.69 (d, J=9.4 Hz, 1H), 1.58 (dd, J=20.3, 10.2 Hz, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12096-3 (286 mg, 1.18 mmol), compound TPD12003-4 (300 mg, 1.08 mmol), DIEA (208 mg, 1.61 mmol) and DMSO (4 ml). The reaction was carried out under nitrogen protection at 130° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (20 ml) and extracted three times with EA (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD12096-4 (130 mg, yellow solid, purity 72.993%), yield: 18.22%. LCMS (ESI) m/z calcd. for C24H32N6O5 [M+H]+ 485.2; found 485.2; 1H NMR (400 MHz, DMSO_d6): δ=10.96 (s, 1H), 8.34 (s, 1H), 7.13 (s, 1H), 6.58 (s, 1H), 5.76 (s, 1H), 5.02 (dd, J=13.3, 5.0 Hz, 1H), 4.32 (d, J=17.4 Hz, 1H), 4.21-4.11 (m 2H), 3.50 (d, J=8.9 Hz, 1H), 3.17 (d, J=5.1 Hz, 1H), 3.10-3.01 (m, 2H), 2.92-2.83 (m, 2H), 2.59 (ddd, J=32.9, 15.3 Hz, 4H), 2.32 (ddd, J=26.4 13.3, 4.3 Hz, 2H), 1.98-1.90 (m, 1H), 1.67 (dd, J=30.9, 10.4 Hz, 2H), 1.38 (s, 10H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12096-4 (130 mg, 0.27 mmol) and 4N HCl/1,4-dioxane (5 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12096-5 (150 mg, yellow solid), yield: 76.84%. LCMS (ESI) m/z calcd. for C19H24N6O3 [M+H]+ 385.2; found 385.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12096-5 (150 mg, 0.39 mol), compound TPD5488-2 (273 mg, 0.47 mmol), sodium triacetoxyborohydride (165 mg, 0.78 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12096 (54.6 mg, yellow solid, purity 95.636%) in the following yield: 14.07%. LCMS (ESI) m/z calcd. for C52H55FN10O7 [M+H]+ 951.4; found 951.4; 1H NMR (400 MHz, DMSO_d6): δ=10.99 (s, 1H), 10.19 (s, 1H), 10.07 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.37 (s, 1H). 8.22 (s, 2H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 7.23-7.12 (m, 5H), 6.60 (s, 1H), 6.39 (d, J=5.2 Hz, 1H), 5.03 (dd. J=13.3, 5.1 Hz, 1H), 4.33 (d, J=17.4 Hz, 1H), 4.19 (d, J=17.4 Hz, 1H), 3.94 (s, 3H), 3.59 (d, J=10.7 Hz, 2H), 3.48 (s, 1H), 3.41 (s, 3H), 2.83-2.57 (m, 9H), 2.39-2.26 (m, 2H), 1.98-1.90 (m, 1H), 1.85 (d, J=11.3 Hz, 2H), 1.69 (s, 2H), 1.56 (s, 1H), 1.47 (s, 4H), 1.35 (s, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound TPD12097-1 (2 g, 0.01 mol), bromoacetonitrile (1.44 g, 0.012 mol), K2CO3 (4.15 g, 0.03 mol), and ACN (30 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (90 ml) and extracted three times with EA (60 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the compound TPD12097-2 (2 g, yellow oil, purity 81.383%), yield: 68%. LCMS (ESI) m/z calcd. for C12H19N3O2 [M+H]+ 238.2; found 237.9; 1H NMR (400 MHz, DMSO_d6): δ=4.20 (d, J=9.6 Hz, 1H), 3.81-3.66 (m, 2H), 3.55 (s, 1H), 3.31 (d, J=10.5 Hz, 1H), 3.10 (t, J=10.2 Hz, 1H), 2.79 (d, J=8.4 Hz, 1H), 2.69 (d, J=9.4 Hz, 1H), 1.80 (d, J=8.8 Hz, 1H), 1.63 (t, J=9.9 Hz, 1H), 1.39 (d, J=3.2 Hz, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compounds TPD12097-2 (2 g, 8.43 mol), Raney Ni (0.59 g), EtOH (24 ml) and NH H2O (3 ml). The reaction was carried out for 16 hours at 40° C. under hydrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature, filtered and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=120/1˜30/1 to afford compound TPD12097-3 (1.8 g, yellow oil, purity 56.329%), yield: 50.00%. LCMS (ESI) m/z calcd. for C12H23N3O2 [M+H]+ 242.2; found 242.1; 1H NMR (400 MHz, DMSO_d6): δ=4.12 (d, J=10.7 Hz, 1H), 3.40 (s, 1H), 3.30 (dd, J=9.0, 6.3 Hz, 1H), 3.09-3.02 (m, 1H), 2.79 (d, J=9.3 Hz, 1H), 2.54-2.38 (m, 6H). 1.69 (d, J=9.5 Hz, 1H), 1.63-1.56 (m, 1H), 1.39 (s, 10H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12097-3 (334 mg, 1.38 mmol), compound TPD12003-4 (350 mg, 1.25 mmol), DIEA (323 mg, 2.50 mmol) and DMSO (4 ml). The reaction was carried out under nitrogen protection at 130° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (20 ml) and extracted three times with EA (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD12097-4 (52 mg, yellow solid, purity 42.921%), yield: 48.91%. LCMS (ESI) m/z calcd. for C23H32N6O5 [M+H]+ 485.2; found 485.1. The compound structure is confirmed.
Compound TPD12097-4 (150 mg, 0.31 mmol) 4N HCl/1,4-dioxane (4 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12097-5 (280 mg, yellow solid), yield: 119.52%. LCMS (ESI) m/z calcd. for C18H24N6O3 [M+H]+ 385.2; found 385.2. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12097-5 (280 mg, 0.73 mmol), compound TPD5488-2 (424 mg, 0.73 mmol), sodium triacetoxyborohydride (309 mg, 1.46 mmol) and DCE (8 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (40 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give compound TPD12097 (6.68 mg, yellow solid, purity 97.125%) in the following yield: 0.93%. LCMS (ESI) m/z calcd. for C51H55FN10O7 [M+H]+ 951.4; found 951.4; 1H NMR (400 MHz, DMSO_d6): δ=10.98 (s, 1H), 10.19 (s, 1H), 10.07 (s, 1H), 8.46-8.17 (m, 3H), 7.76 (d, J=7.7 Hz, 2H), 7.64 (s, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 7.25-7.03 (m, 4H), 6.59 (s, 1H), 6.39 (s, 1H), 5.03 (d, J=8.5 Hz, 1H), 4.33 (d, J=17.6 Hz, 1H), 4.18 (d, J=17.1 Hz, 1H), 3.94 (s, 3H), 3.57 (s, 10H), 2.68 (d, J=12.0 Hz, 8H), 1.95 (s, 2H), 1.84 (s, 2H), 1.61 (s, 2H), 1.47 (s, 5H), 1.24 (s, 5H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound sm-1 (1 g, 0.005 mol), 2-bromoacetamide (0.72 g, 0.0052 mol), K2CO3 (1.04 g, 0.0075 mol), and ACN (10 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with EA (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to obtain the compound TPD12100-1 (1.2 g, white solid, purity 67.57%), yield: 64.00%. LCMS (ESI) m/z calcd. for C12H21N3O3 [M+H]+ 256.16; found 256.1; 1H NMR (400 MHz, DMSO_d6): δ=7.22 (s, 1H), 7.10 (s, 1H), 4.13 (t, J=10.7 Hz, 1H), 3.81 (s, 1H), 3.43 (s, 1H), 3.31 (d, J=4.4 Hz, 1H), 3.04 (dd, J=31.6, 18.1 Hz, 3H), 2.83 (d, J=8.9 Hz, 1H), 1.75 (d, J=9.5 Hz, 1H), 1.63-1.53 (m, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12100-1 (450 mg, 1.76 mmol), compound TPD12003-4 (737 mg, 2.63 mmol), XantPhos (203 mg, 0.35 mmol), Pd(OAc)2 (79 mg, 0.35 mmol), CS2CO3 (1430 mg, 4.39 mmol) and 1,4-dioxane (5 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=12/1 to afford the compound TPD12100-2 (180 mg, yellow solid, purity 98.889%), yield: 20.30%. LCMS (ESI) m/z calcd. for C24H30N6O6 [M+H]+ 499.22; found 499.1; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.30 (s, 1H), 8.70 (s, 1H), 8.33 (s, 1H), 7.17 (d, J=47.5 Hz, 3H), 5.76 (s, 1H), 5.11 (dd, J=13.3, 5.0 Hz, 1H), 4.55 (d, J=18.5 Hz, 1H), 4.41 (d, J=18.6 Hz, 1H), 4.18 (dd, J=23.4, 12.7 Hz, 2H), 3.63-3.37 (m, 5H), 2.63 (d, J=9.3 Hz, 2H), 2.07-1.96 (m, 1H), 1.41 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12100-2 (180 mg, 0.36 mmol) and 4N HCl/1,4-dioxane (5 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12100-3 (200 mg, yellow solid, purity 70.214%), yield: 97.84%. LCMS (ESI) m/z calcd. for C19H22N6O4 [M+H]+ 399.17; found 399.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12100-3 (200 mg, 0.50 mmol), compound TPD5488-2 (273 mg, 0.47 mmol), sodium triacetoxyborohydride (213 mg, 1.00 mmol) and DCE (4 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give the compound TPD12100 (48.8 mg, yellow solid, purity 98.482%) in the following yield: 9.92%. LCMS (ESI) m/z caled. for C52H53FN10O8 [M+H]+ 965.4; found 965.4; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.26 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.71 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.35 (s, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.47 (s, 1H), 7.32 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.39 (d J=5.2 Hz, 1H), 5.11 (dd, J=13.2, 5.1 Hz, 1H), 4.56 (d, J=18.4 Hz, 1H), 4.41 (d, J=18.6 Hz, 1H), 3.95 (s, 4H), 3.60 (d, J=8.6 Hz, 5H), 2.88-2.54 (m, 9H), 2.37 (dd, J=17.8, 9.8 Hz, 4H), 2.04-1.97 (m, 1H), 1.88 (s, 2H), 1.71 (s, 2H), 1.47 (s, 5H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound sm-1 (500 mg, 2.51 mmol), 2-bromoacetamide (363 mg, 2.63 mmol), K2CO3 (520 mg, 3.76 mmol), and ACN (8 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (10 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the compound TPD120101-1a (500 mg, white solid, purity 94.532%), yield: 73.49%. LCMS (ESI) m/z calcd. for C12H21N3O3 [M+H]+ 256.2; found 255.9; 1H NMR (400 MHz, DMSO_d6) 6=7.16 (d, J=46.2 Hz, 2H), 4.15 (d, J=12.1 Hz, 1H), 3.43 (s, 1H), 3.30 (s, 1H), 3.19-2.95 (m, 3H), 2.82 (dd, J=9.4, 1.7 Hz, 1H), 2.48 (s 1H), 1.75 (d, J=9.5 Hz, 1H), 1.65-1.55 (m, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12101-1a (500 mg, 1.97 mmol), compound TPD12003-4 (550 mg, 1.97 mmol), palladium acetate (88 mg, 0.39 mmol), XantPhos (28 mg, 0.39 mmol), Cs2 CO3 (961 mg. 2.95 mmol) and 1,4-dioxane (15 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, filtered with water (50 ml), the filter cake was washed with ethyl ester, the filtrate was extracted with ethyl ester (60 ml) three times, the organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatography column with the ratio of the eluents as DCM/MeOH=100/1˜50/1. The crude product was purified by thin-layer chromatography plate. Purification, the unfolding agent ratio was DCM/MeOH=15/1 to obtain the compound TPD12101-2a (250 mg, yellow solid, purity 85.589%), yield: 21.78%. LCMS (ESI) m/z calcd. for C24H30N6O6 [M+H]+ 499.2; found 499.1; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.30 (s, 1H), 5.76 (s, 1H), 5.10 (dd, J=13.3, 5.1 Hz, 1H), 4.55 (d, J=18.5 Hz, 1H), 4.41 (d, J=18.5 Hz, 1H), 3.59 (s, 1H), 3.44 (t, J=10.3 Hz, 3.44 (t, J=10.3 Hz, 3H), 3.12-2.99 (m, 5H), 2.82 (dd, J=9.4, 1.7 Hz, 2H), 2.63 (d, J=9.4 Hz, 2H), 2.05-1.95 (m, 1H), 1.83 (d, J=9.7 Hz, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12101-2a (250 mg, 0.50 mmol) and 4 N HCl/1,4-dioxane (8 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12101-3a (250 mg, yellow solid, purity 79.403%), yield: 99.54%. LCMS (ESI) m/z calcd. for C19H22N6O4 [M+H]+ 399.2; found 399.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12101-3a (125 mg, 0.31 mmol), compound TPD5488-2 (183 mg, 0.31 mmol), sodium triacetoxyborohydride (133 mg, 0.63 mmol) and DCE (6 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (10 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12101 (20.89 mg, yellow solid, purity 98.574%) in the following yield: 6.79%. LCMS (ESI) m/z calcd. for C52H53FN10O8 [M+H]+ 965.4; found 483.2, 965.4; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.27 (s, 1H), 10.19 (s, 1H), 10.07 (s, 1H), 8.71 (s, 1H). 8.43 (d, J=5.2 Hz, 1H), 8.35 (s, 1H), 8.20 (s, 2H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.47 (s, 1H), 7.32 (s, 1H), 7.25-7.10 (m, 4H), 6.39 (d, J=5.1 Hz, 1H), 5.11 (dd, J=13.2, 5.1 Hz, 1H), 4.56 (d, J=18.7 Hz, 1H), 4.42 (d, J=18.5 Hz, 1H), 3.95 (s, 3H), 3.60 (d, J=8.6 Hz, 2H), 3.44 (s, 4H), 3.05-2.74 (m 5H), 2.63 (s, 4H), 2.00 (d, J=5.2 Hz, 1H), 1.88 (s, 2H), 1.74 (s, 2H), 1.52-1.23 (m, 7H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12100-3a (105 mg, 0.26 mmol), compound TPD12086-9 (150 mg, 0.26 mmol), sodium triacetoxyborohydride (111 mg, 0.53 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (10 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12102 (23.1 mg, yellow solid, purity 96.732%), yield: 8.14%.
