CN117624187A - Efficient HPK1 degradation agent compound and preparation method and application thereof - Google Patents

Efficient HPK1 degradation agent compound and preparation method and application thereof Download PDF

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CN117624187A
CN117624187A CN202311524559.XA CN202311524559A CN117624187A CN 117624187 A CN117624187 A CN 117624187A CN 202311524559 A CN202311524559 A CN 202311524559A CN 117624187 A CN117624187 A CN 117624187A
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cancer
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李汉园
韩晓军
俞智勇
王小寒
夏祥宇
曹泽亚
王友平
许国龙
何南海
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Hangzhou Arnold Biomedical Technology Co ltd
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Abstract

An HPK1 degradation agent compound, a preparation method and application thereof are provided. In particular, the use of compounds having the structure of formula (I) and pharmaceutical compositions thereof in the prevention and/or treatment of HPK 1-related diseases, such as cancer or immune diseases, is provided.

Description

Efficient HPK1 degradation agent compound and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a novel HPK1 degradation agent compound, a pharmaceutical composition containing the same, a preparation method of the same and application of the same in preventing and/or treating HPK1 related diseases.
Background
Targeting protein degradation (Targeted protein degradation) is one of the emerging directions in the field of drug development. The target protein degradation chimera (PROTAC) is used as a novel drug discovery technology, and can be used for connecting target proteins with E3 ligase, so that ubiquitination of the target proteins is induced, and the protease can recognize and degrade the target proteins. Unlike the space-occupying driving mechanism of the traditional small molecule inhibitors, the protein degradation mechanism of the PROTAC molecule does not require high binding force with the active site of the target protein, and can maintain the drug effect at a lower concentration and be longer lasting.
Hematopoietic progenitor kinase 1 (HPK 1, also known as MAP4K 1) is a serine/threonine kinase that is expressed predominantly in hematopoietic cells, including their progenitors, and is highly expressed in T cells, B cells, and dendritic cells. HPK1 is an important immunosuppressive regulatory kinase, whose function on T cells is currently well studied. It inhibits the activity of T Cell Receptor (TCR) signaling pathway by phosphorylating receptor protein SLP76, affecting downstream T cell activation and proliferation. HPK1 binds to a number of adaptor proteins, such as the SLP76 family, gads, HIP-55, GRB2 family, LAT, CRK family, etc., and activates the SAPK/JNK signaling pathway of hematopoietic stem cells, thereby down-regulating the TCR pathway. The study found that HPK1 expression in different cancer patients correlated positively with T cell failure signaling. Inhibition of the HPK1 pathway has potential applications in improving T cell function, antigen presentation, and in combating immunosuppressive tumor microenvironment, among others. In addition to the above-described role mainly through kinase activity, HPK1 has also been reported to have a scaffold function (scaffold), and the HPK1 knockout mice have no abnormality in a resting state, which both indicate that the development of HPK1 degradants can more completely inhibit HPK 1-related functions and have good safety.
Several studies have demonstrated that HPK1 is a potential tumor immunotherapeutic target, and no drugs against HPK1 targets are currently marketed, so more efficient drugs targeting HPK1 need to be developed to meet clinical demands.
Disclosure of Invention
The invention provides a compound with HPK1 degrading activity with the following table structure, and pharmaceutically acceptable salts, prodrugs, isotopic derivatives, metabolites and stereoisomers thereof:
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in one aspect, the invention also provides a pharmaceutical composition comprising a compound as described above, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide, or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
In a further aspect, the invention also provides the use of a compound as described above, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, or a pharmaceutical composition, for the prophylaxis and/or treatment of a disease associated with HPK1 activity. The diseases related to HPK1 activity include cancers or immune diseases; the cancer is preferably lung cancer, thyroid cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, oral cancer, thyroid cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, prostate cancer, cervical cancer, renal cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, cell tumor, glioma, hematological tumor, and lymphoma; the immune diseases are preferably lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis.
In one aspect, the invention also provides a method of preventing and/or treating a disease associated with HPK1 activity comprising administering to a patient in need thereof a therapeutically effective amount of a compound as described above, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, or a pharmaceutical composition. The diseases related to HPK1 activity include cancers or immune diseases; the cancer is preferably lung cancer, thyroid cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, oral cancer, thyroid cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, prostate cancer, cervical cancer, renal cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, cell tumor, glioma, hematological tumor, and lymphoma; the immune diseases are preferably lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis.
In a further aspect, in the above uses or methods, the above compounds, or stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, metabolites, isotopic derivatives, N-oxides, or prodrugs thereof, or the pharmaceutical compositions, may be used alone, or in combination with other types of pharmaceutical formulations and/or treatment methods.
It is particularly noted that, in this context, references to "compounds" of a particular structural formula are also generally intended to encompass stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives thereof.
It is well known to those skilled in the art that salts, solvates, hydrates of a compound are alternative forms of a compound, all of which can be converted to the compound under certain conditions, and therefore, it is of particular note herein that when referring to a compound, generally also pharmaceutically acceptable salts thereof, and further solvates and hydrates thereof, are included.
Similarly, when a compound is referred to herein, prodrugs, metabolites, and nitrogen oxides thereof are also generally included.
"stereoisomers" of the compounds of formula (I) of the present invention mean that when an asymmetric carbon atom is present in a compound of formula (I), an enantiomer is produced; when the compound has a carbon-carbon double bond or a cyclic structure, a cis-trans isomer is produced; tautomers are produced when compounds exist as ketones or oximes, and all enantiomers, diastereomers, racemates, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof of the compounds of formula (I) are included within the scope of the present invention.
The term "pharmaceutically acceptable salt" as used herein refers to addition salts of pharmaceutically acceptable acids and bases or solvates thereof. Such pharmaceutically acceptable salts include the salts of the following acids: hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfurous acid, formic acid, toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, alkanoic acids (such as acetic acid, HOOC- (CH) 2 ) n-COOH (wherein n is 0 to 4)), and the like. Salts of bases: sodium salt, potassium salt, calcium salt, ammonium salt, and the like. A variety of non-toxic pharmaceutically acceptable addition salts are known to those skilled in the art.
The pharmaceutically acceptable salts of the invention may be prepared by conventional methods, for example by dissolving the compounds of the invention in a water miscible organic solvent (e.g. acetone, methanol, ethanol and acetonitrile), adding thereto an excess of an organic or inorganic acid aqueous solution to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.
The precursors or metabolites of the invention may be precursors or metabolites well known in the art, as long as the precursors or metabolites are converted into compounds by in vivo metabolism. For example, "prodrugs" refer to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the formula described above, for example by metabolism in vivo.
The invention provides an application of a compound with a specific formula (I) structure and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs and metabolites thereof in preventing and/or treating HPK1 related diseases.
The HPK1 related diseases include cancers, inflammations, immunological diseases and the like.
The compounds of the invention described herein may be used alone or in combination with other classes of pharmaceutical agents (e.g., PD-1/PD-L1 antagonists, etc.) and/or therapeutic methods (e.g., radiation therapy/chemotherapy, etc.) in the prevention and/or treatment of HPK 1-related diseases.
The invention also provides the use of the compounds of the invention in the manufacture of a medicament for the prophylaxis and/or treatment of HPK 1-associated cancer, inflammation and immune disorders.
Furthermore, the present invention provides a pharmaceutical composition for preventing and/or treating HPK 1-related cancers, inflammations and immunological diseases, comprising the compound of the present invention as an active ingredient.
Detailed Description
Definition of the definition
Terms used in the present application (including the specification and claims) are defined as follows, unless otherwise indicated. It must be noted that, in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Conventional methods of mass spectrometry, nuclear magnetism, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used, if not otherwise indicated. In this application, the use of "or" and "means" and/or "unless otherwise indicated.
In the description and claims, a given formula or name shall encompass all stereoisomers and optical isomers and racemates in which the isomers exist. Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the present invention. Many geometric isomers of c=c double bonds, c=n double bonds, ring systems, etc. may also be present in the compounds, and all such stable isomers are contemplated within the present invention. The present invention describes cis-and trans- (or E-and Z-) geometric isomers of the compounds of the present invention, and which may be separated into mixtures of isomers or separate isomeric forms. The compounds of the invention may be isolated in optically active or racemic forms. All processes for preparing the compounds of the invention and intermediates prepared therein are considered part of the present invention. When preparing the enantiomeric or diastereomeric products, they can be separated by conventional methods, for example by chromatography or fractional crystallization. Depending on the process conditions, the end products of the invention are obtained in free (neutral) or salt form. Both the free form and the salt of these end products are within the scope of the invention. If desired, one form of the compound may be converted to another form. The free base or acid may be converted to a salt; the salt may be converted to the free compound or another salt; mixtures of the isomeric compounds of the invention may be separated into the individual isomers. The compounds of the invention, free forms and salts thereof, may exist in various tautomeric forms in which hydrogen atoms are transposed to other parts of the molecule and thereby the chemical bonds between the atoms of the molecule are rearranged. It is to be understood that all tautomeric forms that may exist are included within the invention.
In the case where nitrogen atoms (e.g., amines) are present on the compounds of the present invention, these nitrogen atoms may be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxide) to obtain other compounds of the present invention. Thus, the nitrogen atoms shown and claimed are considered to both encompass the nitrogen shown and its N-oxides to obtain the derivatives of the invention.
In the present invention, the term "patient" refers to an organism treated by the method of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian/monkey, equine, bovine, porcine, canine, feline, etc.) and most preferably refer to humans.
In the present invention, the term "effective amount" means an amount of a drug or pharmaceutical agent (i.e., a compound of the present invention) that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of: such amounts result in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such amounts. An effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. The term also includes within its scope an effective amount to enhance normal physiological function.
In the present invention, the term "treatment" is intended to include its broad meaning, covering therapeutic treatment and/or prophylactic treatment of a subject. In particular, the "treatment" includes any treatment that results in alleviation, inhibition, elimination, and amelioration and/or prevention of a condition, disease, disorder, etc., such as alleviation, reduction, regulation, amelioration, elimination, prevention, or amelioration of a symptom thereof. The therapeutic treatment includes alleviation, inhibition, or amelioration of the symptoms or conditions of the disease; inhibit the occurrence of complications; improving underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or treating signs caused by diseases or symptoms. The prophylactic treatment includes prior treatment to prevent, block or delay, slow the occurrence or progression of, or attenuate the severity of a disease or disorder.
Likewise, "therapeutic agent" also includes an agent or reagent that has therapeutic and/or prophylactic treatment of a subject.
In the present invention, the term "pharmaceutically" or "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are: it is suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic response, and/or other problems or complications commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.
In the present invention, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" means a pharmaceutical substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, which involves carrying or transporting the subject compound from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient.
In the present invention, the term "pharmaceutical composition" means a composition comprising a compound of the present invention and at least one other pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" refers to a medium commonly accepted in the art for delivery of biologically active agents to animals, particularly mammals, and includes (i.e., adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow control agents, disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, antibacterial agents, antifungal agents, lubricants, and dispersing agents, depending upon the mode of administration and the nature of the dosage form.
Specific pharmaceutical and medical terminology
In the present invention, the term "acceptable" as used herein, means that a prescription component or active ingredient does not unduly adversely affect the health of the general therapeutic objective.
In the present invention, the term "cancer", as used herein, refers to an abnormal growth of cells that cannot be controlled, and is capable of metastasis (spread) under certain conditions. Cancers of this type include, but are not limited to, solid tumors (e.g., bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovary, pancreas, or other endocrine organ (e.g., thyroid), prostate, skin (melanoma), or hematological tumors (e.g., non-leukemia).
In the present invention, the term "co-administration" or similar terms, as used herein, refers to administration of several selected therapeutic agents to a patient, in the same or different modes of administration, at the same or different times.
In the present invention, the term "enhance" or "potentiating", as used herein, means that the intended result can be increased or prolonged in either potency or duration. Thus, in enhancing the therapeutic effect of a drug, the term "enhancing" refers to the ability of a drug to increase or prolong the potency or duration of the drug in the system. As used herein, "potentiating value" refers to the ability of an additional therapeutic agent to be maximally enhanced in an ideal system.
In the present invention, the term "immunological disease" refers to a disease or condition that is an adverse or detrimental reaction to an endogenous or exogenous antigen. As a result, the cells are often dysfunctional, or thus destroyed and dysfunctional, or destroy organs or tissues that may develop immune symptoms.
In the present invention, the term "kit" is synonymous with "product package".
In the present invention, the term "subject", "subject" or "patient" includes mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as gorillas, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, pigs; domestic animals such as rabbits and dogs; laboratory animals include rodents such as rats, mice, guinea pigs, and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like. In a preferred embodiment, the mammal selected is a human.
As used herein, a compound or pharmaceutical composition, upon administration, may result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay of onset, slow progression, or decrease in duration. Whether stationary or temporary, continuous or intermittent, may be due to or associated with administration.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention may be better understood by reference to the following specific examples which are intended to illustrate, but are not to be limiting of, the present invention.
When no preparation route is mentioned in the present invention, both the relevant raw materials and intermediates are purchased from commercial reagents (e.g., from pichia, medical stones, etc.).
Abbreviations in the present invention have the following meanings:
in the examples below, the reaction temperature was room temperature (15-30 ℃ C.) unless otherwise specified.
The compounds of the present invention are isolated and purified by preparative TLC, silica gel column chromatography, prep-HPLC and/or Flash column chromatography (Flash column chromatography), the structure of which is described by 1 H NMR and/or MS. Reaction monitoring was performed by TLC or LC-MS.
1 H-NMR spectra were recorded on a Bruker instrument at 500 MHz. Chemical shift values are expressed in parts per million, i.e., delta values. The following abbreviations are used for multiplicity of NMR signals: s=singlet, brs=broad, d=doublet, t=triplet, m=multiplet. Coupling constants are listed as J values, measured in Hz. LC-MS experimental conditions were: instrument: thermo U3000, ALLtech ELSD, MSQ, UV detector combined ELSD and MSD (outflow ratio 4:1). Column: waters X-Bridge C-18,3.5 μm,4.6X50 mm; column temperature: 30 ℃. Gradient [ time (min)/solvent B in A (%) ]:0.00/5.0,1.40/95,2.80/95,2.82/5,3.00/5. (solvent a=0.01% trifluoroacetic acid in water; solvent b=0.01% trifluoroacetic acid in acetonitrile). UV detection: 214/254/280/300nm; DAD detection: 210-350nm; flow rate: 2mL/min; MS: ESI,100-1500m/z.
Preparative HPLC uses an alkaline method or an acidic method (alkaline method mobile phase: acetonitrile/0.05% ammonium bicarbonate aqueous solution, acidic method mobile phase: acetonitrile/0.05% formic acid aqueous solution); the instrument is Thermo U3000 AFC-3000; column: globalsil C-18 12nm,250x 20mm,10 μm, or equivalent; flow rate: and (3) carrying out gradient elution separation at 20 mL/min.