LCMS (ESI) m/z calcd. for C51H50F2N10O7 [M+H]+ 953.38; found 953.3; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.23 (d, J=23.6 Hz, 2H), 10.06 (s, 1H), 8.71 (s, 1H). 8.56 (d, J=5.2 Hz, 1H), 8.35 (s, 1H), 7.86 (d, J=13.5 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.45 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.45 (d, J=5.2 Hz, 1H), 5.11 (dd, J=13.2, 5.1 Hz, 1H), 4.56 (d, J=18.6 Hz, 1H), 4.41 (d, J=18.4 Hz, 1H), 3.58 (d, J=10.1 Hz, 2H), 3.42 (s, 3H), 2.95-2.84 (m, 2H), 2.77 (t, J=8.4 Hz, 3H), 2.69 (d, J=10.0 Hz, 1H), 2.65-2.53 (m, 2H), 2.48-2.30 (m, 4H), 2.03-1.96 (m, 1H), 1.92 (d, J=4.6 Hz, 2H). 1.70 (s, 2H), 1.58 (s, 1H), 1.47 (s, 4H), 1.36 (s, 2H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound sm-1 (300 mg, 1.51 mmol), acrylamide (139 mg, 1.96 mmol), potassium carbonate (312 mg, 2.26 mmol) and methanol (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was concentrated to dryness and the residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=15/1 to afford the compound TPD12110-1 (400 mg, colorless oily substance) in the yield: 88.45%. LCMS (ESI) m/z calcd. for C13H23N3O3 [M+H]+ 270.2; found 270.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12110-1 (300 mg, 1.11 mmol), compound TPD12003-4 (310 mg, 1.11 mmol), palladium acetate (50 mg, 0.22 mmol), XantPhos (128 mg, 0.22 mmol), cesium carbonate (542 mg, 1.66 mmol) and 1,4-dioxane (6 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature and concentrated to dryness. The residue was purified by column chromatography with eluent and ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12110-2 (100 mg, yellow solid, purity 88.457%), yield: 11.64%. LCMS (ESI) m/z calcd. for C25H32N6O6 [M+H]+ 513.2; found 513.1; 1H NMR (400 MHz, DMSO_d6): δ=11.80 (s, 1H), 11.58 (s, 1H), 8.76 (s, 1H), 8.36 (s, 1H), 8.10 (s, 1H), 5.19 (dd, J=13.3, 5.1 Hz, 1H), 4.42 (dd, J=62.6, 16.8 Hz, 2H), 3.75-3.59 (m, 2H), 3.27 (d, J=10.1 Hz, 1H), 3.09-2.78 (m, 5H), 2.56-2.48 (m, 2H), 2.36 (qd, J=13.1, 4.8 Hz, 1H), 2.27-2.16 (m, 1H), 2.11-1.95 (m, 1H), 1.91-1.80 (m, 1H), 1.54-1.40 (m, 10H). The compound structure is confirmed.
Compound TPD12110-2 (100 mg, 0.19 mmol) and 4N HCl/1,4-dioxane (6 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12110-3 (100 mg, off-white solid), yield: 96.97%. LCMS (ESI) m/z calcd. for C20H24N6O4 [M+H]+ 413.2; found 413.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12110-3 (85 mg, 0.206 mmol), compound TPD5488-2 (120 mg, 0.206 mmol), sodium triacetoxyborohydride (87 mg, 0.412 mmol) and DCE (4 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml). The organic phase was separated and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was separated by preparative plate (DCM/MeOH=10/1) to afford the compound TPD12110 (16.3 mg, yellow solid, purity 98.666%), yield: 7.09%. LCMS (ESI) m/z calcd. for C53H55FN10O8 [M+H]+ 979.4; found 490.2; 979.4; 1H NMR (400 MHz, DMSO_d6): δ=11.37-11.24 (m, 1H), 11.03 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.69 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.34 (s, 1H), 7.76 (d, J=8.7 Hz, 2H)), 7.64 (dd, J=8.7, 5.1 Hz, 2H), 7.47 (s, 1H), 7.32 (s, 1H), 7.27-7.10 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 5.10 (dd, J=13.2, 5.0 Hz, 1H), 4.47 (dd, J=58.2, 18.4 Hz 2H), 3.94 (s, 3H), 3.67-3.40 (m, 5H), 3.02-2.72 (m, 7H), 2.69-2.55 (m, 6H), 2.44-2.25 (m, 1H), 2.06-1.95 (m, 1H), 1.94-1.81 (m, 2H), 1.80-1.67 (m, 2H). 1.67-1.55 (m, 1H), 1.47 (s, 4H), 1.43-1.28 (m, 2H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound sm-1 (500 mg, 2.51 mmol), acrylamide (232 mg, 3.26 mmol), potassium carbonate (520 mg, 3.76 mmol) and methanol (15 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (50 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12112-1 (340 mg, white solid, purity 97.539%) in the yield: 48.89%. LCMS (ESI) m/z calcd. for C13H23N3O3 [M+H]+ 270.2; found 269.9; 1H NMR (400 MHz, DMSO_d6): δ=7.33 (s, 1H), 6.76 (s, 1H), 3.94 (d, J=5.8 Hz, 2H), 3.00-2.89 (m, 2H), 2.78-2.66 (m, 4H). 2.29-2.19 (m, 3H), 1.52 (d, J=7.9 Hz, 1H), 1.38 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-1 (300 mg, 1.11 mmol), compound TPD12003-4 (310 mg, 1.11 mmol), palladium acetate (50 mg, 0.22 mmol), XantPhos (128 mg, 0.22 mmol), cesium carbonate (542 mg, 1.66 mmol) and 1,4-dioxane (5 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with EA (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with unfolding agent ratio DCM/MeOH=15/1 to afford compound TPD12112-2 (90 mg, brown solid, purity 98.588%), yield: 15.57%. LCMS (ESI) m/z calcd. for C25H32N6O [M+H]+ 513.2; found 513.2; 1H NMR (400 MHz, DMSO_d6): δ=11.07 (s, 1H), 11.01 (s, 1H), 8.67 (s, 1H), 8.33 (s, 1H), 5.10 (dd, J=13.3, 5.1 Hz, 1H), 4.53 (d, J=18.4 Hz, 1H), 4.38 (d, J=18.4 Hz, 1H). 3.97 (d, J=5.7 Hz, 2H), 2.95-2.81 (m, 7H), 2.63-2.59 (m, 3H), 2.43-2.33 (m, 1H), 2.28 (dd, J=13.5, 6.2 Hz, 1H), 2.02-1.97 (m, 1H), 1.61 (d, J=7.9 Hz, 1H). 1.38 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-2 (90 mg, 0.18 mmol), 1,4-dioxane (1 ml) and 4N HCl/1,4-dioxane (4 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12112-3 (100 mg, off-white solid), yield: 111.99%. LCMS (ESI) m/z calcd. for C20H24N6O4 [M+H]+ 413.2; found 413.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-3 (100 mg, 0.24 mmol), compound TPD5488-2 (141 mg, 0.24 mmol), sodium triacetoxyborohydride (103 mg, 0.485 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: T3 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-35/7 min to afford the compound TPD12112 (21.3 mg, yellow solid, purity 97.980%) in the following yield: 8.25%. LCMS (ESI) m/z calcd. for C53H55FN10O8 [M+H]+ 979.4; found 490.3; 979.3; 1H NMR (400 MHz, DMSO_d6): δ=11.18 (s, 1H), 11.04 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.68 (s, 1H), 8.43 (d J=5.2 Hz, 1H), 8.36 (s, 1H), 8.21 (s, 1.4H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.46 (s, 1H), 7.31 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.39 (d, J=5.2 Hz, 1H), 5.09 (dd, J=13.3, 5.0 Hz, 1H), 4.53 (d, J=18.5 Hz, 1H), 4.39 (d, J=18.4 Hz, 1H), 3.94 (s, 3H), 3.56 (d, J=12.3 Hz, 5H), 2.99 (dd, J=17.8, 8.9 Hz, 4H), 2.93-2.85 (m, 3H), 2.67-2.58 (m, 5H), 2.41-2.33 (m, 4H), 1.99-1.94 (m, 1H), 1.89 (d, J=7.9 Hz, 1H), 1.80 (d, J=11.2 Hz, 2H), 1.47 (s 4H), 1.40-1.32 (m, 2H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound sm-1 (600 mg, 3.01 mmol), 3-bromopropionamide (595 mg, 3.91 mmol), K2CO3 (624 mg, 4.52 mmol), and ACN (15 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (10 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the compound TPD120113-1 (650 mg, white solid, purity 74.165%), yield: 59.22%. LCMS (ESI) m/z calcd. for C13H23N3O3 [M+H]+ 270.2; found 269.9; 1H NMR (400 MHz, DMSO_d6): δ=6.75 (s, 1H), 3.54-3.38 (m, 6H), 3.24-3.16 (m, 2H), 2.47 (s, 1H), 2.32 (dd, J=13.8, 6.0 Hz, 1H), 2.06 (t, J=7.1 Hz, 2H), 1.42 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12113-1 (650 mg, 2.32 mmol), compound TPD12003-4 (628 mg, 2.32 mmol), palladium acetate (104 mg, 0.46 mmol), XantPhos (268.94 mg, 0.46482 mmol), Cs2 CO3 (1136 mg, 3.49 mmol) and 1,4-dioxane (15 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, filtered with water (50 ml), the filter cake was washed with ethyl ester, the filtrate was extracted with ethyl ester (60 ml) three times, the organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatography column with the ratio of the eluents as DCM/MeOH=100/1˜50/1. The crude product was purified by thin-layer chromatography plate. Purification, the unfolding agent ratio was DCM/MeOH=15/1 to obtain the compound TPD12113-2 (250 mg, yellow solid, purity 94.269%), yield: 21.32%. LCMS (ESI) m/z calcd. for C25H32N6O6 [M+H]+ 513.2; found 513.3; 1H NMR (400 MHz. DMSO_d6): δ 11.03 (s, 2H), 8.69 (s, 1H), 8.33 (s, 1H), 5.75 (s, 1H), 5.09 (dd, J=13.3, 5.1 Hz, 1H), 4.59-4.33 (m, 2H), 3.53 (dd, J=20.2, 12.3 Hz, 4H), 3.28-3.20 (m, 2H), 2.98-2.86 (m, 1H), 2.72-2.57 (m, 3H), 2.46-2.26 (m, 3H), 2.04-1.95 (m, 1H), 1.43 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12113-2 (270 mg, 0.53 mmol) and 4N HCl/1,4-dioxane (8 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12113-3 (270 mg, yellow solid, purity 94.118%), yield: 117.2.%. LCMS (ESI) m/z calcd. for C20H24N6O4 [M+H]+ 413.2; found 413.1. as seen. the structure of the compound is correct.
To a 25 ml single-necked vial were sequentially added compound TPD12113-3 (135 mg, 0.33 mmol), compound TPD5488-2 (191 mg, 0.3273 mmol), sodium triacetoxyborohydride (139 mg, 0.65 mmol) and DCE (6 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12113 (7.46 mg, yellow solid, purity 97.940%) in the following yield: 2.29%. LCMS (ESI) m/z calcd. for C53H55FN10O8 [M+H]+ 979.4; found 490.3, 979.4; 1H NMR (400 MHz, DMSO_d6): δ=11.19 (s, 1H), 11.02 (s, 1H), 10.19 (s, 1H), 10.07 (s, 1H), 8.69 (s, 1H). 8.43 (d, J=5.1 Hz, 1H), 8.34 (s, 1H), 8.22 (s, 2H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 7.23-7.12 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 5.10 (dd, J=13.2, 5.1 Hz, 1H), 4.54 (d, J=18.3 Hz, 1H), 4.39 (d, J=18.6 Hz, 1H), 3.94 (s, 3H), 3.17 (s, 1H), 2.87-2.68 (m, 5H), 2.79-2.53 (m, 6H). 2.33-2.40 (m, 4H), 2.09-1.94 (m, 2H), 1.91-1.68 (m, 5H), 1.47 (s, 4H), 1.34-1.23 (m, 3H). The compound structure is confirmed.
Compound sm-1 (500 mg, 2.51 mmol), acrylonitrile (160 mg, 3.01 mmol) and methanol (10 ml) were added sequentially in a 50 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (50 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford the compound TPD12116-1 (560 mg, colorless oil, purity 93.352%) in the yield: 82.57%. LCMS (ESI) m/z calcd. for C13H21N3O2 [M+H]+ 252.2; found 251.8; 1H NMR (400 MHz, DMSO_d6): δ=4.14 (d, J=9.9 Hz, 1H), 3.51 (s, 1H), 3.25 (dd, J=10.0, 5.2 Hz, 2H), 3.10-3.04 (m, 1H). 2.82-2.79 (m, 1H), 2.74-2.68 (m, 2H), 2.58 (t, J=6.5 Hz, 2H), 1.71-1.59 (m, 2H), 1.39 (s, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added the compounds TPD12116-1 (560 mg, 2.22 mmol), EtOH/NH H32 O=10/1 (10 ml) and Raney nickel (130 mg, 2.22 mmol). The reaction was carried out for 16 h at 40° C. under the protection of hydrogen (1 atm). After completion of the reaction, the reaction solution was filtered and concentrated to dryness to give the crude compound TPD12116-2 (530 mg, colorless oil, purity 83.869%), yield: 78.13%. LCMS (ESI) m/z calcd. for C13H25N3O2 [M+H]+ 256.2; found 255.8. The compound structure is confirmed.
To a 25 ml three-necked vial was sequentially added compound TPD12116-2 (500 mg, 1.95 mmol), compound TPD12003-4 (545 mg, 1.95 mmol), DIEA (504 mg, 3.90 mmol) and DMSO (5 ml). The reaction was carried out under nitrogen protection at 130° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (30 ml) and extracted three times with EA (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=8/1 to afford the compound TPD12116-3 (200 mg, brown solid, purity 83.106%), yield: 17.06%. LCMS (ESI) m/z calcd. for C25H34N6O5 [M+H]+ 499.2; found 499.1; 1H NMR (400 MHz, DMSO_d6): δ=10.97 (s, 1H), 8.35 (s, 1H), 7.21 (t, J=5.0 Hz, 1H), 6.54 (s, 1H), 5.02 (dd, J=13.3, 5.0 Hz, 1H), 4.32 (d, J=17.3 Hz, 1H), 4.20-4.12 (m, 2H)), 3.35-3.30 (m, 4H), 3.05 (t, J=11.3 Hz, 1H), 2.94-2.79 (m, 2H), 2.60-2.53 (m, 2H), 2.42-2.27 (m, 3H), 1.96-1.93 (m, 1H), 1.71 (d, J=9.1 Hz, 1H), 1.65-1.58 (m, 3H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12116-3 (200 mg, 0.40 mmol), 1,4-dioxane (1 ml) and 4N HCl/1,4-dioxane (4 ml). The reaction was carried out under nitrogen protection at 25° C. for 2 hours. After completion of the reaction it was concentrated to dryness to give the crude compound TPD12116-4 (200 mg, off-white solid), yield: 90.98%. LCMS (ESI) m/z calcd. for C20H26N6O [M+H]+ 399.2; found 399.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12116-4 (100 mg, 0.25 mmol), compound TPD5488-2 (146 mg, 0.25 mmol), sodium triacetoxyborohydride (106 mg, 0.50 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 5-40/7 min to afford compound TPD12116 (4.65 mg, yellow solid, purity 98.518%) in 1.71% yield. LCMS (ESI) m/z calcd. for C53H57FN10O7 [M+H]+ 965.4; found 483.3; 966.4; 1H NMR (400 MHz, DMSO_d6): δ=11.00 (s, 1H), 10.20 (s, 1H), 10.07 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.36 (s, 1H). 8.25 (s, 2H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.46 (s, 1H), 7.31 (d, J=16.8 Hz, 2H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.57 (s 1H), 6.39 (d, J=5.2 Hz, 1H), 5.03 (dd, J=13.3, 5.1 Hz, 1H), 4.33 (d, J=17.3 Hz, 2H), 4.19 (d, J=17.3 Hz, 2H), 3.94 (s, 3H), 3.59 (d, J=11.3 Hz, 5H), 3.44 (s, 1H). 3.36 (s, 2H), 2.94-2.80 (m, 5H), 2.74 (d, J=9.1 Hz, 1H), 2.66-2.60 (m, 3H), 2.38-2.27 (m, 2H), 1.95-1.92 (m, 1H), 1.85 (d, J=10.7 Hz, 2H), 1.76 (s, 3H), 1.55 (br. s, 1H), 1.47 (s, 4H), 1.35 (br. s, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound sm-1 (1 g, 0.005 mol), acrylonitrile (0.27 g, 0.005 mol) and MeOH (10 ml). The reaction was carried out under nitrogen protection at 65° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (30 ml) and extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the compound TPD12118-1 (0.9 g, white solid, purity 79.943%), yield: 56.00%. LCMS (ESI) m/z calcd. for 13H21N3O2 [M+H]+ 252.16; found 252.1; 1H NMR (400 MHz, DMSO_d6): δ=3.59-3.40 (m, 4H), 3.23 (dd, J=18.2, 12.1 Hz, 2H), 2.52 (d, J=13.1 Hz, 5H), 2.38 (d, J=7.5 Hz, 1H), 1.43 (s, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compounds TPD12118-1 (0.9 g, 0.0036 mol), Raney Ni (0.63 g, 0.0108 mol), EtOH (20 ml) and NH H32 O (2 ml). The reaction was carried out for 8 hours at 40° C. under hydrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature, filtered and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to obtain the compound TPD12118-2 (0.7 g, yellow oil, purity 81.874%), yield: 61.11%. LCMS (ESI) m/z calcd. for C13H25N3O2 [M+H]+ 256.19; found 256.1; 1H NMR (400 MHz, CDCl3): δ=7.27 (s, 1H), 3.56 (s, 2H), 3.35 (dd, J=18.5, 6.3 Hz, 3H), 2.77 (t, J=6.8 Hz, 2H), 2.47 (t, J=7.2 Hz, 2H), 1.75 (s, 6H), 1.49 (d, J=4.2 Hz, 9H). The compound structure is confirmed.
Compound TPD12118-2 (500 mg, 2.06 mmol), compound TPD12003-4 (582 mg, 2.27 mmol), DIEA (400 mg, 3.09 mmol), and DMSO (6 ml) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 130° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (30 ml) and extracted three times with EA (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=8/1 to afford the compound TPD12118-3 (65 mg, yellow solid, purity 93.786%), yield: 5.91%. LCMS (ESI) m/z calcd. for C25H34N6O5 [M+H]+ 499.26; found 499.1; 1H NMR (400 MHz, DMSO_d6): δ=10.96 (s, 1H), 8.34 (s, 1H), 7.21 (d, J=7.1 Hz, 1H), 6.54 (s, 1H), 5.76 (s, 2H), 5.02 (dd, J=13.3, 5.0 Hz, 1H), 4.24 (dd, J=58.0, 17.4 Hz, 2H), 3.43 (dd, J=29.2, 10.7 Hz, 3H), 3.19 (d, J=12.3 Hz, 2H), 2.90-2.82 (m, 1H), 2.63 (d, J=30.7 Hz, 2H), 2.34 (dd, J=14.9, 10.0 Hz, 4H), 1.95 (s, 2H), 1.58 (d, J=7.3 Hz, 2H), 1.44-1.38 (m, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12118-3 (65 mg, 0.13 mmol) and 4N HCl/1,4-dioxane (3 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12118-4 (80 mg, yellow solid), yield: 77.09%. LCMS (ESI) m/z calcd. for C20H26N6O3 [M+H]+ 399.21; found 399.0. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12118-4 (80 mg, 0.20 mmol), compound TPD5488-2 (117 mg, 0.20 mmol), sodium triacetoxyborohydride (85 mg, 0.40 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12118 (6.21 mg, yellow solid, purity 98.746%) in the following yield: 2.89%. LCMS (ESI) m/z calcd. for C53H57FN10O7 [M+H]+ 965.44; found 965.4; 1H NMR (400 MHz, CD3 OD): δ=8.57-8.46 (m, 2H), 7.81 (d, J=8.9 Hz, 2H), 7.76 (s, 1H), 7.56 (dd, J=8.9, 4.8 Hz, 2H). 7.36-7.28 (m, 3H), 7.07 (t, J=8.7 Hz, 2H), 6.80 (d, J=6.6 Hz, 2H), 5.09 (dd, J=13.4, 5.0 Hz, 1H), 4.55-4.30 (m, 4H), 4.09 (s, 3H), 3.94 (d, J=12.2 Hz, 2H), 3.62-3.36 (m, 5H), 3.30-3.13 (m, 4H), 2.97-2.58 (m, 7H), 2.49-2.35 (m, 2H), 2.04 (dd, J=75.8, 6.6 Hz, 7H), 1.65 (s, 5H), 1.44 (d, J=30.5 Hz, 2H), 1.30 (d, J=8.9 Hz, 1H). The compound structure is confirmed.
To a 250 ml triple-necked flask was sequentially added compound sm-1 (25 g, 0.1295 mol), concentrated sulfuric acid (125 ml) and concentrated nitric acid (12.5 ml). The reaction was carried out under nitrogen protection at 50° C. for 2 hours. After the reaction was completed, the reaction solution was reduced to room temperature, adjusted to weak basicity with NaOH solution and extracted three times with DCM (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=20/1˜5/1 to obtain the compound int-P-1 (15 g, off-white solid, purity 99.721%), yield: 48.57%. LCMS (ESI) m/z calcd. for C6H2BrF2NO2 [M+H]+ 237.9; found 353.1; 1H NMR (400 MHz, CDCl3): δ=8.20-8.09 (m, 1H), 7.16-7.11 (m, 1H). The compound structure is confirmed.
Compound int-P-1 (14 g, 58.8 mmol) and methanol (200 ml) were added sequentially in a 500 ml three-necked flask. Under nitrogen protection, it was lowered to 0° C. and sodiummethanol (7.94 g, 147 mmol) was added in batches and reacted at 0° C. for 3 hours. After the reaction was completed, the reaction solution was poured into water (600 ml) and extracted three times with EA (200 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=20/1˜5/1 to obtain the compound int-P-2 (6 g, white solid, purity 99%) in the yield of 40.48%. LCMS (ESI) m/z calcd. for C7H5BrFNO3 [M+H]+ 249.9; found 263.9; 1H NMR (400 MHz, CDCl3): δ=8.14 (dd, J=9.2, 8.5 Hz, 1H), 6.81 (dd, J=9.4, 1.5 Hz, 1H), 4.04 (s, 3H). The compound structure is confirmed.
To a 250 ml triple-necked flask were sequentially added compound int-P-2 (6 g, 24.0 mmol), EtOH/H2O=3/1 (120 ml), iron powder (6.70 g, 120 mmol) and ammonium chloride (6.42 g, 120 mmol). The reaction was carried out under nitrogen protection at 80° C. for 4 hours. After the reaction was completed, the reaction solution was reduced to room temperature, filtered, and the filtrate was poured into water (300 ml) and extracted three times with EA (200 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=20/1˜5/1 to obtain the compound int-P-3 (4.7 g, yellow oil, purity 99.619%) in the yield: 88.75%. LCMS (ESI) m/z caled. for C7H7BrFNO [M+H]+ 219.9; found 219.9; 1H NMR (400 MHz, DMSO_d6): δ=6.77-6.70 (m, 2H), 4.91 (s, 2H), 3.73 (s, 3H). The compound structure is confirmed.
To a 250 ml triple-necked vial were sequentially added compound int-P-3 (4.7 g, 21.4 mmol), EtOH (100 ml) and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (4.78 g, 25.7 mmol). The reaction was refluxed under nitrogen protection for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature and filtered by extraction to give compound int-P-4 (7.8 g, off-white solid, purity 99.659%), yield: 97.20%. LCMS (ESI) m/z calcd. for C14H13BrFNO5 [M+H]+ 374.0; found 317.8; 1H NMR (400 MHz, DMSO_d6): δ=11.25 (d, J=14.3 Hz, 1H), 8.55 (d, J=14.3 Hz, 1H), 7.82 (t, J=9.1 Hz, 1H), 7.05 (d, J=8.3 Hz, 1H), 3.91 (s, 3H), 1.68 (s, 6H). The compound structure is confirmed.
Compound int-P-4 (7.8 g, 20.8 mmol) and diphenyl ether (100 ml) were added sequentially in a 250 ml single-necked vial. The reaction was carried out at 240° C. for 1 h under nitrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature and the compound int-P-5 (3.7 g, brown solid, purity 99.415%) was obtained by filtration, yield: 64.90%. LCMS (ESI) m/z calcd. for C10H7BrFNO2 [M+H]+ 271.9; found 271.9; 1H NMR (400 MHz, DMSO_d6). 6=11.95 (s, 1H), 7.83 (t, J=6.3 Hz, 1H), 7.39 (d, J=7.8 Hz, 1H), 6.09 (d, J=7.2 Hz, 1H), 3.95 (s, 3H). The compound structure is confirmed.
To a 100 ml triple-necked flask was added sequentially compound int-P-5 (3.6 g, 13.2 mmol), toluene (50 ml) and POCl3 (6.07 g, 39.5 mmol). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, concentrated and adjusted to weak basicity with saturated NaHCO3 solution and extracted three times with DCM (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with the eluent ratio of PE/EtOAc=50/1˜30/1 to obtain the compound int-P-6 (2.7 g, off-white solid, purity 99.539%) in the yield of 70.45%. LCMS (ESI) m/z calcd. for C10H6BrClFNO [M+H]+ 289.9; found 289.9; 1H NMR (400 MHz, DMSO_d6): δ=8.73 (d, J=4.7 Hz, 1H), 7.86 (d, J=4.7 Hz, 1H), 7.30 (s, 1H), 4.06 (s, 3H), which shows that the compound is structurally correct.
To a 100 ml triple-necked flask were sequentially added compounds int-P-6 (1.8 g, 6.2 mmol), int-A-2 (1.95 g, 6.2 mmol), cesium carbonate (4.04 g, 12.4 mmol) and DMSO (40 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, decanted into water (200 ml) and extracted three times with DCM (100 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of PE/EtOAc=5/1˜2/1 to give compound int-P (0.9 g, off-white solid, purity 92.536%), yield: 24.19%. LCMS (ESI) m/z calcd. for C27H20BrF2N3O4 [M+H]+ 568.1; found 568.0; 1H NMR (400 MHz, DMSO_d6): δ=10.23 (s, 1H), 10.05 (s, 1H), 8.59 (d, J=5.1 Hz, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.65 (dd, J=8.9, 5.1 Hz, 2H), 7.52 (s, 1H), 7.28 (d, J=8.9 Hz, 2H), 7.16 (t, J=8.9 Hz, 2H), 6.66 (d, J=5.1 Hz, 1H), 4.05 (s, 3H), 1.49 (s, 4H). The compound structure is confirmed.
To a 100 ml single-necked vial was sequentially added compound int-P (1.2 g, 2.11 mmol), 4-hydroxymethylpiperidine (486 mg, 4.22 mmol), Pd2 (dba)3 (290 mg, 0.32 mmol), BINAP (394 mg, 0.63 mmol), cesium carbonate (1.38 g, 4.22 mmol), and 1,4-dioxane (30 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (100 ml) and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12139-1 (380 mg, brown solid, purity 91.919%), yield: 27.45%. LCMS (ESI) m/z calcd. for C33H32F2N4O5 [M+H]+ 603.2; found 603.1. The compound structure is confirmed.