Example 1
The first step: 5-fluoro-2-nitroaniline (5.0 g,32.03 mmol) and 1-t-butoxycarbonyl piperazine (6.3 g,33.63 mmol) were dissolved in NMP (30 mL), DIPEA (11.2 mL,64.06 mmol) was added and reacted at 120℃overnight. The reaction solution was diluted with ethyl acetate, which was then washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and concentrated by filtration to give 1a (9.6 g, yield 93%) as a yellow solid. ESI-MS (m/z): 323.7[ M+H ]] + 。
And a second step of: 1a (9.6 g,29.78 mmol) was dissolved in absolute ethanol (40 mL), 10% palladium on carbon (0.361 g,2.98 mmol) was added, the reaction system replaced hydrogen with a hydrogen balloon, and stirred overnight at 40℃under hydrogen balloon pressure. Then, ethyl 3-ethoxy-3-iminopropionate hydrochloride (8.7 g,44.63 mmol) was added to the reaction solution, and the temperature was raised to 80℃and stirring was continued for 4 hours. After the completion of the reaction, the system was filtered through celite, washed with methylene chloride, and the filtrate was concentrated to give 1b (10 g, yield 87%) as a yellow solid. ESI-MS (m/z): 389.5[ M+H ] ] + 。
And a third step of: 1b (10 g,25.74 mmol) and 2-aminothiophene-3-carbonitrile (3.20 g,25.74 mmol) were dissolved in tetrahydrofuran (50 mL), a solution of 2M LDA in THF (64 mL,128.71 mmol) was added under nitrogen, and the system was warmed to 40℃and stirred for 2 hours. After the reaction, the system was quenched with saturated ammonium chloride, extracted three times with dichloromethane, and the organic phase was washed successively with water and saturated sodium chloride solution. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 1c as a brown solid (6.8 g, yield 56%). ESI-MS (m/z): 467.3[ M+H ]] + 。
Fourth step: 1c (6.8 g,14.6 mmol) was dissolved in dioxane (30 mL) and dichloromethane (10 mL)) To this solution, 4M HCl dioxane solution (20 mL) was added and the mixture was stirred at room temperature for 2 hours. The reaction system was concentrated to give 1d (5.2 g, yield 98%). ESI-MS (m/z): 367.7[ M+H ]] + 。
Fifth step: 2- (2, 6-Dioxopiperidin-3-yl) -4-hydroxy-iso-1, 3-dione (500 mg,2 mmol), potassium iodide (30 mg,0.15 mmol) and sodium bicarbonate (305 mg,2 mmol) were dissolved in DMF (5 mL), 8-bromo-1-octanol (502 mg,2.4 mmol) was added to the reaction system, after which the reaction system was heated to 80℃and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate, and extracted 3 times with saturated brine, and the organic phase was taken, dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol) to give final product 1e (425 mg, yield 44%). ESI-MS (m/z): 403.1[ M+H ] ] + 。
Sixth step: 1e (100 mg,0.25 mmol) was dissolved in dichloromethane (4 mL), and dess-martin oxidant (106 mg,0.25 mmol) was added to the reaction system, after which the reaction system was stirred at room temperature for 1 hour, and LCMS detected the completion of the substrate reaction. The reaction system was filtered, washed with methylene chloride, and the organic phase was washed with saturated sodium bicarbonate solution, saturated brine, then dried over anhydrous sodium sulfate, filtered, and concentrated to give the final product 1f (91 mg, yield 86%) which was directly used for the next reaction. ESI-MS (m/z): 401.3[ M+H ]] + 。
Seventh step: 1d (50 mg,0.14 mmol), 1f (56 mg,0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath conditions, sodium acetate (19 mg,0.14 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hour, and then sodium triacetoxyborohydride (59 mg,0.28 mmol) was added. The system was stirred overnight at room temperature and LCMS monitored reaction was complete. The reaction mixture was concentrated, and the residue was dissolved in methanol and purified by HPLC to give Compound 1 (23 mg, yield 29%). ESI-MS (m/z): 751.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(d,J＝6.2Hz,1H),12.08(s,1H),11.11(s,1H),10.85–10.53(m,1H),8.25(s,1H),7.98(s,1H),7.81(dd,J＝8.5,7.3Hz,1H),7.59(d,J＝5.8Hz,1H),7.54–7.42(m,2H),7.19–7.08(m,2H),6.91–6.86(m,1H),5.08(dd,J＝12.8,5.5Hz,1H),4.21(t,J＝6.4Hz,2H),3.09(t,J＝4.9Hz,4H),2.96–2.80(m,2H),2.32(t,J＝7.4Hz,2H),2.06–1.95(m,2H),1.77(p,J＝6.6Hz,2H),1.53–1.28(m,12H),1.26–1.14(m,2H).
Example 2
The compound 2 can be obtained by a similar method and reaction procedure by replacing the 8-bromo-1-octanol of the fifth step in example 1 with 9-bromo-1-nonanol. ESI-MS (m/z): 765.9[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝6.3Hz,1H),12.07(s,1H),11.10(s,1H),10.86–10.53(m,1H),8.07–7.90(m,1H),7.79(t,J＝7.9Hz,1H),7.57(d,J＝5.8Hz,1H),7.54–7.40(m,3H),7.23–7.02(m,2H),6.92–6.83(m,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.19(t,J＝6.4Hz,2H),3.08(t,J＝4.7Hz,4H),2.91–2.82(m,1H),2.66–2.52(m,3H),2.30(t,J＝7.4Hz,2H),2.06–1.98(m,1H),1.75(t,J＝7.3Hz,3H),1.51–1.18(m,14H).
Example 3
The compound 3 can be obtained by a similar method and reaction procedure, substituting 11-bromo-1-undecanol for 8-bromo-1-octanol in the fifth step of example 1. ESI-MS (m/z): 793.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝5.9Hz,1H),12.07(s,1H),11.09(s,1H),10.83–10.45(m,1H),8.05–7.89(m,1H),7.79(t,J＝7.9Hz,1H),7.57(d,J＝5.8Hz,1H),7.36–7.29(m,3H),7.25–7.03(m,2H),6.88(td,J＝7.0,5.8,2.4Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.18(t,J＝6.5Hz,2H),3.09(s,4H),2.95–2.77(m,1H),2.78–2.21(m,3H),2.30(t,J＝7.0Hz,2H),2.05–1.98(m,1H),1.96–1.51(m,2H),1.49–1.41(m,5H),1.35–1.20(m,14H).
Example 4
The compound 4 can be obtained by a similar method and reaction procedure, substituting 7-bromo-1-heptanol for 8-bromo-1-octanol in the fifth step of example 1. ESI-MS (m/z): 737.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.63(s,1H),12.06(s,1H),11.09(s,1H),10.95–10.35(m,1H),7.95(s,1H),7.80(t,J＝7.9Hz,1H),7.57(d,J＝5.7Hz,1H),7.53–7.41(m,3H),7.21–7.06(m,2H),6.87(t,J＝7.5Hz,1H),5.07(dd,J＝12.7,5.4Hz,1H),4.20(t,J＝6.5Hz,2H),3.08(t,J＝4.7Hz,4H),2.91–2.82(m,1H),2.65–2.53(m,2H),2.31(t,J＝7.3Hz,2H),2.05–1.99(m,1H),1.80–1.73(m,2H),1.56–1.13(m,12H).