Compound oxalyl chloride (160 mg, 1.26 mmol) and DCM (4 ml) were sequentially added to a 50 ml triple-necked vial, cooled down to −78° C. A DCM (2 ml) solution of DMSO (197 mg, 2.52 mmol) was added and −78° C. stirred for 0.5 hours. Then DCM (4 ml) solution of compound TPD12139-1 (380 mg, 0.63 mmol) was added and −78° C. stirred for 0.5 hr. Finally, TEA (510 mg, 5.04 mmol) was added and the reaction was slowly returned to room temperature and protected by nitrogen for 1 hour. After completion of the reaction, the reaction solution was poured into saturated NaHCO3 solution (30 ml) and extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by preparative plates with an eluent ratio of DCM/MeOH=12/1 to afford the compound TPD12139-2 (160 mg, yellow solid, purity 92.928%), yield: 39.26%. LCMS (ESI) m/z calcd. for C33H30F2N4O5 [M+H]+ 601.2; found 601.1; 1H NMR (400 MHz, DMSO_d6): δ=10.20 (s, 1H), 10.05 (s, 1H), 9.68 (s, 1H), 8.50 (d, J=5.1 Hz, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.64 (dd. J=8.7, 5.1 Hz, 2H), 7.33 (s, 1H), 7.23 (d, J=8.8 Hz, 2H), 7.15 (t, J=8.8 Hz, 2H), 6.50 (d, J=5.1 Hz, 1H), 3.96 (s, 3H), 3.37 (br. s, 2H), 3.19 (t, J=11.1 Hz, 2H). 2.58-2.53 (m, 1H), 1.95 (d, J=10.2 Hz, 2H), 1.70-1.60 (m, 2H), 1.48 (s, 4H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-3 (60 mg, 0.15 mmol), compound TPD12139-2 (87 mg, 0.15 mmol), sodium triacetoxyborohydride (62 mg, 0.29 mmol) and DCE (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 5-40/7 min to afford the compound TPD12139 (26.35 mg, yellow solid, purity 96.683%) in the following yield: 16.29%. LCMS (ESI) m/z calcd. for C53H54F2N10O8 [M+H]+ 997.4; found 499.4; 997.3; 1H NMR (400 MHz, DMSO_d6): δ=11.17 (s, 1H), 11.01 (s, 1H), 10.21 (s, 1H), 10.06 (s, 1H), 8.68 (s, 1H), 8.49 (d, J=5.1 Hz, 1H), 8.36 (s, 1H), 8.23 (s, 1.7H), 7.77 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.32 (s, 1H), 7.23 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H). 6.49 (d, J=5.1 Hz, 1H), 5.09 (dd, J=13.2, 5.0 Hz, 1H), 4.53 (d, J=18.4 Hz, 1H), 4.39 (d, J=18.3 Hz, 2H), 3.95 (s, 3H), 3.67 (s, 2H), 3.31 (d, J=10.9 Hz, 2H), 3.13-2.85 (m, 10H), 2.67-2.54 (m, 4H), 2.41-2.33 (m, 2H), 1.99-1.91 (m, 2H), 1.75 (d, J=10.9 Hz, 2H), 1.55 (br. s, 1H), 1.48 (s, 4H), 1.32 (d, J=11.0 Hz, 2H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12140-4 (60 mg, 0.146 mmol), compound TPD12139-2 (88 mg, 0.146 mmol), sodium triacetoxyborohydride (62 mg, 0.292 mmol) and DCE (5 ml). The reaction was carried out for 16 hours at 25° C. under nitrogen protection. After the reaction was completed, the reaction solution was decanted into water (15 ml) and appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: T3 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 20-35/7 min to afford the compound TPD12140 (21.1 mg, yellow solid, purity 98.864%) in the following yield: 13.31%. LCMS (ESI) m/z calcd. for C54H55F2N9O8 [M+H]+ 996.4; found 498.8; 996.3; 1H NMR (400 MHz, DMSO_d6): δ=11.05 (s, 1H), 10.21 (s, 1H), 10.06 (s, 1H), 9.97 (s, 1H), 8.49 (d, J=5.1 Hz, 1H), 7.81-7.76 (m, 3H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.54-7.48 (m, 2H), 7.32 (s, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.49 (d, J=5.1 Hz 1H), 5.16 (dd, J=13.3, 5.1 Hz, 1H), 4.40 (d, J=17.5 Hz, 1H), 4.32 (d, J=17.4 Hz, 1H), 3.95 (s, 3H), 3.67 (s, 2H), 3.31 (d, J=11.4 Hz, 2H), 3.13-2.88 (m, 10H). 2.67-2.54 (m, 3H), 2.33-2.28 (m, 3H), 2.05-2.02 (m, 1H), 1.84 (d, J=7.3 Hz, 1H), 1.75 (d, J=11.4 Hz, 2H), 1.56 (s, 1H), 1.48 (s, 4H), 1.37-1.32 (m, 2H). The compound structure is confirmed.
Compound sm-1 (25 g, 0.0976 mol), EtOH (300 ml) and sm-2 (18.17 g, 0.0976 mol) were added sequentially in a 500 ml triple-necked vial. The reaction was refluxed under nitrogen protection for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature and filtered by extraction to afford the compound int-Q-1 (35 g, yellow solid, purity 98.883%), yield: 86.48%. LCMS (ESI) m/z calcd. for C14H11BrF3NO5 [M+H]+ 409.98, 411.98; found 351.8, 353.8; 1H NMR (400 MHz, DMSO_d6): δ=11.26 (s, 1H), 8.58 (s, 1H), 8.16 (d, J=2.7 Hz, 1H), 7.72 (dd, J=9.0, 2.7 Hz, 1H), 7.63-7.52 (m, 1H), 1.68 (s, 6H). The compound structure is confirmed.
Compound int-Q-1 (12 g, 0.0292 mol) and diphenyl ether (120 ml) were added sequentially in a 250 ml single-necked vial. The reaction was carried out at 240° C. for 30 min under nitrogen protection. The other two batches were operated identically. After completion of the reaction, the reaction solution was reduced to room temperature and the compound int-Q-2 (17 g, white solid, purity 93.766%) was obtained by filtration, yield: 60.61%. LCMS (ESI) m/z calcd. for C10H5BrF3NO2 [M+H]+ 307.95, 309.95; found 307.9, 309.9; 1H NMR (400 MHz, DMSO_d6): δ 11.99 (s, 1H), 8.05-8.00 (m, 1H), 8.00-7.88 (m, 1H), 7.70 (dd, J=35.0, 9.1 Hz, 1H), 6.11 (dd, J=14.7, 7.4 Hz, 1H). The compound structure is confirmed.
To a 500 ml triple-necked flask was sequentially added compound int-Q-2 (17 g, 0.0551 mol), toluene (200 ml) and POCl3 (25.37 g, 0.165 mol). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, concentrated and adjusted to alkaline with saturated NaHCO3 solution and extracted three times with DCM (200 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of PE/EtOAc=50/1˜30/1 to obtain the compound int-Q-3 (4.2 g, white solid, purity 99.055%) in the yield: 25.49%. LCMS (ESI) m/z calcd. for C10H4BrClF3NO [M+H]+ 325.91, 327.91; found 325.8, 327.8; 1H NMR (400 MHz, DMSO_d6): δ=8.95 (d, J=4.8 Hz, 1H), 8.61 (s, 1H), 8.16 (d, J=1.0 Hz, 1H), 7.93 (d, J=4.7 Hz, 1H). The compound structure is confirmed.
Compound int-A-1 (34 g, 0.152 mol), 4-aminophenol (23.27 g, 0.213 mol), HATU (81.07 g, 0.213 mol), DIEA (49.21 g, 0.381 mol), and DMF (300 ml) were added sequentially in a 1000 ml single-necked vial. The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to afford compound int-A-2 (22 g, brown oil, purity 94.060%), yield: 43.20%. LCMS (ESI) m/z calcd. for C17H15FN2O3 [M+H]+ 315.11; found 315.0; 1H NMR (400 MHz, DMSO_d6): δ=10.18 (s, 1H), 9.75 (s, 1H), 9.28 (s, 1H), 7.72-7.54 (m, 2H), 7.35 (d, J=8.7 Hz, 2H), 7.14 (t, J=8.9 Hz, 2H), 6.70 (d, J=8.8 Hz, 2H). 1.45 (s, 4H). The compound structure is confirmed.
Compounds int-Q-3 (4.2 g, 0.0129 mol), int-A-2 (4.87 g, 0.0154 mol), cesium carbonate (8.41 g, 0.0258 mol) and DMSO (50 ml) were added sequentially to a 100 ml triple-necked vial. The reaction was carried out under nitrogen protection at 120° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, decanted into water (300 ml) and extracted three times with DCM (200 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of PE/EtOAc=5/1˜2/1 to give compound int-Q (7 g, yellow solid, purity 96.576%), yield: 86.82%. LCMS (ESI) m/z calcd. for C27H18BrF4N3O4 [M+H]+ 604.04, 606.04; found 603.9, 605.9; 1H NMR (400 MHz, DMSO_d6): δ=10.23 (s, 1H), 10.05 (s, 1H), 8.77 (d, J=5.2 Hz, 1H), 8.53 (s, 1H), 8.31 (s, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.65 (dd, J=8.7, 5.1 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.15 (t, J=8.8 Hz, 2H), 6.69 (d, J=5.2 Hz, 1H), 1.48 (s, 4H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound int-Q (3.5 g, 0.0058 mol), 4-hydroxymethylpiperidine (1.34 g, 0.0116 mol), Pd2 (dba)3 (0.53 g, 0.0005 mol), BINAP (0.72 g, 0.0011 mol), cesium carbonate (3.78 g, 0.0116 mol) and 1,4-dioxane (70 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (200 ml) and extracted three times with EA (150 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=80/1˜30/1 to afford the compound TPD12148-1 (2 g, yellow solid, purity 68.172%), yield: 36.21%. LCMS (ESI) m/z calcd. for C33H30F4N4O5 [M+H]639.22; found 639.0; 1H NMR (400 MHz, DMSO_d6): δ=10.21 (s, 1H), 10.06 (s, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.04 (d, J=1.0 Hz, 1H), 7.77 (d, J=8.9 Hz, 2H), 7.65 (dd, J=9.0, 5.1 Hz, 2H). 7.54 (s, 1H), 7.26 (d, J=9.0 Hz, 2H), 7.16 (t, J=8.9 Hz, 2H), 6.47 (d, J=5.2 Hz, 1H), 4.55 (t, J=5.3 Hz, 1H), 3.52 (d, J=11.8 Hz, 2H), 3.33-3.28 (m, 2H), 2.77 (t J=11.2 Hz, 2H), 1.83 (d, J=11.5 Hz, 2H), 1.58 (s, 1H), 1.48 (s, 4H), 1.34 (dd, J=20.7, 11.7 Hz, 2H). The compound structure is confirmed.
Compound oxalyl chloride (0.41 g, 0.0032 mol) and DCM (15 ml) were sequentially added to a 50 ml triple-necked vial, cooled down to −78° C. A DCM (5 ml) solution of DMSO (0.5 g, 0.0064 mol) was added and stirred for 0.5 hr at −78° C. The mixture was then stirred for 0.5 hr. Then DCM (10 ml) solution of compound TPD12148-1 (1 g, 0.0016 mol) was added and −78° C. stirred for 0.5 hours. Finally, TEA (1.3 g, 0.0128 mol) was added and the reaction was slowly restored to room temperature and protected by nitrogen for 1 hour. After completion of the reaction, the reaction solution was poured into saturated NaHCO3 solution (60 ml) and extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=80/1˜30/1 to afford the compound TPD12148-2 (0.7 g, yellow solid, purity 70.18%) in 50.00% yield. LCMS (ESI) m/z calcd. for C33H28F4N4O5 [M+H]+ 637.2; found 637.1; 1H NMR (400 MHz, DMSO_d6): δ=10.21 (s, 1H), 10.06 (s, 1H), 8.64 (t, J=5.0 Hz, 1H), 8.05 (d, J=7.6 Hz, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.54 (d, J=11.2 Hz, 1H), 7.26 (d, J=8.8 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.48 (t, J=5.6 Hz, 1H), 3.57-3.45 (m, 3H). 3.26 (s, 2H), 3.04-2.82 (m, 1H), 2.72 (t, J=12.1 Hz, 1H), 2.06 (dd, J=19.6, 11.2 Hz, 1H), 1.85 (d, J=12.3 Hz, 1H), 1.68 (dd, J=20.3, 10.4 Hz, 1H), 1.48 (s, 4H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-3 (150 mg, 0.36 mmol), compound TPD12148-2 (231 mg, 0.36 mmol), sodium triacetoxyborohydride (154 mg, 0.72 mmol) and DCE (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 5-40/7 min to give compound TPD12148 (40.0 mg, yellow solid, purity 98.899%), yield: 9.98%. LCMS (ESI) m/z calcd. for C53H52F4N10O8 [M+H]+ 1033.4; found 517.3; 1033.3; 1H NMR (400 MHz, DMSO_d6): δ=11.18 (s, 1H), 11.03 (s, 1H), 10.21 (s, 1H), 10.06 (s, 1H), 8.68 (s, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.36 (s, 1H), 8.21 (s, 1.2H), 8.04 (s, 1H), 7.77 (d, J=8.8 Hz, 2H), 7.64 (dd, J=8.9, 5.1 Hz, 2H), 7.53 (s, 1H),
7.26 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.47 (d, J=5.2 Hz, 1H), 5.10 (dd, J=13.3, 5.0 Hz, 1H), 4.54 (d, J=18.5 Hz, 1H), 4.39 (d, J=18.4 Hz, 1H), 3.60 (s, 2H), 3.49 (d, J=11.2 Hz, 2H), 3.02-2.86 (m, 7H), 2.74 (t, J=11.2 Hz, 2H), 2.66-2.61 (m, 3H), 2.41-2.34 (m, 4H), 2.00-1.97 (m, 1H), 1.92-1.85 (m, 3H), 1.53 (s, 1H), 1.48 (s, 4H), 1.32 (dd, J=22.0, 11.1 Hz, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound sm-1 (2 g, 0.01 mol), methyl acrylate (1.29 g, 0.015 mol) and MeOH (30 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After completion of the reaction, the reaction solution was concentrated to dryness to give the compound TPD12140-1 (2.9 g, colorless oil, purity 92.339%), yield: 81%. LCMS (ESI) m/z calcd. for C14H24N2O4 [M+H]+ 285.17; found 285.1; 1H NMR (400 MHz, DMSO_d6): δ=3.93 (d, J=5.8 Hz, 2H), 3.60 (d, J=10.8 Hz, 3H), 3.05-2.86 (m, 2H), 2.80-2.63 (m, 4H), 2.46 (t, J=7.1 Hz, 2H), 2.30-2.21 (m, 1H), 1.49 (d, J=7.9 Hz, 1H), 1.38 (s, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound TPD12140-1 (2.9 g, 0.0102 mol), lithium hydroxide monohydrate (1.28 g, 0.0306 mol), MeOH (20 ml) and H2O (10 ml). The reaction was carried out for 16 hours at 25° C. under hydrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature, filtered and concentrated to dryness to afford the compound TPD 12140-2 (1 g, yellow oil, purity 70.663%), yield: 25.49%. LCMS (ESI) m/z calcd. for C13H22N2O4 [M+H]+ 271.16; found 271.1; 1H NMR (400 MHz. DMSO_d6): δ=3.95-3.89 (m, 3H), 3.03-2.82 (m, 3H), 2.70-2.63 (m, 4H), 2.19 (dd, J=16.0, 8.3 Hz, 3H), 1.38 (s, 9H). The compound structure is confirmed.
Compound TPD12140-2 (1020 mg, 3.76 mmol), lenalidomide (650 mg, 2.51 mmol), DIEA (648 mg, 5.01 mmol), HATU (1097 mg, 5.01 mmol), and DMF (25 ml) were added sequentially to a 25 ml single mouth vial. The reaction was carried out under nitrogen protection at 50° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (100 ml) and extracted three times with EA (90 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by column chromatography with eluent ratio of DCM/MeOH=100/1˜20/1 to give compound TPD12140-3 (770 mg, red solid, purity 77.576%), yield: 46.48%. LCMS (ESI) m/z calcd. for C26H33N5O [M+H]+ 512.24; found 512.3; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 9.88 (s, 1H), 7.81-7.76 (m, 1H), 7.54-7.47 (m, 2H), 5.19-5.11 (m, J=13.3, 5.1 Hz, 1H), 4.41-4.29 (m, 2H). 3.99 (t, J=17.3 Hz, 3H), 3.01-2.75 (m, 8H), 2.35-2.23 (m, 2H), 1.43-1.29 (m, 12H). The compound structure is confirmed.
Compound TPD12140-3 (110 mg, 0.21 mmol) 4N HCl/1,4-dioxane (12 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12140-4 (110 mg, yellow solid), yield: 99.44%. LCMS (ESI) m/z calcd. for C21H25N5O4 [M+H]+ 412.19; found 412.0. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12140-4 (110 mg, 0.27 mmol), compound TPD12148-2 (171 mg, 0.27 mmol), sodium triacetoxyborohydride (113 mg, 0.53 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (5 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12149 (31.5 mg, yellow solid, purity 98.694%) in the following yield: 11.45%. LCMS (ESI) m/z calcd. for C54H53F4N9O8 [M+H]+ 1032.07; found 1032.3; 1H NMR (400 MHz, DMSO_d6): δ=11.05 (s, 1H), 10.22 (s, 1H), 10.06 (s, 1H), 9.97 (s, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.23 (s, 2H), 8.04 (s, 1H), 7.78 (t, J=8.3 Hz, 3H), 7.70-7.58 (m, 2H), 7.58-7.40 (m, 3H), 7.26 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.47 (d, J=5.2 Hz, 1H), 5.20-5.12 (m, 1H), 4.40-4.31 (m, J=12.9 Hz, 2H), 3.59 (s, 2H), 3.49 (d, J=10.9 Hz, 2H), 3.02-2.88 (m, 6H), 2.75 (t, J=11.1 Hz, 2H), 2.68-2.54 (m, 3H), 2.42-2.25 (m, 4H), 2.07-2.00 (m, J=5.1 Hz, 1H), 1.90-1.75 (m, 3H), 1.47 (s, 4H), 1.38-1.16 (m, 4H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12148-2 (160 mg, 0.25 mmol), compound TPD12141-4 (99 mg, 0.25 mmol), sodium triacetoxyborohydride (107 mg, 0.50 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (10 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12150 (60.7 mg, yellow solid, purity 99.902%) in the following yield: 22.09%. LCMS (ESI) m/z calcd. for C55H52F4N8O7 [M+H]+ 1013.39; found 1013.4; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 10.21 (s, 1H), 10.06 (s, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.04 (s, 1H)), 7.77 (d, J=8.9 Hz, 2H), 7.72 (d, J=7.3 Hz, 1H), 7.67-7.62 (m, 3H), 7.55-7.50 (m, 2H), 7.26 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.47 (d, J=5.2 Hz, 1H). 5.17 (dd, J=13.3, 5.1 Hz, 1H), 4.46 (d, J=17.7 Hz, 1H), 4.31 (d, J=17.7 Hz, 1H), 3.61 (s, 2H), 3.48 (d, J=10.4 Hz, 3H), 3.07 (d, J=10.9 Hz, 2H), 2.98-2.88 (m, 4H), 2.80-2.65 (m, 4H), 2.60 (d, J=17.2 Hz, 1H), 2.45-2.30 (m, 4H), 2.06-1.99 (m, 1H), 1.85 (br. s, 3H), 1.54 (br. s, 1H), 1.47 (s, 4H), 1.37-1.28 (m, 2H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12112-3 (150 mg, 0.36 mmol), compound TPD12086-9 (208 mg, 0.36 mmol), sodium triacetoxyborohydride (154 mg, 0.73 mmol) and DCE (5 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was decanted into water (15 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted twice with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: sunfire 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 5-40/7 min to afford the compound TPD12162 (52.9 mg, yellow solid, purity 99.423%) in the following yield: 14.96%. LCMS (ESI) m/z calcd. for C52H52F2N10O7 [M+H]+ 967.4; found 484.4; 967.3; 1H NMR (400 MHz, DMSO_d6): δ=11.15 (s, 1H), 11.04 (s, 1H), 10.21 (s, 1H), 10.06 (s, 1H), 8.68 (s, 1H), 8.56 (d J=5.2 Hz, 1H), 8.36 (s, 1H), 8.21 (s, 2.3H), 7.86 (d, J=13.4 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.64 (dd, J=9.0, 5.1 Hz, 2H), 7.44 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.45 (d, J=5.2 Hz, 1H), 5.10 (dd, J=13.3, 5.0 Hz, 1H), 4.53 (d, J=18.5 Hz, 1H), 4.39 (d, J=18.4 Hz, 1H), 3.73 (s, 2H), 3.55 (d J=11.3 Hz, 2H), 3.06-2.86 (m, 8H), 2.75 (t, J=11.3 Hz, 2H), 2.67-2.54 (m, 4H), 2.41-2.30 (m, 2H), 1.99-1.92 (m, 2H), 1.84 (d, J=11.3 Hz, 2H), 1.59 (s, 1H). 1.47 (s, 4H), 1.42-1.37 (m, 2H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound sm-1 (1 g, 0.005 mol), acrylamide (0.36 g, 0.005 mol), K2CO3 (1.04 g, 0.0075 mol), and MeOH (10 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, decanted into water (30 ml) and extracted three times with EA (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=80/1˜30/1 to obtain the compound TPD12163-1 (0.8 g, white solid, purity 73.888%), yield: 44.00%. LCMS (ESI) m/z calcd. for C13H23N3O3 [M+H]+ 270.17; found 270.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12163-1 (800 mg, 2.73 mmol), compound TPD12003-4 (1241 mg, 4.44 mmol), XantPhos (342 mg, 0.59 mmol), Pd(OAc)2 (133 mg, 0.59 mmol), Cs2 CO3 (2410 mg, 7.40 mmol) and 1,4-dioxane (10 ml). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After completion of the reaction, the reaction solution was brought to room temperature, decanted into water (40 ml) and extracted three times with DCM (30 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate with the unfolding agent ratio of DCM/MeOH=10/1 to afford the compound TPD12163-2 (240 mg, yellow solid, purity 86.393%), yield: 13.64%. LCMS (ESI) m/z calcd. for C25H32N6O6 [M+H]+ 513.24; found 513.1; 1H NMR (400 MHz, DMSO_d6): δ=11.18-11.01 (m, 2H), 8.67 (s, 1H), 8.33 (s, 1H), 5.10 (dd, J=13.3, 5.0 Hz, 1H), 4.53 (d, J=18.4 Hz, 1H), 4.39 (d, J=18.4 Hz, 1H), 4.15 (d, J=10.1 Hz, 1H), 3.52 (s, 1H), 3.22 (d, J=14.2 Hz, 1H), 3.07 (dd, J=15.9, 8.9 Hz, 1H), 2.96-2.87 (m, 1H), 2.81 (d, J=7.3 Hz, 3H), 2.64-2.52 (m, 3H), 2.37 (dd, J=13.0, 4.4 Hz, 1H), 2.03-1.96 (m, 1H), 1.71 (d, J=9.2 Hz, 1H), 1.62 (t, J=9.6 Hz, 1H), 1.38 (s, 9H), 1.20 (d, J=24.3 Hz, 1H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12163-2 (240 mg, 0.47 mmol) and 4 N HCl/1,4-dioxane (8 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12163-3 (240 mg, yellow solid, purity 75.179%), yield: 93.62%. LCMS (ESI) m/z calcd. for C20H24N6O4 [M+H]+ 413.19; found 413.1. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12163-3 (108 mg, 0.26 mmol), compound TPD12086-9 (150 mg, 0.26 mmol), sodium triacetoxyborohydride (111 mg, 0.53 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (10 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to afford the compound TPD12163 (9.04 mg, yellow solid, purity 92.832%) in the following yield: 3.08%. LCMS (ESI) m/z calcd. for C52H52F2N10O7 [M+H]+ 967.40; found 967.3; 1H NMR (400 MHz, DMSO_d6): δ=11.36 (s, 1H), 11.02 (s, 1H), 10.20 (s, 1H), 10.07 (s, 1H), 8.68 (s, 1H), 8.56 (d, J=5.1 Hz, 1H), 8.34 (s, 1H), 7.85 (d, J=13.5 Hz, 1H), 7.76 (d, J=8.7 Hz, 2H), 7.68-7.56 (m, 2H), 7.44 (d, J=8.3 Hz, 1H), 7.28-7.06 (m, 4H), 6.45 (d, J=5.1 Hz, 1H). 5.14-5.04 (m, 1H), 4.54 (d, J=18.3 Hz, 1H), 4.39 (d, J=18.1 Hz, 1H), 3.33 (s, 3H), 2.96-2.87 (m, 2H), 2.77 (d, J=7.1 Hz, 4H), 2.71-2.60 (m, 3H), 2.54 (s, 2H). 2.44-2.31 (m, 3H), 2.09-1.83 (m, 4H), 1.60 (d, J=32.7 Hz, 3H), 1.48-1.23 (m, 7H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound sm-1 (1000 mg, 5.02 mmol), K2CO3 (1040 mg, 7.53 mmol),methyl bromoacetate (768 mg, 5.02 mmol) and ACN (15 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, poured into water (20 ml), extracted with EtOAc (60 ml) for three times, the reaction solution was concentrated to dryness, and the crude product was purified by silica gel chromatographic column, the ratio of the unfolding agent was D/M=100/1˜70/1, to obtain the compound TPD12216-1 (1200 mg, white solid, purity 73.225%), the yield: 64.53%; LCMS (ESI) m/z calcd. for C13H22N2O4 [M+H]+ 271.16; found 271.2; 1H NMR (400 MHz, DMSO_d6): δ=4.13 (d, J=11.9 Hz, 1H), 3.60 (s, 3H), 3.49 (d, J=3.5 Hz, 1H), 3.38 (s 2H), 3.29-3.23 (m, 1H), 3.10-3.03 (m, 1H), 2.87-2.80 (m, 1H), 2.55-2.51 (m, 1H), 1.75-1.70 (m, 1H), 1.63-1.54 (m, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 1000 ml single-necked vial was sequentially added compound TPD 12216-1 (1300 mg, 4.79 mmol), lithium hydroxide monohydrate (603 mg, 14.37 mmol) and MeOH/H2O=2/1 (15 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was brought to room temperature, spun dry as a solid, decanted into water (12 mL), adjusted to a pH of 7 with hydrochloric acid, saturated with salt, and then extracted eight times with EA (60 ml) and THF (60 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The compound TPD12216-2 (1100 mg, yellow oil, purity 46.144%) was obtained, yield: 41.17%; LCMS (ESI) m/z calcd. for C12H20N2O4 [M+H]+ 257.14; found 257.3; 1H NMR (400 MHz, DMSO_d6): δ=4.15 (d, J=12.9 Hz, 1H), 3.66 (d, J=11.4 Hz, 2H), 3.18-3.06 (m, 3H), 2.94-2.87 (m, 1H), 2.67 (t, J=11.4 Hz, 1H), 1.89-1.81 (m, 1H), 1.71-1.59 (m, 1H), 1.39 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12216-2 (1.00 g, 3.89 mmol), compound TPD12215-3 (1.01 g, 3.89 mmol), HATU (2.96 g, 7.77 mmol), DIEA (1.01 g, 7.77 mmol), and DMF (20 ml). The reaction was carried out at 50° C. for 16 h. After the reaction was completed, it was reduced to room temperature, poured into water (100 ml) and extracted with EtOAc (100 ml) for five times, and the reaction solution was concentrated to dryness, and the crude product was purified by silica gel chromatographic column, the ratio of unfolding reagent was DCM/MeOH=50/1˜15/1, and the crude was prepared by high-performance liquid chromatography (HPLC), with the relevant parameters as follows: chromatographic column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min, after the crude compound TPD12216-3 (40 mg, yellow solid, purity 83.183%) was obtained, yield: 1.72%. LCMS (ESI) m/z calcd. for C25H31N5O6 [M+H]+ 498.23; found 498.1. found 498.1. The compound structure is confirmed.
Compound TPD12216-3 (30 mg, 0.060 mmol) 4 N HCl/1,4-dioxane (4 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to obtain the crude compound TPD12216-4 (25 mg, yellow solid, purity 95.206%) in 99.50% yield; LCMS (ESI) m/z calcd. for C20H23N5O4 [M+H]+ 399.18; found 399.6. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12215-5 (40 mg, 0.101 mmol), compound TPD12086-9 (57 mg, 0.101 mmol), sodium triacetoxyborohydride (43 mg, 0.201 mmol) and DCE (1 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (3 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give compound TPD12205 (3.41 mg, yellow solid, purity 96.597%), yield: 3.28%; LCMS (ESI) m/z calcd. for C52H51F2N9O7 [M+H]+ 952.39; found 952.3; 1H NMR (400 MHz, DMSO_d6): δ=10.97 (s, 1H), 10.18 (s, 1H), 10.04 (s, 1H), 9.98 (s, 1H), 8.56 (d, J=5.2 Hz, 1H). 8.21 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.85 (d, J=13.4 Hz, 1H), 7.80-7.71 (m, 3H), 7.70-7.59 (m, 3H), 7.44 (d, J=8.5 Hz, 1H), 7.23-7.11 (m, 4H), 6.45 (d, J=5.1 Hz, 1H), 5.09 (dd, J=13.2, 4.9 Hz, 1H), 4.43 (d, J=17.1 Hz, 1H), 4.30 (d, J=17.4 Hz, 1H), 3.68-3.47 (m, 4H), 2.96-2.85 (m, 1H), 2.83-2.53 (m, 7H), 2.42-2.24 (m, 5H), 2.03-1.95 (m, 1H), 1.92-1.86 (m, 2H), 1.74 (d, J=9.9 Hz, 3H), 1.65 (d, J=8.1 Hz, 1H), 1.56 (s, 1H), 1.47 (s, 4H), 1.39-1.30 (m, 2H). The compound structure is confirmed.