Example 5
The compound 5 can be obtained by a similar method and reaction procedure, substituting 5-bromo-1-pentanol for 8-bromo-1-octanol in the fifth step of example 1. ESI-MS (m/z): 709.2[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝6.0Hz,1H),12.07(s,1H),11.09(s,1H),10.85–10.44(m,1H),7.97(s,1H),7.80(t,J＝7.8Hz,1H),7.58(d,J＝5.8Hz,1H),7.54–7.41(m,3H),7.20–7.06(m,2H),6.92–6.83(m,1H),5.07(dd,J＝12.6,5.5Hz,1H),4.22(t,J＝6.5Hz,2H),3.11–3.05(m,4H),2.89–2.83(m,1H),2.54–2.53(m,4H),2.38–2.32(m,2H),2.07–1.75(m,4H),1.60–1.43(m,5H).
Example 6
The compound 6 can be obtained by a similar method and reaction procedure by substituting 6-bromo-1-hexanol for 8-bromo-1-octanol in the fifth step of example 1. ESI-MS (m/z): 723.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝5.6Hz,1H),11.09(s,1H),10.81–10.54(m,1H),8.03–7.75(m,2H),7.58(d,J＝5.8Hz,1H),7.55–7.41(m,3H),7.23–7.05(m,2H),6.90–6.55(m,2H),5.07(dd,J＝12.6,5.5Hz,1H),4.21(t,J＝6.3Hz,2H),3.28–2.86(m,6H),2.39–2.29(m,2H),2.05–1.95(m,2H),1.77(s,2H),1.54–1.13(m,10H).
Example 7
The first step: 2- (2, 6-Dioxopiperidin-3-yl) -4-fluoro-iso-1, 3-dione 7a (100 mg,0.36 mmol) and 4-hydroxymethylpiperidine (42 mg,0.36 mmol) were dissolved in DMF (5 mL), N-diisopropylethylamine (93 mg,0.72 mmol) was added to the reaction, after which the reaction was heated to 80℃and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate, and extracted 3 times with saturated brine, and the organic phase was taken, dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol) to give final product 7b (130 mg, yield 97%). ESI-MS (m/z): 372.8[ M+H ] ] + 。
And a second step of: 7b (100 mg,0.27 mmol) was dissolved in methylene chloride (4 mL), and after adding dess-Martin oxidant (125 mg,0.29 mmol) to the reaction system, the reaction system was stirred at room temperature for 1 hour, and LCMS detected the completion of the substrate reaction. The reaction system was filtered, washed with methylene chloride, and the organic phase was washed with saturated sodium hydrogencarbonate solution, saturated brine, then dried over anhydrous sodium sulfate, filtered and concentrated to give final product 7c (76 mg, yield 77%) which was directly used in the next reaction. ESI-MS (m/z): 370.4[ M+H ]] + 。
And a third step of: 1d (50 mg,0.14 mmol) and 7c (52 mg,0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath, sodium acetate (19 mg,0.14 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hour, and then sodium triacetoxyborohydride (59 mg,0.28 mmol) was added. The system was stirred overnight at room temperature and LCMS monitored reaction was complete. The reaction mixture was concentrated, and the residue was dissolved in methanol, and purified by HPLC to give Compound 7 (19 mg, yield 14%). ESI-MS (m/z): 720.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.61(d,J＝4.5Hz,1H),12.10(s,1H),11.09(s,1H),10.89–10.46(m,1H),7.98(s,1H),7.68(t,J＝7.8Hz,1H),7.59(d,J＝5.7Hz,1H),7.31–7.16(m,1H),7.33(dd,J＝12.0,7.8Hz,2H),7.23–7.09(m,2H),6.98–6.84(m,1H),5.09(dd,J＝12.7,5.6Hz,1H),3.71(d,J＝11.7Hz,2H),3.12(d,J＝6.1Hz,4H),2.94–2.83(m,3H),2.65–2.55(m,5H),2.27(d,J＝7.0Hz,2H),2.10–1.70(m,5H),1.38–1.28(m,2H).
Example 8
Compound 8 can be obtained by a similar procedure and reaction steps substituting 2- (2, 6-dioxopiperidin-3-yl) -5-fluoro-iso-1, 3-dione for 2- (2, 6-dioxopiperidin-3-yl) -4-fluoro-iso-1, 3-dione in the first step of example 7. ESI-MS (m/z): 720.8[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.61(s,1H),12.05(s,1H),11.08(s,1H),10.79–10.38(m,1H),7.98(s,1H),7.65(d,J＝8.5Hz,1H),7.59(d,J＝5.7Hz,1H),7.52–7.40(m,1H),7.32(s,1H),7.27–7.08(m,3H),6.96–6.83(m,1H),5.07(dd,J＝13.1,5.3Hz,1H),4.06(d,J＝12.9Hz,2H),3.12(s,4H),3.05–2.83(m,4H),2.66–2.58(m,1H),2.22(d,J＝7.0Hz,2H),2.07–1.79(m,5H),1.37–1.09(m,5H).
Example 9
The fifth step of 8-bromo-1-octanol in example 1 was replaced with bromoethanol, and compound 9 was obtained by a similar method and reaction procedure. ESI-MS (m/z): 667.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(d,J＝5.6Hz,1H),12.09(s,1H),11.11(s,1H),10.74–10.59(m,1H),7.97(d,J＝13.6Hz,1H),7.87–7.79(m,1H),7.62–7.40(m,4H),7.19(dd,J＝12.4,4.0Hz,1H),7.13–6.85(m,2H),5.10(dd,J＝12.8,5.4Hz,1H),4.39(t,J＝5.7Hz,2H),3.11(t,J＝4.9Hz,5H),2.90–2.83(m,3H),2.75(t,J＝4.9Hz,4H),2.09–1.94(m,2H).
Example 10
The first step of 4-hydroxymethylpiperidine in example 7 was replaced with (S) -pyrrolidine-3-methanol and a similar procedure and reaction procedure was followed to give compound 10.ESI-MS (m/z): 706.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.62(s,1H),12.08(s,1H),11.05(s,1H),10.83–10.50(m,1H),7.95(s,1H),7.61–7.54(m,2H),7.52–7.43(m,1H),7.32–6.97(m,4H),6.90(d,J＝8.3Hz,1H),5.09–5.04(m,1H),3.69–3.54(m,3H),3.12(s,3H),2.65–2.56(m,6H),2.42(d,J＝7.3Hz,2H),2.14–1.94(m,4H),1.78–1.40(m,3H).
Example 11
The compound 11 can be obtained by a similar method and reaction procedure, substituting 1-bromo-1-butanol for 8-bromo-1-octanol in the fifth step of example 1.ESI-MS (m/z): 695.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.08(s,1H),11.11(s,1H),10.95–10.29(m,1H),7.98(d,J＝11.2Hz,1H),7.81(dd,J＝8.5,7.3Hz,1H),7.61–7.43(m,4H),7.23–7.04(m,2H),6.93–6.85(m,1H),5.09(dd,J＝12.7,5.4Hz,1H),4.27(t,J＝6.3Hz,2H),3.10(s,4H),2.93–2.82(m,1H),2.61–2.53(m,6H),2.42(t,J＝7.0Hz,2H),2.07–1.99(m,1H),1.87–1.76(m,2H),1.74–1.61(m,2H).