To a 500 ml single-necked vial were sequentially added compound sm-2 (20.0 g, 0.10 mol), NBS (27.4 g, 0.15 mol), AIBN (1.68 g, 10.3 mmol) and carbon tetrachloride (250 ml). The reaction was carried out at 70° C. for 16 h under nitrogen protection. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (200 ml), extracted twice with DCM (200 ml) and the reaction solution was concentrated to dryness to give compound TPD12215-1 (34 g, yellow solid, purity 58.534%), yield: 70.83%; LCMS (ESI) m/z calcd. for C9H8BrNO4 [M+H]+ 273.96/275.96; found 275.0/276.0; 1H NMR (400 MHz, DMSO_d6): δ=8.16-7.97 (m, 3H), 5.10 (s, 2H), 3.96 (s, 3H). The compound structure is confirmed.
To a 1000 ml single-necked vial was sequentially added compound TPD12215-1 (42 g crude, 0.10 mol), 3-amino-2,6-piperidinedione hydrochloride (30.26 g, 0.18 mol), DIEA (39.6 g, 0.31 mol) and DMSO (450 mL). The reaction was carried out under nitrogen protection at 100° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, decanted into water (2500 ml) and extracted eight times with EA (3000 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by column chromatography with eluent ratio of DCM/MeOH=100/1˜20/1 to afford compound TPD12215-2 (11.75 g, green solid, purity 90.781%), yield: 24.09%; LCMS (ESI) m/z calcd. for C13H11N3O5 [M+H]+ 290.07; found 289.9; 1H NMR (400 MHz, DMSO_d6): δ=11.06 (s, 1H), 8.53 (s, 1H), 8.36 (dd, J=8.3, 1.7 Hz, 1H), 7.98 (d, J=8.3 Hz, 1H), 5.18 (dd, J=13.3, 5.1 Hz, 1H), 4.61 (d, J=18.1 Hz, 1H), 4.50 (d, J=18.1 Hz, 1H), 2.98-2.87 (m, 1H), 2.50-2.38 (m, 2H), 2.11-2.00 (m, 1H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compound TPD12215-2 (3 g, 0.0104 mol), iron powder (1.16 g, 0.0208 mol), ammonium chloride (1.11 g, 0.0208 mmol), isopropanol (180 ml) and H2 O (60 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After the reaction was completed, the reaction solution was reduced to room temperature, filtered, concentrated to dryness, and the residue was purified by chromatographic column with eluent ratio of DCM/MeOH=100/1˜20/1 to obtain the compound TPD12215-3 (770 mg, yellow solid, purity 94.466%), yield: 26.92%; LCMS (ESI) m/z calcd. for C13H13N3O3 [M+H]+ 260.10; found 260.1; 1H NMR (400 MHz, DMSO_d6): δ=10.93 (s, 1H), 7.41-7.26 (m, 1H), 6.64-6.61 (m, 1H), 5.92-5.64 (m, 2H), 5.01 (dd, J=13.3, 5.0 Hz. 1H). 4.37-4.02 (m, 2H), 2.97-2.83 (m, 1H), 2.60-2.53 (m, 2H), 2.40-2.26 (m, 1H), 1.99-1.88 (m, 1H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD12140-2 (610 mg, 2.25 mmol), compound TPD12215-3 (583 mg, 2.25 mmol), HATU (1.71 g, 4.50 mmol), DIEA (581 mg, 4.50 mmol) and DMF (10 ml). The reaction was carried out at 50° C. for 16 h. After completion of the reaction, it was reduced to room temperature, decanted into water (60 ml) and extracted with EA (160 ml) for eight times, and the residue was purified by a column with the eluent ratio of DCM/MeOH=100/1˜20/1 to give the crude compound TPD12215-4 (500 mg, yellow solid, purity 86.189%), yield: 37.39%; LCMS (ESI) m/z calcd. for C21H25N5O4 [M+H]+ 512.24; found 512.2; 1H NMR (400 MHz, DMSO_d6): δ=11.03-10.90 (m, 1H), 10.41-10.28 (m, 1H), 8.76 (d, J=4.2 Hz, 1H), 8.53 (d, J=8.4 Hz, 1H), 7.55-7.47 (m, 1H). 4.54-4.22 (m, 3H), 4.14-4.07 (m, 3H), 4.05-3.94 (m, 3H), 2.95-2.89 (m, 5H), 2.69-2.57 (m, 3H), 2.37-2.29 (m, 2H), 1.39 (s, 9H). The compound structure is confirmed.
Compound TPD12215-4 (110 mg, 0.21 mmol) 4 N HCl/1,4-dioxane (8 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12215-5 (100 mg, yellow solid, purity 89.832%), yield: 86.44%; LCMS (ESI) m/z calcd. for C21H25N5O4 [M+H]+ 412.19; found 412.2. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12215-5 (120 mg, 0.29 mmol), compound TPD12086-9 (166 mg, 0.29 mmol), sodium triacetoxyborohydride (124 mg, 0.58 mmol) and DCE (3 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (3 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give compound TPD12215 (16 mg, yellow solid, purity 97%), yield: 5%; LCMS (ESI) m/z calcd. for C54H53F4N9O8 [M+H]+ 966.40; found 966.3; 1H NMR (400 MHz, DMSO_d6): δ=11.01 (s, 1H), 10.39 (s, 1H), 10.20 (s, 1H), 10.06 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 8.21 (s, 2H)), 7.99 (s, 1H), 7.86 (d, J=13.4 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.70-7.56 (m, 4H), 7.44 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.19-7.11 (m, 2H), 6.45 (d, J=5.2 Hz, 1H). 5.12-5.03 (m, 1H), 4.42 (d, J=17.3 Hz, 1H), 4.29 (d, J=17.3 Hz, 1H), 3.67 (s, 2H), 3.54 (d, J=11.4 Hz, 2H), 3.09-2.65 (m, 10H), 2.63-2.53 (m, 3H), 2.43-2.26 (m, 3H), 2.02-1.80 (m, 4H), 1.56 (s, 1H), 1.47 (s, 4H), 1.41-1.32 (m, 2H). The compound structure is confirmed.
Compound TPD12216-3 (55 mg, 0.11 mmol) 4 N HCl/1,4-dioxane (8 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 1.5 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12216-4 (50 mg, white solid, purity 83.291%) in 80.24% yield. LCMS (ESI) m/z calcd. for C20H23N5O4 [M+H]+ 398.18; found 398.1. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12216-4 (70 mg, 0.18 mmol), compound TPD5488-2 (103 mg, 0.18 mmol), sodium triacetoxyborohydride (75 mg, 0.35 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (3 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give compound TPD12216 (2.17 mg, yellow solid, purity 87%) in 0.19% yield. LCMS (ESI) m/z calcd. for C53H54FN9O8 [M+H]+ 963.41; found 482.9; 1H NMR (400 MHz, DMSO_d6): δ=10.97 (s, 1H), 10.18 (s, 1H), 10.07-9.97 (m, 1H), 8.43 (d, J=4.9 Hz, 1H), 8.27 (s, 2H). 8.07-7.93 (m, 1H), 7.79-7.70 (m, 3H), 7.68-7.58 (m, J=15.3, 6.8 Hz, 3H), 7.46 (s, 1H), 7.32 (s, 1H), 7.23-7.10 (m, 4H), 6.39 (d, J=4.7 Hz, 1H), 5.14-5.03 (m 1H), 4.43 (d, J=17.7 Hz, 1H), 4.35-4.24 (m, 1H), 3.94 (s, 3H), 3.63-3.55 (m, 3H), 3.49-3.40 (m, 5H), 3.37-3.30 (m, 5H), 2.75-2.62 (m, 5H), 2.42-2.31 (m, 3H), 2.03-1.95 (m, 1H), 1.88-1.85 (m, 1H), 1.74 (d, J=9.7 Hz, 1H), 1.66-1.60 (m, 1H), 1.47 (s, 3H), 1.39-1.32 (m, 2H). The compound structure is confirmed.
Compounds sm-1 (1.0 g, 5.02 mmol), sm-2 (1.22 g, 7.53 mmol) and methanol (15 ml) were added sequentially in a 50 ml single-necked vial. The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was concentrated to dryness and the residue was purified by column chromatography with eluent ratio of DCM/MeOH=100/1-60/1 to afford the compound TPD12218-1 (950 mg, colorless oily substance, purity 97.200%), yield: 50.91%. LCMS (ESI) m/z calcd. for C20H28N2O4 [M+H]+ 361.2; found 361.2; 1H NMR (400 MHz, CDCl3): δ=7.41-7.28 (m, 5H), 5.19-5.09 (m, 2H), 4.27 (d, J=51.6 Hz, 1H), 3.54-3.38 (m, 2H), 3.13 (t, J=10.3 Hz, 1H), 2.96-2.82 (m, 3H), 2.65-2.44 (m, 3H), 1.82-1.74 (m, 1H), 1.71-1.65 (m, 1H), 1.45 (s, 9H). The compound structure is confirmed.
To a 25 ml vial was added compound TPD12218-1 (900 mg, 2.49 mmol), palladium carbon (100 mg, 10%) and ethyl acetate (10 ml) in sequence. 1 atm hydrogen pressure reacted at 20° C. for 16 h. After completion of the reaction, the filtrate was spun dry to give compound TPD12218-2 (500 mg, colorless oil, purity: 70.31%) in yield: 70.31%. After completion of the reaction, the filtrate was filtered and the filtrate was spun dry to give the compound TPD12218-2 (500 mg, colorless oil, purity 95%), yield: 70.31%. LCMS (ESI) m/z calcd. for C13H22N2O4 [M+H]+ 271.2; found 271.2; 1H NMR (400 MHz, DMSO_d6): δ=4.14 (d, J=11.5 Hz, 1H), 3.50 (s, 1H), 3.32-3.22 (m, 1H), 3.17 (s, 1H), 3.12-3.01 (m, 1H), 2.84-2.64 (m, 3H), 2.49-2.42 (m, 1H), 2.29 (t, J=7.0 Hz, 2H), 1.75-1.57 (m, 2H), 1.39 (s, 8H).
To a 25 ml single-necked vial were sequentially added compound TPD12218-2 (400 mg, 1.47 mmol), compound TPD12215-3 (382 mg, 1.47 mmol), HATU (1.12 g, 2.95 mmol), DIEA (572 mg, 2.95 mmol) and DMF (5 ml). The reaction was carried out under nitrogen protection at 70° C. for 16 hours. After the reaction was completed saturated aqueous sodium bicarbonate solution (20 ml) was added and the aqueous phase was extracted three times with EA (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by column chromatography with an eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12218-3 (60 mg, yellow solid, purity 81.748%), yield: 4.33%. LCMS (ESI) m/z calcd. for C26H33N5O6 [M+H]+ 512.2; found 512.1. The compound structure is confirmed.
Compound TPD12218-3 (60 mg, 0.117 mmol) and 1,4-dioxane (4 ml, 4 N) were added sequentially in a 25 ml single-necked vial. The reaction was carried out under nitrogen protection at 20° C. for 4 hours. The reaction was completed and concentrated to dryness to give compound TPD12218-4 dihydrochloride (60 mg, yellow solid, purity 92.356%), yield: 97.69%. LCMS (ESI) m/z calcd. for C21H25N5O4 [M+H]+ 411.2; found 411.2. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12218-4 (58 mg, 0.12 mmol), compound TPD5488-2 (70 mg, 0.12 mmol), sodium triacetoxyborohydride (76 mg, 0.36 mmol) and DCE (4 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 ml). The organic phase was separated and the aqueous phase was extracted three times with DCM (10 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was separated by preparative plate (DCM/MeOH=8/1) to afford compound TPD12218 (23.5 mg, yellow solid, purity 97.638%), yield: 17.32%. LCMS (ESI) m/z calcd. for C54H56FN9O8 [M+H]+ 978.4; found 490.2; 979.4; 1H NMR (400 MHz, DMSO_d6): δ=11.01 (s, 1H), 10.54 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.97 (s, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.71-7.57 (m, 4H). 7.46 (s, 1H), 7.32 (s, 1H), 7.27-7.11 (m, 4H), 6.39 (d, J=5.2 Hz, 1H), 5.09 (dd, J=13.2, 5.0 Hz, 1H), 4.43 (d, J=17.4 Hz, 1H), 4.29 (d, J=17.4 Hz, 1H), 3.94 (s 3H), 3.59 (d, J=10.9 Hz, 2H), 3.47 (d, J=26.9 Hz, 3H), 2.99-2.72 (m, 7H), 2.70-2.54 (m, 5H), 2.44-2.29 (m, 2H), 2.04-1.95 (m, 1H), 1.91-1.81 (m, 2H), 1.71 (s, 2H), 1.63-1.52 (m, 1H), 1.47 (s, 4H), 1.42-1.29 (m, 2H). The compound structure is confirmed.
To a 250 ml three-necked vial was added sequentially compound sm-1 (5 g, 71.3 mmol), DCM (60 ml) and TEA (8.66 g, 85.6 mmol). MsCl (8.94 g, 78.5 mmol) was added dropwise at 0° C. The reaction was raised to 20° C. for 2 hours. After the reaction was completed, the reaction was quenched with water (100 ml), the organic phase was separated and washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with an eluent ratio of PE/EtOAc=30/1˜10/1 to afford the compound TPD12141-1a (1.6 g, colorless oily substance), yield: 13.62%. LCMS (ESI) m/z calcd. for C5H8O3S [M+H]+ 149.02; found 149.0; 1H NMR (400 MHz, CDCl3): δ=4.32 (t, J=6.7 Hz, 2H), 3.07 (s, 3H), 2.67 (td, J=6.7, 2.6 Hz, 2H), 2.08 (t, J=2.6 Hz, 1H). The compound structure is confirmed.
To a 100 ml single-necked vial were sequentially added compounds TPD12141-1a (1.19 g, 8.03 mmol), ACN (20 ml), sm-2 (1.6 g. 8.03 mmol) and potassium carbonate (1.66 g, 12.04 mmol). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, water (80 ml) was added and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12141-2a (0.8 g, yellow solid, purity 81.061%), yield: 32.13%. LCMS (ESI) m/z calcd. for C14H22N2O2 [M+H]+ 251.17; found 250.9; 1H NMR (400 MHz, CDCl3): δ=4.05 (br. s, 2H), 3.30-2.97 (m, 2H), 2.93-2.71 (m, 5H), 2.43-2.32 (m, 3H), 1.97 (t, J=2.6 Hz, 1H), 1.65 (d, J=7.9 Hz, 1H), 1.45 (s, 9H). The compound structure is confirmed.
To a 100 ml single-necked vial was sequentially added compound TPD12141-1a (747 mg, 2.97 mmol), DMF (16 ml), sm-3 (800 mg, 2.48 mmol), Pd(PPh)32 Cl2 (174 mg, 0.25 mmol), iodinated ketone (24 mg, 0.12 mmol) and DIEA (640 mg, 4.95 mmol). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, water (80 ml) was added and extracted three times with EA (50 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatographic column with eluent ratio of DCM/MeOH=50/1˜30/1 to afford the compound TPD12141-3 (460 mg, yellow solid, purity 90.450%) in the yield: 34.05%. LCMS (ESI) m/z calcd. for C27H32N4O5 [M+H]+ 493.24; found 493.1; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 7.71 (d, J=7.4 Hz, 1H), 7.63 (d, J=7.4 Hz, 1H), 7.52 (t, J=7.6 Hz, 1H). 5.16 (dd, J=13.3, 5.0 Hz, 1H), 4.46 (d, J=17.8 Hz, 1H), 4.30 (d, J=17.7 Hz, 1H), 3.97 (d, J=5.7 Hz, 2H), 3.18-2.99 (m, 2H), 2.97-2.76 (m, 5H), 2.68-2.56 (m, 3H), 2.46-2.36 (m, 1H), 2.34-2.23 (m, 1H), 2.06-1.97 (m, 1H), 1.54 (d, J=7.9 Hz, 1H), 1.37 (s, 9H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD12141-3 (60 mg, 0.12 mmol) and HCl(g)/1,4-dioxane (4 ml). the reaction was carried out at 20° C. for 2 h. After the reaction was completed, the reaction solution was concentrated to dryness to give compound TPD12141-4 (65 mg, yellow solid), purity: 92.52%. After completion of the reaction, the reaction solution was concentrated to dryness to obtain compound TPD12141-4 (65 mg, yellow solid, purity 80.563%), yield: 92.52%. LCMS (ESI) m/z calcd. for C22H24N4O3 [M+H]+ 393.19; found 393.1. The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD5488-2 (70 mg, 0.12 mmol), compound TPD12141-4 (56 mg, 0.12 mmol), sodium triacetoxyborohydride (76 mg, 0.36 mmol) and DCE (4 ml). The reaction was carried out under nitrogen protection at 20° C. for 16 hours. After completion of the reaction, the reaction solution was decanted into aqueous saturated NaHCO3 solution (10 ml). It was extracted three times with DCM (20 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was separated by preparative plate with unfolding agent ratio DCM/MeOH=10/1 to give compound TPD12234 (11.5 mg, off-white solid, purity 99.127%), yield: 9.91%. LCMS (ESI) m/z calcd. for C55H55FN8O7 [M+H]+ 959.42; found 480.3. 961.3; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.26 (s, 1H), 7.81-7.68 (m, 3H), 7.68-7.59 (m, 3H), 7.53 (t, J=7.5 Hz, 1H), 7.46 (s, 1H), 7.30 (s, 1H), 7.25-7.10 (m, 4H), 6.39 (d, J=5.1 Hz, 1H), 5.16 (dd, J=13.3, 5.1 Hz, 1H), 4.46 (d, J=17.7 Hz, 1H), 4.31 (d, J=17.6 Hz, 1H), 3.94 (s, 3H), 3.59-3.49 (m, 6H), 3.05 (d, J=10.7 Hz, 2H), 2.98-2.81 (m, 5H), 2.70 (t, J=7.1 Hz, 2H), 2.65-2.55 (m, 3H), 2.46-2.37 (m, 2H), 2.35-2.28 (m, 2H), 2.06-1.97 (m, 1H), 1.84-1.79 (m, 2H), 1.47 (s, 4H), 1.41-1.30 (m, 2H). The compound structure is confirmed.
Compound TPD12141-3 (70 mg, 1.14 mmol) 4 N HCl/1,4-dioxane (6 ml) was added sequentially in a 25 ml single-necked vial. The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12141-4 (65 mg, yellow solid, purity 85.176%), yield: 99.51%. LCMS (ESI) m/z calcd. for C22H24N4O3 [M+H]+ 393.18; found 393.1. The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12141-4 (70 mg, 0.18 mmol), compound TPD12086-9 (102 mg, 0.18 mmol), sodium triacetoxyborohydride (76 mg, 0.36 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (3 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give the compound TPD12235 (15.8 mg, yellow solid, purity 98.969%) in the following yield: 8.52%. LCMS (ESI) m/z calcd. for C54H52F2N8O [M+H]+ 947.40; found 948.3; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.19 (s, 1H), 10.05 (s, 1H). 8.56 (d, J=5.2 Hz, 1H), 8.20 (s, 2H), 7.85 (d, J=13.4 Hz, 1H), 7.79-7.69 (m, 3H), 7.67-7.58 (m, 3H), 7.53 (t, J=7.6 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.45 (d, J=5.2 Hz, 1H). 5.16 (dd, J=13.3, 5.1 Hz, 1H), 4.46 (d, J=17.7 Hz, 1H), 4.31 (d, J=17.7 Hz, 1H), 3.57-3.49 (m, 4H), 3.08 (d, J=10.8 Hz, 2H), 2.98-2.84 (m, 5H), 2.79-2.67 (m 4H), 2.61 (d, J=17.4 Hz, 1H), 2.46-2.30 (m, 5H), 2.14-1.95 (m, 2H), 1.90-1.80 (m, 3H), 1.58-1.44 (m, 5H), 1.42-1.25 (m, 3H). The compound structure is confirmed.
To a 25 ml single-necked vial was sequentially added compound TPD12236-2 (40 mg, 0.102 mmol), compound TPD5488-2 (59 mg, 0.102 mmol), sodium triacetoxyborohydride (43 mg, 0.204 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give the compound TPD12236 (10.67 mg, yellow solid, purity 95.057%) in the following yield: 9.72%. LCMS (ESI) m/z calcd. for C55H55FN8O7 [M+H]+ 959.09; found 959.4; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.18 (s, 1H), 10.06 (s, 1H), 8.43 (d, J=5.2 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.69 (d, J=7.9 Hz, 1H), 7.64 (s, 3H), 7.51 (d, J=7.9 Hz, 1H), 7.46 (s, 1H), 7.31 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.39 (d, J=5.1 Hz, 1H), 5.10 (dd, J=13.2, 5.1 Hz, 1H), 4.43 (d, J=17.6 Hz, 1H), 4.31 (d, J=17.5 Hz, 1H), 3.93 (s, 3H), 3.55 (s, 4H), 3.11 (d, J=10.5 Hz, 3H), 2.95-2.80 (m, 5H). 2.72-2.66 (m, 2H), 2.59 (d, J=10.5 Hz, 2H), 2.42-2.28 (m, 5H), 2.00-1.93 (m, 1H), 1.82 (d, J=7.9 Hz, 2H), 1.75 (s, 1H), 1.47 (s, 4H), 1.41-1.31 (m, 2H), 1.23 (s, 1H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12236-2 (40 mg, 0.102 mmol), compound TPD12086-9 (58 mg, 0.102 mmol), sodium triacetoxyborohydride (43 mg, 0.204 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give the compound TPD12237 (7.60 mg, yellow solid, purity 99.509%) in the following yield: 7.07%. LCMS (ESI) m/z calcd. for C54H52F2N8O [M+H]+ 947.4; found 947.4; 1H NMR (400 MHz, DMSO_d6): δ=11.03 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 7.85 (d, J=13.5 Hz, 1H), 7.76 (d, J=8.9 Hz, 2H), 7.69 (d, J=7.9 Hz, 1H), 7.66-7.61 (m, 3H), 7.51 (d, J=8.1 Hz, 1H), 7.43 (d, J=8.5 Hz, 1H), 7.22 (d, J=8.9 Hz, 2H), 7.15 (t, J=8.9 Hz, 2H), 6.45 (d, J=5.2 Hz, 1H), 5.11 (dd, J=13.2, 5.1 Hz, 1H), 4.44 (d, J=17.6 Hz, 1H), 4.32 (d, J=17.5 Hz, 1H), 3.54 (d, J=11.3 Hz, 4H), 3.10 (d, J=10.7 Hz, 3H), 2.94-2.84 (m, 5H), 2.77-2.67 (m, 4H), 2.57 (d, J=17.4 Hz, 1H), 2.38-2.32 (m, 3H), 2.01-1.95 (m, 1H), 1.84 (t, J=11.5 Hz, 3H), 1.75 (s, 1H), 1.47 (s, 4H). 1.42-1.33 (m, 2H). The compound structure is confirmed.
To a 50 ml single-necked vial was sequentially added compound TPD12141-2a (1 g, 3.58 mmol), compound TPD12003-4 (1.08 g, 4.29 mmol), tetrakis(triphenylphosphine)palladium (413 mg, 0.36 mmol), cuprous iodide (34 mg, 0.18 mmol), DIEA (924 mg, 7.15 mmol) and DMF (15 ml). The reaction was carried out under nitrogen protection at 80° C. for 16 hours. After completion of the reaction, the reaction solution was reduced to room temperature, decanted into water (60 ml) and extracted three times with EA (15 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel column with eluent ratio of DCM/MeOH=100/1˜30/1 to give compound TPD12238-1 (500 mg, yellow solid, purity 98.367%), yield: 27.87%. LCMS (ESI) m/z calcd. for C26H31N5O5 [M+H]+ 494.2; found 494.3; 1H NMR (400 MHz, CDCl3): δ=9.06 (s, 1H), 8.15 (s, 1H), 7.52 (s, 1H), 5.21 (dd, J=13.3, 5.2 Hz, 1H), 4.54 (d, J=17.1 Hz, 1H), 4.35 (d, J=17.1 Hz, 1H), 4.07 (s, 2H), 3.19 (d, J=65.4 Hz, 2H), 2.93-2.80 (m, 6H), 2.41-2.21 (m, 4H), 1.65 (d, J=6.8 Hz, 2H), 1.45 (s, 9H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12238-1 (150 mg, 0.30 mmol) and 4 N HCl/1,4-dioxane (10 ml). The reaction was carried out at 25° C. for 2 h. After completion of the reaction, it was concentrated to dryness to give the crude compound TPD12238-2 (150 mg, yellow solid, purity 72.993%), yield: 60.406%. LCMS (ESI) m/z calcd. for C21H23N5O3 [M+H]+ 394.18; found 394.2. It is seen that the compound is structurally correct.
To a 25 ml single-necked vial was sequentially added compound TPD12238-2 (150 mg, 0.38 mol), compound TPD5488-2 (222 mg, 0.38 mmol), sodium triacetoxyborohydride (162 mg, 0.76 mmol) and DCE (2 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (6 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: XBridge-1 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 10-55/8 min to give the compound TPD12238 (10.5 mg, yellow solid, purity 82.478%) in the following yield: 2.12%. LCMS (ESI) m/z calcd. for C54H54FN9O7 [M+H]+ 962.41; found 962.4; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 10.18 (s, 1H), 10.05 (s, 1H), 8.93 (s, 1H), 8.43 (d, J=5.1 Hz, 1H). 7.75 (d, J=8.7 Hz, 2H), 7.68-7.58 (m, 3H), 7.46 (s, 1H), 7.32 (s, 1H), 7.27-7.07 (m, 4H), 6.61 (d, J=11.9 Hz, 1H), 6.39 (d, J=5.2 Hz, 1H), 6.14-5.98 (m, 1H). 5.13 (dd, J=13.4, 5.4 Hz, 1H), 4.52 (t, J=15.9 Hz, 1H), 4.44-4.35 (m, 1H), 3.94 (s, 4H), 3.66-3.52 (m, 5H), 3.04-2.76 (m, 10H), 2.71-2.55 (m, 6H), 2.45-2.22 (m, 3H), 2.09-1.72 (m, 4H), 1.48 (s, 5H), 1.43-1.25 (m, 2H). The compound structure is confirmed.
To a 25 ml single-necked vial were sequentially added compound TPD12238-2 (100 mg, 0.25 mmol), compound TPD12086-9 (145 mg, 0.25 mmol), sodium triacetoxyborohydride (108 mg, 0.51 mmol) and DCE (4 ml). The reaction was carried out under nitrogen protection at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (10 ml) and an appropriate amount of saturated NaHCO3 solution was added to make the reaction solution alkaline. It was extracted three times with DCM (5 ml). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was prepared by high performance liquid chromatography (HPLC) with the following parameters: column: T3 5 μm 19-150 mm; mobile phase: acetonitrile-water (0.1% FA); gradient: 15-35/9 min to give the compound TPD12239 (11.33 mg, white solid, purity 99.700%, time to front: 8.74 min), the compound TPD12239a (1.59 mg, white solid, purity 96.635%, time to peak: 8.51 min).
Compound TPD12239: LCMS (ESI) m/z calcd. for C53H53F2N9O6 [M+H]+ 950.4; found 950.4; 1H NMR (400 MHz, DMSO_d6): δ=11.04 (s, 1H), 10.19 (s, 1H), 10.05 (s, 1H), 8.93 (s, 1H). 8.56 (d, J=5.2 Hz, 1H), 7.85 (d, J=13.5 Hz, 1H), 7.76 (d, J=8.7 Hz, 2H), 7.69-7.56 (m, 3H), 7.44 (d, J=8.4 Hz, 1H), 7.21 (d, J=8.7 Hz, 2H), 7.15 (t, J=8.8 Hz, 2H) (m, 1H), 6.60 (d, J=11.9 Hz, 1H), 6.45 (d, J=5.1 Hz, 1H), 6.13-6.01 (m, 1H), 5.13 (dd, J=13.2, 5.0 Hz, 1H), 4.54 (d, J=18.3 Hz, 1H), 4.41 (d, J=18.2 Hz, 1H), 3.65-3.50 (m, 4H), 3.03-2.84 (m, 6H), 2.81-2.64 (m, 4H), 2.44-2.27 (m, 5H), 2.02-1.95 (m, 1H), 1.83 (d, J=12.3 Hz, 3H), 1.75 (s, 1H), 1.55 (s, 1H), 1.47 (s, 4H), 1.40-1.32 (m, 2H). The compound structure is confirmed.