Example 12
The first step: 2- (2, 6-Dioxopiperidin-3-yl) -4-fluoro-iso-1, 3-dione 7a (100 mg,0.36 mmol) and (R) -pyrrolidine-3-methanol (40 mg,0.36 mmol) were dissolved in DMF (5 mL), N-diisopropylethylamine (93 mg,0.72 mmol) was added to the reaction, after which the reaction was heated to 80℃and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate and extracted with saturated brineThe organic phase was taken 3 times, dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol) to give the final product 12a (126 mg, yield 95%). ESI-MS (m/z): 358.6[ M+H ] ] + 。
And a second step of: 12a (100 mg,0.30 mmol) was dissolved in methylene chloride (4 mL), and after adding dess-martin oxidant (125 mg,0.30 mmol) to the reaction system, the reaction system was stirred at room temperature for 2 hours. The reaction system was filtered, washed with methylene chloride, and the organic phase was washed with saturated sodium hydrogencarbonate solution, saturated brine, then dried over anhydrous sodium sulfate, filtered and concentrated to give the final product 12b (73 mg, yield 78%) which was directly used in the next reaction. ESI-MS (m/z): 356.4[ M+H ]] + 。
And a third step of: 1d (50 mg,0.14 mmol) and 12b (48 mg,0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath, sodium acetate (19 mg,0.14 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hour, and then sodium triacetoxyborohydride (59 mg,0.28 mmol) was added. The system was stirred overnight at room temperature. The reaction was concentrated, and the residue was dissolved in DMF and purified by HPLC to give compound 12 (14 mg, yield 18%). ESI-MS (m/z): 706.1[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.61(d,J＝4.7Hz,1H),12.10(s,1H),11.05(s,1H),10.88–10.57(m,1H),7.97(s,1H),7.61–7.53(m,2H),7.50–7.36(m,1H),7.22–7.09(m,4H),6.93–6.87(m,1H),5.07(ddd,J＝12.7,5.4,1.5Hz,1H),3.67–3.62(m,2H),3.59–3.53(m,2H),3.12(t,J＝5.3Hz,5H),2.91–2.83(m,1H),2.66–2.55(m,6H),2.44–2.41(m,2H),2.11–2.06(m,1H),2.03–1.97(m,1H),1.74–1.68(m,1H).
Example 17
The first step: 7c (70 mg,0.19 mmol) of potassium hydrogen persulfate complex salt (oxone) (116 mg,0.19 mmol) was dissolved in DMF (3 mL), stirred overnight at room temperature, ethyl acetate was added to the reaction system to dilute and extracted 3 times with water and saturated brine, the organic phase was added to anhydrous sodium sulfate to dry, filtered, Concentration gave final product 17a (45 mg, 62% yield). ESI-MS (m/z): 386.8[ M+H ]] + 。
And a second step of: compound 17a (35 mg,0.09 mmol) was dissolved in DMF (2 mL), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (68 mg,0.18 mmol) was added with stirring at 0deg.C, after 10 min DIPEA (57 mg,0.45 mmol) and compound 1d (43 mg,0.09 mmol) were added sequentially, and the mixture was warmed to room temperature and stirred overnight. The reaction was filtered and purified by HPLC to give compound 17 (12 mg, yield 18%). ESI-MS (m/z): 734.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.65(s,1H),12.32–11.78(br,1H),11.09(s,1H),10.84–10.38(m,1H),8.11–7.89(m,1H),7.69(dd,J＝8.5,7.1Hz,1H),7.59(d,J＝5.8Hz,1H),7.550–7.39(m,1H),7.35(dd,J＝11.6,7.8Hz,2H),7.27–7.15(m,2H),6.98–6.91(m,1H),5.10(dd,J＝12.8,5.4Hz,1H),3.78–3.65(m,6H),3.16–3.06(m,4H),3.03–2.85(m,4H),2.65–2.54(m,2H),2.03(ddd,J＝10.7,5.6,3.1Hz,1H),1.88–1.77(m,4H).
Example 18
The first step: 2- (2, 6-Dioxopiperidin-3-yl) -5-fluoro-iso-1, 3-dione (100 mg,0.36 mmol) and 4-hydroxymethylpiperidine (42 mg,0.36 mmol) were dissolved in DMF (5 mL), N-diisopropylethylamine (93 mg,0.72 mmol) was added to the reaction, after which the reaction was heated to 80℃and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate, extracted 3 times with saturated brine, the organic phase was taken, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give the final product 18a (124 mg, yield 95%). ESI-MS (m/z): 372.7[ M+H ]] + 。
And a second step of: 18a (70 mg,0.19 mmol) and a potassium hydrogen persulfate complex salt (oxone) (116 mg,0.19 mmol) were dissolved in DMF (3 mL), stirred overnight at room temperature, ethyl acetate was added to the reaction system to dilute and extracted 3 times with water and saturated brine, and the organic phase was added to anhydrous sodium sulfate to dry, filtered and concentrated to give the final product 18b (52 mg, yield 71%). ESI-MS(m/z):370.5[M+H] + 。
And a third step of: 18b (35 mg,0.09 mmol) was dissolved in DMF (2 mL), stirred at 0deg.C, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (68 mg,0.18 mmol) was added, after 10 min DIPEA (57 mg,0.45 mmol) and Compound 1d (43 mg,0.09 mmol) were added sequentially, warmed to room temperature and stirred overnight. The reaction was filtered and purified by HPLC to give compound 18 (16 mg, yield 24%). ESI-MS (m/z): 734.7[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.31–11.60(m,1H),11.01(s,1H),10.87–10.30(m,1H),8.10–7.77(m,1H),7.60(d,J＝8.5Hz,1H),7.52(d,J＝5.7Hz,1H),7.49–7.39(m,1H),7.27(d,J＝2.3Hz,1H),7.21–7.07(m,3H),6.91–6.84(m,1H),5.00(dd,J＝12.8,5.5Hz,1H),4.02(d,J＝13.1Hz,2H),3.90–3.25(m,4H),3.11–2.97(m,7H),2.86–2.78(m,1H),2.59–2.47(m,2H),1.98–1.91(m,1H),1.72–1.54(m,4H).