Compound TPD12239a: LCMS (ESI) m/z calcd. for C53H53F2N9O6 [M+H]+ 951.4; found 951.5; 1H NMR (400 MHz, DMSO_d6): δ=11.02 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 8.85 (s, 1H), 8.56 (d, J=5.3 Hz, 1H), 7.85 (d, J=13.4 Hz, 1H), 7.76 (d, J=8.6 Hz, 2H), 7.64 (t, J=6.7 Hz, 3H), 7.43 (d, J=8.5 Hz, 1H), 7.21 (d, J=8.6 Hz, 2H), 7.15 (t, J=8.6 Hz, 2H), 7.07-6.91 (m, 1H), 6.70 (d, J=15.8 Hz, 1H), 6.45 (d, J=5.2 Hz, 1H), 5.11 (dd, J=13.3, 5.0 Hz, 1H), 4.51 (d, J=18.2 Hz, 1H), 4.38 (d, J=18.0 Hz, 1H), 3.51 (dd, J=22.2, 8.0 Hz, 8H), 2.97 (d, J=11.0 Hz, 2H), 2.84-2.72 (m, 5H), 2.34-2.23 (m, 4H), 2.02-1.97 (m, 1H), 1.82 (dd, J=20.1, 9.9 Hz, 3H), 1.75 (s, 1H), 1.47 (s, 5H), 1.38-1.32 (m, 2H). The compound structure is confirmed.
Inoculate gastric cancer cells (SNU5 or MKN45) into a 96 well plate at a concentration of 4×10 Å cells/ml, with 100 μl added to each well. Incubate for 30 minutes. Observe the cell status under a microscope. Prepare compounds, weigh a certain amount of the series compounds or reference drug cabozantinib of this patent, first prepare a high concentration mother liquor with pure DMSO, and use gradient dilution method to prepare a concentration of 200 μM, 60 μM, 20 μM, 6 μM, 2 μM, 600 nM, 200 nM, 0 nM drug solution, shake to make it fully mixed. The control group was prepared with cabozantinib using the same solvent and method.
After preparation, add 1 μl of drug solution to each well of the 96 well plate, and then add 100 μl of culture medium. Repeat each dose three times. Gently shake to allow the cells and medication to come into full contact and incubate in the incubator for 72 hours. 72 hours later, remove and color with CCK-8 reagent kit. Add 20 μl of CCK-8 reagent to each well, gently shake well, and incubate in the incubator for 1 hour. After 1 hour, remove and measure the absorbance of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.
According to the formula: Cell growth inhibition rate=[1−(absorbance of experimental group absorbance of culture medium control group)/(absorbance of blank control group−absorbance of culture medium control group)]×100%. It can be concluded that the compound has the ability to inhibit the growth of gastric cancer cells at the current dose and time.
Draw a graph based on the gradient of concentration and use GraphPad Prism 9 software (GraphPad Software, La Jolla, CA) to calculate the maximum inhibitory concentration (IC50) at 50%. The results are shown in Table 2 and
Western blot experiment: Take gastric cancer cells (MKN45) and inoculate them into a 6-well plate at a concentration of 2×10 Å cells/ml, adding 2 ml to each well and incubating for 24 hours.
The next day, observe the cells under a microscope to confirm their status. Prepare compounds, weigh a certain amount of the series compounds or reference drug cabozantinib from this patent, first prepare a high concentration mother liquor with pure DMSO, and use gradient dilution method to prepare compound concentrations of 2 mM, 200 μM, 100 μM, 20 μM, 2 μM, 200 nM, and 0 nM. The final concentration of DMSO is 0.5%. Shake to thoroughly mix. Add 10 μl of solution to each well. After incubation for 24 hours, remove the cells and use a pipette to aspirate the suspended cells into a 1.5 ml centrifuge tube. Centrifuge at 5000 rpm for 10 minutes to precipitate the cells. Carefully remove the cell precipitate from the culture medium, add 1 ml of PBS and shake slightly. After repeated cleaning for 3 times, dry the residue. Add 150 μl of RIPA lysate pre mixed with 1% PMSF, blow to allow the lysate to fully contact the cells, and incubate on ice for 10 minutes. Centrifuge at 12000 rpm for 10 minutes, extract the supernatant, use the BCA protein quantification kit to determine and homogenize the sample loading.
Mix BCA-A in a 50:1 ratio Prepare working solution with B solution, dilute the sample by 5 times, then pipette 20 μl onto a 96 well ELISA plate. Add 200 μl of pre mixed BCA working solution and incubate at 37° C. for 20 minutes. Measure the A562 nm absorbance value using an ELISA reader and substitute it into the protein standard curve formula to obtain the sample concentration. After calculating the required volume for loading 20 g of sample, divide the sample into 1.5 ml centrifuge tubes. Add 10 μl of sample buffer to each tube and mix well. Heat in a 95° C. metal bath for 5 minutes to denature the protein.
Take 4-20% gradient pre adhesive and fix it on the adhesive rack. Add electrophoresis solution to the slot and remove the comb, Marker and protein samples were loaded, and after 30 minutes of electrophoresis at 80V, the voltage was adjusted to 120V for electrophoresis, The marker band stops electrophoresis at the forefront of the gel. Take out the adhesive block and cut it to the appropriate size, The PVDF membrane was cut and activated in methanol, and after activation, the PVDF membrane was equilibrated in a membrane transfer equilibrium solution for 1 minute. Using sponge pads The sequence of PVDF film, adhesive, and sponge pad is placed in the splint and placed in the eBlot equipment for rapid wet rotation. Remove the membrane and seal it in 5% skim milk powder dissolved in 1×TBST for 1 hour. Cut the membrane according to the molecular weight of the target protein and internal reference protein, and place it in primary antibody diluted with 1×TBST. Shake overnight at 4° C.
The next day, transfer the band to 1×TBST for 3 washes, then add a specific dilution of the secondary antibody and shake it for 1.5 hours. Transfer the band again to 1×TBST for 3 washes, prepare ECL color solution, place the membrane in the imaging system, cover the surface with ECL color solution, and take photos of the membrane using the imaging system.
Using the SHST analysis system to analyze the results, measure the grayscale values of each band of the target protein and internal reference protein. Normalize the grayscale values of the target protein/corresponding lane GAPDH grayscale values, and perform quantitative analysis according to the protein amount=experimental group grayscale value/blank control group grayscale value. The results are shown in Table 2 and
It showed that the compound of the present invention significantly degrades the target protein DDR1 in tumor cells.
Referring to steps 1 and 2 of this embodiment, the inhibitory effect of the compound of the present invention on the growth of cancer cells and the degradation effect of target proteins were measured, and the following results were obtained (see Table 2 below).
The growth inhibitory effects of the compound of the present invention on tumor cell lines MKN45 and SNU5 were measured. The activity is expressed as half inhibitory concentration (C50). The results are shown in Table 2 below; When the half inhibitory concentration is less than or equal to 100 nM, the compound activity is labeled as “++++”; When the half inhibitory concentration is greater than 100 nM and less than 500 nM, the compound activity is labeled as “++”; When the half inhibitory concentration is greater than 500 nM and less than 1000 nM, the compound activity is labeled as “++”; When the half inhibitory concentration is greater than 1000 nM and less than 3000 nM, the compound activity is marked as “+”; When the half inhibitory concentration is greater than 3000 nM, the compound activity is marked as
The activity of the compound of the present invention in degrading DDR in tumor cell line MKN45 was measured. The activity is expressed as half degradation concentration (DC50) and maximum degradation percentage (Dmax); The results are shown in Table 2 below; When the half degradation concentration is less than or equal to 1 nM, the compound activity is marked as “++++”; When the half degradation concentration is greater than 100 nM and less than 500 nM, the compound activity is marked as “++”; When the half degradation concentration is greater than 500 nM and less than 1000 nM, the compound activity is marked as “++”; When the half degradation concentration is greater than 1000 nM and less than 3000 nM, the compound activity is marked as “+”; When the half degradation concentration is greater than 3000 nM, the compound activity is marked as “-”. When the maximum degradation percentage is greater than or equal to 85%, the compound activity is marked as “++++”; When the maximum degradation percentage is greater than 75% and less than 85%, the compound activity is marked as “++”; When the maximum degradation percentage is greater than 65% and less than 75, the compound activity is marked as “++”; When the maximum degradation percentage is greater than 50% and less than 65%, the compound activity is marked as “+”; When the maximum degradation percentage is less than 50%, the compound activity is marked as “-”
It showed that the growth inhibitory activity of the compounds of the present invention on the tumor cell lines MKN45 and SNU5 was in the range of 1-3000 nM expressed as half inhibitory concentration (IC50). The degradation activity of the compounds against DDR1 in the MKN45 cell line was in the range of 1-3000 nM expressed as half degradation concentration (DC50); the maximum percentage of degradation was in the range of 50%-95%.
Using healthy male BALB/c Nude mice as a model, human gastric cancer MKN45 cells were subcutaneously implanted. After 7 days of feeding, the tumor volume reached 75-100 mm3. Six mice were randomly divided into 5 groups: blank group, positive drug control group, drug 10 mg/kg group, drug 30 mg/kg group, and drug 50 mg/kg group.
Precisely weigh the subject compound TPD12140 or positive control drug cabozantinib, dissolve the samples in 5% DMSO/10% PEG400/10% Tween-80/75% Saline, dilute to a low concentration in a gradient, store at 4° C. and reserve for use, and dispense once a week. The drug was administered once a day for 14 consecutive days. Tumor volume was measured, and mice were weighed every two days, and the results are shown in A-B in Supplementary
As can be seen, in vivo experiments showed that the drug of the present invention had a significant inhibitory effect on tumors in all dose groups and there was no significant change in the average body weight.
Tumor cells were inoculated into 6-well plates with 1-3×105 cells per well, and the cell growth status was observed after 24 hours. After the cells grew to about 80% confluence, compound TPD12140 (working concentration 100 nM) was added to the cell culture medium in the experimental group, and an equal volume of DMSO was added to the control group, and the incubation was continued for 24 hours.
RNA extraction: Discard the culture medium from the well plate, add 1 mL Trizol, and let it sit at room temperature for 10 minutes; Blow the cells several times with a gun, transfer the lysate to a 1.5 ml EP tube treated with DEPC, add 200 μl of chloroform, and shake well for 15 seconds. After standing at room temperature for 2-3 minutes, centrifuge at 4° C. and 12000 rpm for 15 minutes. Then take the upper colorless aqueous phase into the EP tube treated with DEPC, being careful not to suck into the middle protein layer. Add 600 μl of isopropanol and let it stand at room temperature for 10 minutes or −80° C. for 60 minutes. 4° C., 12000 rpm, centrifuge for 15 minutes. Discard the supernatant, add 1.0 ml of 75% ethanol freshly prepared with DEPC water, mix upside down, centrifuge at 4° C. 7500 rpm for 5 minutes, and repeat the above operation once. Abandon the upper clean and use a small gun to suck up the liquid. Dry the precipitate in the air, add DEPC water, mix well to dissolve RNA. Detect the OD 260/280 value of RNA samples. Store the RNA sample in a −80° C. freezer.
Fluorescence quantitative PCR: Using the Invitrogen fluorescence quantitative reagent kit Platinum SYBR green qPCR Super Mix UDG, each sample is equipped with 3 complex wells for each primer pair, and a 20 μl system can be used for each well. After PCR is completed, running the corresponding program can test the reliability of PCR amplification. The fusion curve detection demonstrated the specificity of the amplified target gene and internal reference gene (GAPDH) PCR product. Finally, use the 2-ΔΔΔCt algorithm to perform relative quantitative analysis on the results. The 20 μl PCR amplification reaction system and reaction conditions are: 1 μl of the first strand of cDNA diluted 4 times, 10 μl of 2×SYBR green qPCR Super Mix UDG, 0.5 μl of upstream and downstream primers, and supplementation of dH2O to 20 μl. Reaction conditions: 50° C. for 2 minutes, 95° C. for 2 minutes, 95° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for 30 seconds, 40 cycles. The primer sequences for fluorescence quantitative PCR are as follows:
The experiment was repeated three times and more, and the experimental data were statistically analyzed using GraphPad Prism software. The results are shown in
Tumor cells were taken and inoculated into 6-well plates with 1×3×105 cells per well, and the cell growth status was observed on the next day. When the cells reached 80% confluence, TPD12140 (working concentration 100 nM) and different concentrations of cabozantinib or lenalidomide were added to the cell culture medium of the experimental group, and the control group was added with an equal volume of DMSO, and the incubation was continued for 24 h. Western Blot was performed to detect the expression of DDR1 protein (the same operation as above). The experiments were repeated three times or more, and the experimental data were statistically analyzed using GraphPad Prism software.
The experimental results are shown as B-C in
7. siRNA Knockdown Assay
Tumor cells were inoculated into 6-well plates with 1×3×105 cells per well, and cell growth was observed the next day. Transfection was performed when the cell density was about 30-50%. Add CRBN siRNA in a certain volume of antibiotic-free and serum-free medium and mix well, mix the transfection reagent with a certain volume of serum-free and antibiotic-free medium, mix well and leave it at room temperature for 5 min, mix the diluted siRNA and the transfection reagent, mix gently, and leave it at room temperature for 20 min. add the complex slowly into the cell culture medium and mix well by shaking gently. After incubation for 12 h, the cells were replaced with medium and incubated at 37° C. in a C02 incubator for 36 h. TPD12140 (working concentration 100 nM) was added to the cell culture medium of the experimental group, and an equal volume of DMSO was added to the control group, and the cells were further incubated for 24 h. Western Blot was performed to detect the expression of DDR1 protein (same as the above procedure). The experiments were repeated three times and more, and the experimental data were statistically analyzed using GraphPad Prism software.
The experimental results are shown in
8. Experiments with Inhibitors of the Ubiquitin-Proteasome System
Tumor cells were inoculated into 6-well plates with 1-3×105 cells per well, and the cell growth status was observed the next day. When the cells grew to about 80% confluence, the proteasome inhibitor MG132 (working concentration 10 uM) or the protein ubiquitination inhibitor MLN4924 (working concentration 1 uM) was added to the cell culture medium of the experimental group, and an equal volume of DMSO was added to the control group, and the incubation was continued for 6 hours. The medium in the well plates was discarded, washed twice with PBS, fresh medium was added, and compound TPD12140 (working concentration 100 nM) or an equal volume of DMSO was added to each group of cells, and the incubation was continued for 18 h. Western Blot was performed to detect the expression of DDR1 protein (same operation as above). The experiments were repeated three times or more, and the experimental data were statistically analyzed using GraphPad Prism software.
The experimental results are shown as E in
Obviously, the above embodiments are merely examples for the purpose of clear illustration and are not a limitation of the embodiments. For those of ordinary skill in the art, other variations or changes in different forms may be made based on the above description. It is neither necessary nor possible to exhaust all the embodiments herein. The obvious variations or changes derived therefrom remain within the scope of protection of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2023102574885 | Mar 2023 | CN | national |