Example 19
The first step: 19a (1 g,5.79 mmol) and 19a-1 (1.97 g,6.37 mmol) were added to a mixed solvent of 1, 4-dioxane (16 mL) and water (4 mL), followed by addition of potassium carbonate solid (2.40 g,17.38 mmol) and Pd (dppf) Cl2 (424.01 mg,0.58 mmol). The reaction system was replaced with nitrogen three times with a nitrogen ball and reacted at 90℃for 3 hours under the protection of the nitrogen ball. After the reaction, the reaction solution was cooled to room temperature. The reaction solution was diluted with ethyl acetate, which was then washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 19b (1.41 g, yield 76.3%) as a yellow solid. ESI-MS (m/z): 320.6[ M+H ]] + 。
And a second step of: 19b (1.41 g,4.42 mmol) was dissolved in anhydrous methanol (30 mL), 10% palladium on carbon (0.56 g,5.30 mmol) was added, the reaction system was replaced with hydrogen gas by hydrogen balloon, the reaction was carried out at 25℃for 3 hours under the pressure of hydrogen balloon, then ethyl 3-ethoxy-3-iminopropionate hydrochloride (1.29 g,4.43 mmol) was added to the reaction solution, and the temperature was raised to 70℃C Stirring was continued for 4 hours. The system was filtered through celite, washed with dichloromethane, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 19c as a brown solid (1.72 g, yield 100%). ESI-MS (m/z): 388.6[ M+H ]] + 。
And a third step of: 19c (0.5 g,1.29 mmol) and 2-aminothiophene-3-carbonitrile (160 mg,1.29 mmol) were dissolved in tetrahydrofuran (10 mL), and a 1M solution of LiHMDS in THF (12.9 mL,12.9 mmol) was added under nitrogen and the reaction stirred at 35℃for 2 hours. The system was quenched with saturated ammonium chloride, extracted three times with ethyl acetate, and the organic phase was washed successively with water and saturated sodium chloride solution. The organic phase was concentrated and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 19d as a brown solid (457 mg, yield 76.1%). ESI-MS (m/z): 466.7[ M+H ]] + 。
Fourth step: 19d (457 mg,0.98 mmol) was dissolved in dioxane (5 mL) and dichloromethane (5 mL), 4M HCl dioxane solution (5 mL) was added, and the mixture was stirred at 35℃for 3 hours. The reaction system was concentrated to give compound 19e (429 mg, yield 99%). ESI-MS (m/z): 366.2[ M+H ]] + 。
Fifth step: 19e (100 mg,0.27 mmol) and 7c (101 mg,0.27 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath conditions, sodium acetate (37 mg,0.27 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hours, then sodium triacetoxyborohydride (35 mg,0.55 mmol) was added and the system was stirred at room temperature overnight. The reaction was concentrated, and the residue was dissolved in DMF and purified by HPLC to give compound 19 (21 mg, yield 11%). ESI-MS (m/z): 719.4[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.72(s,1H),12.11(s,1H),11.09(s,1H),10.85–10.56(m,1H),8.19(s,1H),7.68(dd,J＝8.5,7.1Hz,1H),7.62–7.53(m,2H),7.52–7.44(m,1H),7.33(dd,J＝13.1,7.8Hz,2H),7.19(d,J＝5.7Hz,1H),7.05(d,J＝8.5Hz,1H),5.10(dd,J＝12.7,5.5Hz,1H),3.71(d,J＝11.6Hz,2H),3.02(d,J＝10.7Hz,2H),2.93–2.86(m,3H),2.63–2.56(m,2H),2.27(d,J＝7.0Hz,2H),2.10–2.01(m,3H),1.89–1.71(m,8H),1.40–1.31(m,2H).
Example 20
The compound 20 can be obtained by a similar method and reaction procedure, substituting trans-4-aminocyclohexylmethanol for the 4-hydroxymethylpiperidine of the first step of example 7. ESI-MS (m/z): 734.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.61(s,1H),12.10(s,1H),11.10(s,1H),10.80–10.51(m,1H),7.98(s,1H),7.67–7.41(m,3H),7.26–6.99(m,4H),6.95–6.86(m,1H),6.19(d,J＝8.2Hz,1H),5.05(dd,J＝12.8,5.5Hz,1H),3.11(t,J＝4.7Hz,6H),2.94–2.79(m,2H),2.64–2.55(m,2H),2.54–2.53(m,2H),2.19(d,J＝7.1Hz,2H),2.07–1.99(m,3H),1.87(d,J＝12.8Hz,2H),1.64–1.52(m,1H),1.32–1.22(m,2H),1.07(q,J＝12.1Hz,2H).
Example 21
The first step of 4-hydroxymethylpiperidine in example 7 was replaced with 4-piperidineethanol and compound 21 was obtained in a similar manner and reaction procedure. ESI-MS (m/z): 734.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.10(s,1H),11.09(s,1H),10.71–10.54(m,1H),7.98(s,1H),7.71–7.56(m,2H),7.44(d,J＝8.7Hz,1H),7.33(t,J＝8.5Hz,2H),7.23–7.05(m,2H),6.93–6.85(m,1H),5.09(dd,J＝12.7,5.5Hz,1H),3.69(d,J＝11.6Hz,3H),3.12(t,J＝4.9Hz,5H),2.91–2.83(m,3H),2.60–2.55(m,4H),2.45–2.40(m,2H),2.06–1.96(m,1H),1.81(d,J＝12.0Hz,2H),1.53–1.33(m,5H).
Example 22
The first step of 4-hydroxymethylpiperidine in example 7 was replaced with (R) -3-piperidinemethanol and compound 22 was obtained in a similar manner and reaction steps. ESI-MS (m/z): 720.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.69–12.53(m,1H),12.09(s,1H),11.14–11.04(m,1H),10.81–10.50(m,1H),8.05–7.92(m,1H),7.74–7.65(m,1H),7.63–7.56(m,1H),7.54–7.41(m,1H),7.38–7.29(m,2H),7.21–7.03(m,2H),6.96–6.81(m,1H),5.17–5.03(m,1H),3.84–3.55(m,2H),3.09(s,4H),2.96–2.84(m,2H),2.72–2.54(m,4H),2.33–2.16(m,2H),2.08–1.96(m,6H),1.87–1.66(m,3H).
Example 23
Compound 23 was obtained by a similar procedure and reaction steps using (S) -3-piperidinemethanol instead of 4-hydroxymethylpiperidine as in the first step of example 7. ESI-MS (m/z): 720.7[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(d,J＝8.6Hz,1H),12.08(s,1H),11.21–11.00(m,1H),10.83–10.53(m,1H),8.10–7.91(m,1H),7.73–7.65(m,1H),7.60(dd,J＝7.9,5.8Hz,1H),7.54–7.41(m,1H),7.39–7.27(m,2H),7.21–7.02(m,2H),6.93–6.83(m,1H),5.18–5.01(m,1H),3.84–3.56(m,2H),3.14–3.00(m,4H),2.93–2.84(m,2H),2.70–2.55(m,4H),2.48–2.39(m,2H),2.02–1.92(m,6H),1.88–1.64(m,3H).
Example 24
In a similar manner and reaction procedure, compound 24 can be obtained by substituting 9-bromo-1-nonanol for 8-bromo-1-octanol in the fifth step of example 1, and (2, 6-dioxopiperidin-3-yl) -5-hydroxyiso-1, 3-dione for 2- (2, 6-dioxopiperidin-3-yl) -4-hydroxyiso-1, 3-dione in the fifth step of example 1.ESI-MS (m/z): 765.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(d,J＝6.1Hz,1H),12.09(s,1H),11.11(s,1H),10.79–10.53(m,1H),7.97(d,J＝13.0Hz,1H),7.82(d,J＝8.3Hz,1H),7.59(d,J＝5.7Hz,1H),7.53–7.39(m,2H),7.34(dd,J＝8.3,2.3Hz,1H),7.21–7.07(m,2H),6.92–6.82(m,1H),5.12(dd,J＝12.8,5.4Hz,1H),4.17(t,J＝6.5Hz,2H),3.09(t,J＝4.9Hz,4H),2.92–2.84(m,1H),2.66–2.53(m,3H),2.31(t,J＝7.4Hz,2H),2.11–1.96(m,2H),1.76(p,J＝6.8Hz,2H),1.48–1.26(m,14H).
Example 25
Compound 25 was obtained by a similar procedure and reaction steps, substituting 12b for 7c of the fifth step of example 19. ESI-MS (m/z): 705.9[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.75(s,1H),12.12(d,J＝8.7Hz,1H),11.07(s,1H),10.78–10.58(m,1H),8.06(s,1H),7.62–7.45(m,4H),7.19(d,J＝5.6Hz,1H),7.14(t,J＝8.3Hz,2H),7.06(d,J＝8.3Hz,1H),5.11–5.04(m,1H),3.86–3.40(m,5H),2.95–2.81(m,2H),2.70–2.54(m,3H),2.29–1.65(m,10H),1.27–1.20(m,2H).
Example 26
The first step: tert-butyl 2, 6-diazaspiro [3.4 ]]Octane-6-carboxylate (1.0 g,4.7 mmol) and 5-fluoro-2-nitroaniline (0.74 g,4.7 mmol) were dissolved in NMP (10 mL), DIPEA (1.64 mL,9.4 mmol) was added and reacted at 120℃overnight. The reaction solution was diluted with ethyl acetate, which was then washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and concentrated by filtration to give 26a (1.58 g, yield 96%) as a yellow solid. ESI-MS (m/z): 349.6[ M+H ]] + 。
And a second step of: 26a (1.6 g,4.6 mmol) was dissolved in absolute ethanol (20 mL), 10% palladium on carbon (50 mg,0.46 mmol) was added, the reaction was replaced with hydrogen balloon and stirred overnight at 40℃under hydrogen balloon pressure, and LCMS monitored substrate reaction was complete. Then, ethyl 3-ethoxy-3-iminopropionate hydrochloride (1.35 g,6.9 mmol) was added to the reaction solution, and the temperature was raised to 80℃and stirring was continued for 4 hours. The system was filtered through celite, washed with dichloromethane, and the filtrate was concentrated to give 26b as a yellow solid (1.47 g, 77% yield). ESI-MS (m)/z):415.5[M+H] + 。
And a third step of: 26b (1.47 g,3.55 mmol) and 2-aminothiophene-3-carbonitrile (0.44 g,3.55 mmol) were dissolved in tetrahydrofuran (10 mL), a solution of 2M LDA in THF (14 mL,28.4 mmol) was added under nitrogen, and the system was warmed to 40℃and stirred for 2 hours. The reaction was quenched with saturated ammonium chloride and extracted three times with dichloromethane, and the organic phase was washed successively with water and saturated sodium chloride solution. The organic phase was concentrated and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 26c as a brown solid (381 mg, yield 22%). ESI-MS (m/z): 493.7[ M+H ] ] + 。
Fourth step: 26c (300 mg,0.6 mmol) was dissolved in TFA (2 mL) and dichloromethane (4 mL) and stirred for 2 h under ice-bath conditions. The reaction system was concentrated to give 26d (300 mg, yield 97%). ESI-MS (m/z): 393.6[ M+H ]] + 。
Fifth step: 26d (70 mg,0.14 mmol) and 12b (49 mg,0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath, sodium acetate (37 mg,0.28 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hour, then sodium triacetoxyborohydride (88 mg,0.42 mmol) was added and the system was stirred at room temperature overnight. After the completion of the reaction, the reaction mixture was concentrated, and the residue was dissolved in DMF and purified by HPLC to give compound 26 (10 mg, yield 10%). ESI-MS (m/z): 732.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.64–12.40(m,1H),12.09(s,1H),11.07(s,1H),10.84–10.41(m,1H),8.21(s,2H),7.95(s,1H),7.63–7.51(m,2H),7.50–7.36(m,1H),7.18(d,J＝5.6Hz,1H),7.16–7.04(m,2H),6.72–6.58(m,1H),6.35(d,J＝8.4Hz,1H),5.12–5.01(m,1H),3.81–3.67(m,9H),2.95–2.75(m,4H),2.64–2.53(m,4H),2.18–1.91(m,6H).
Example 27
Compound 27 was obtained by a similar procedure and reaction procedure as in example 26, substituting 15b for 12b in the fifth step. ESI-MS (m/z): 718.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.63–12.30(m,1H),12.07(s,1H),11.07(s,1H),10.87–10.52(m,1H),8.19(s,2H),7.96(d,J＝13.0Hz,1H),7.62–7.52(m,2H),7.49–7.28(m,1H),7.18(d,J＝5.7Hz,1H),7.10(d,J＝6.9Hz,1H),6.79(d,J＝8.5Hz,1H),6.72–6.59(m,1H),6.36(d,J＝8.7Hz,1H),5.05(dd,J＝12.7,5.5Hz,1H),4.35–4.28(m,2H),3.90–3.67(m,6H),2.98–2.35(m,2H),2.78(s,2H),2.70(d,J＝7.5Hz,2H),2.63–2.50(m,4H),2.08–1.97(m,3H).
Example 28
With 3, 9-diazaspiro [5.5 ]]Undecane-3-carboxylic acid tert-butyl ester replacing tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, compound 28 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 774.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.58(d,J＝10.3Hz,1H),12.08(s,1H),11.06(s,1H),10.80–10.53(m,1H),7.97(s,1H),7.63–7.52(m,2H),7.50–7.40(m,1H),7.23–7.14(m,2H),7.13–7.10(m,2H),6.93–6.86(m,1H),5.07(dd,J＝12.8,5.5Hz,1H),3.63–3.54(m,2H),3.08(s,4H),2.92–2.84(m,1H),2.42–2.34(m,6H),2.06–1.97(m,4H),1.73–1.56(m,6H),1.53–1.43(m,6H).
Example 29
With 3, 9-diazaspiro [5.5 ]]Undecane-3-carboxylic acid tert-butyl ester replacing tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26 ]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can afford compound 29 using similar methods and reaction steps. ESI-MS (m/z): 760.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.07(s,1H),11.07(s,1H),10.62(s,1H),8.32(s,1H),7.60–7.53(m,1H),7.51–7.40(m,1H),7.26–7.04(m,4H),6.91–6.63(m,2H),5.05(dd,J＝12.8,5.5Hz,1H),4.29(s,2H),3.93–3.62(m,1H),3.29–2.99(m,4H),2.92–2.83(m,2H),2.39–2.36(m,4H),2.02–1.97(m,4H),1.67–1.45(m,10H).
Example 30
With 2, 8-diazaspiro [4.5 ]]Tert-butyl decane-2-carboxylate substitution of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, compound 30 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 760.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝8.2Hz,1H),12.08(s,1H),11.07(s,1H),10.83–10.54(m,1H),8.05–7.91(m,1H),7.61–7.53(m,2H),7.51–7.41(m,1H),7.26–7.09(m,3H),6.94–6.77(m,2H),5.34–5.02(m,1H),4.51–4.21(m,2H),3.88(s,2H),3.29–3.01(m,4H),2.94–2.53(m,8H),2.21–1.96(m,2H),187–1.46(m,6H),1.44–1.12(m,3H).
Example 31
With 2, 8-diazaspiro [4.5 ]]Tert-butyl decane-2-carboxylate substitution of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can give compound 31 in a similar manner and reaction procedure. ESI-MS (m/z): 746.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.58(d,J＝9.0Hz,1H),12.08(s,1H),11.06(s,1H),10.80–10.56(m,1H),8.16–7.91(m,1H),7.60–7.54(m,2H),7.50–7.41(m,1H),7.21–7.10(m,4H),6.91–6.86(m,1H),5.06(dd,J＝12.9,5.5Hz,1H),3.67–3.52(m,3H),3.15–2.82(m,6H),2.70–2.53(m,4H),2.12–1.97(m,3H),1.76–1.41(m,9H).
Example 32
With 2-tert-butoxycarbonyl-2, 7-diazaspiro [3.5 ]]Nonane alternative example 26 first step tert-butyl 2, 6-diazaspiro [3.4 ]]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can afford compound 32 in a similar manner and reaction procedure. ESI-MS (m/z): 731.7[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.13(s,1H),11.09(s,1H),10.89–10.52(m,1H),7.98(s,1H),7.60–7.53(m,2H),7.50–7.36(m,1H),7.23–7.08(m,3H),6.88(d,J＝8.5Hz,1H),6.77(d,J＝8.4Hz,1H),5.04(dd,J＝12.8,5.5Hz,1H),4.25(t,2H),3.84(t,3H),3.03(s,7H),2.93–2.80(m,2H),2.69(s,3H),2.60–2.55(m,1H),2.02–1.95(m,1H),1.82(t,J＝5.4Hz,4H).
Example 33
With 2-tert-butoxycarbonyl-2, 7-diazaspiro [3.5 ]]Nonane alternative example 26 first step tert-butyl 2, 6-diazaspiro [3.4 ] ]Octane-6-carboxylate, compound 33 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 746.6[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝8.8Hz,1H),12.07(s,1H),11.06(s,1H),10.62(s,1H),7.97(s,1H),7.61–7.53(m,2H),7.50–7.30(m,1H),7.22–7.08(m,4H),6.91–6.85(m,1H),5.06(ddd,J＝12.5,5.5,1.9Hz,1H),3.66–3.47(m,3H),3.35(s,4H),3.05–2.99(m,7H),2.90–2.84(m,1H),2.61–2.55(m,1H),2.48–2.47(m,1H),2.27–2.21(m,1H),2.06–1.98(m,2H),1.85–1.76(m,4H),1.68–1.61(m,1H).
Example 34
With 2, 7-diazaspiro [3.5 ]]T-butyl nonane-7-carboxylate replaces t-butyl 2, 6-diazaspiro [3.4 ] as in example 26, first step]Octane-6-carboxylate, compound 34 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 746.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60–12.42(m,1H),12.10(s,1H),11.06(s,1H),10.72–10.53(m,1H),7.95(s,1H),7.67–7.52(m,2H),7.40(dd,J＝8.5Hz,1H),7.22–7.10(m,3H),6.80–6.45(m,1H),6.35(d,J＝8.1Hz,1H),5.07(dd,J＝12.8,5.5Hz,1H),3.64–3.59(m,3H),3.56–3.51(m,6H),3.02–2.80(m,2H),2.61–2.54(m,2H),2.42–2.28(m,5H),2.09–1.98(m,2H),1.81–1.63(m,5H).
Example 35
With 2, 6-diazaspiro [3.4 ]]octane-2-Carbonic acid tert-butyl ester instead of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can afford compound 35 using similar methods and reaction steps. ESI-MS (m/z): 718.2[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.53–12.38(m,1H),12.07(s,1H),11.07(s,1H),10.80–10.59(m,1H),8.20(s,1H),8.00–7.88(m,1H),7.59–7.53(m,2H),7.48–7.38(m,1H),7.17(d,J＝5.6Hz,1H),7.10(d,J＝6.9Hz,1H),6.80–6.75(m,2H),6.54–6.47(m,1H),5.04(dd,J＝12.7,5.5Hz,1H),4.25(s,2H),3.86(s,2H),3.23–3.18(m,7H),2.92–2.82(m,2H),2.74–2.54(m,5H),2.15(t,J＝6.8Hz,2H),2.06–1.95(m,2H).
Example 36
By 2, 6-diaza-spiro [3.4 ]]octane-2-Carbonic acid tert-butyl ester instead of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, compound 36 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 732.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.52(s,1H),12.08(s,1H),11.06(s,1H),10.83–10.50(m,1H),8.02–7.78(m,1H),7.59–7.53(m,2H),7.47–7.38(m,1H),7.17–7.10(m,3H),6.78–6.67(m,1H),6.50(d,J＝8.4Hz,1H),5.06(dd,J＝12.8,5.3Hz,1H),3.64–3.48(m,4H),3.23–3.16(m,6H),2.91–2.84(m,1H),2.65–2.55(m,2H),2.32–2.21(m,2H),2.15(t,J＝6.3Hz,2H),2.07–1.97(m,4H),1.75–1.58(m,2H).
Example 37
The first step: 2- (2, 6-Dioxopiperidin-3-yl) -5-fluoro-iso-1, 3-dione (100 mg,0.36 mmol) and azetidin-3-yl-methanol (31 mg,0.36 mmol) were dissolved in DMF (5 mL), DIPEA (93 mg,0.72 mmol) was added to the reaction system, after which the reaction system was heated to 80℃and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate, and extracted 3 times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol) to give the final product 37a (120 mg, yield 97%). ESI-MS (m/z): 343.8[ M+H ] ] + 。
And a second step of: 37a (120 mg,0.34 mmol) was dissolved in methylene chloride (4 mL), and dess-martin oxidant (158 mg,0.37 mmol) was added to the reaction system, followed by stirring at room temperature for 1 hour. The reaction system was filtered, washed with methylene chloride, and the organic phase was washed with a saturated sodium hydrogencarbonate solution and a saturated brine in this order, then dried over anhydrous sodium sulfate, filtered, and concentrated to give the final product 37b (98 mg, yield 84%) which was directly used for the next reaction. ESI-MS (m/z): 341.2[ M+H ]] + 。
And a third step of: 26d (80 mg,0.16 mmol) and 37b (54 mg,0.16 mmol) were dissolved in 1, 2-dichloroethane (4 mL) and methanol (2 mL) under ice-bath, sodium acetate (43 mg,0.32 mmol) was added to the reaction system, after which the reaction system was stirred for 0.5 hour, then sodium triacetoxyborohydride (100 mg,0.47 mmol) was added and the system was stirred at room temperature overnight. The reaction solution was concentrated, and the residue was dissolved in methanol, and purified by HPLC to give Compound 37 (9 mg, yield 8%). ESI-MS (m/z): 718.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.62–12.41(m,1H),12.09(s,1H),11.07(s,1H),10.79–10.53(m,1H),8.30(s,2H),7.96(s,1H),7.63(d,J＝8.2Hz,1H),7.58(dd,J＝5.9,2.2Hz,1H),7.49–7.37(m,1H),7.18(d,J＝5.6Hz,1H),6.78(d,J＝2.1Hz,1H),6.71–6.61(m,2H),6.40–6.32(m,1H),5.05(dd,J＝12.7,5.5Hz,1H),4.15(t,J＝8.2Hz,2H),3.78–3.69(m,6H),2.98–2.85(m,3H),2.78–2.58(m,6H),2.06–1.98(m,4H).
Example 38
With 2, 6-diazaspiro [3.4 ]]octane-2-Carbonic acid tert-butyl ester instead of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 37b for 12b of the fifth step of example 26, can afford compound 38 in a similar manner and reaction procedure. ESI-MS (m/z): 718.4[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.54–12.42(m,1H),12.06(s,1H),11.07(s,1H),10.81–10.55(m,1H),8.31(s,1H),8.01–7.84(m,1H),7.63(d,J＝8.3Hz,1H),7.61–7.56(m,1H),7.49–7.37(m,1H),7.17(d,J＝5.7Hz,1H),6.79–6.68(m,2H),6.63(dd,J＝8.4,2.1Hz,1H),6.55–6.47(m,1H),5.05(dd,J＝12.8,5.5Hz,1H),4.09(t,J＝8.0Hz,2H),3.70(dd,J＝8.4,5.2Hz,2H),3.27–3.18(m,7H),2.91–2.84(m,1H),2.77–2.54(m,5H),2.18–2.12(m,2H),2.06–1.94(m,2H).
Example 39
With 2, 8-diazaspiro [4.5 ]]Tert-butyl decane-2-carboxylate substitution of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 37b for 12b of the fifth step of example 26, can afford compound 39 in a similar manner and reaction procedure. ESI-MS (m/z): 746.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.59(d,J＝9.0Hz,1H),12.07(s,1H),11.07(s,1H),10.80–10.54(m,1H),8.44–7.84(m,1H),7.74–7.60(m,2H),7.58–7.35(m,1H),7.22–7.10(m,2H),6.89(dt,J＝9.0,3.0Hz,1H),6.78(d,J＝2.2Hz,1H),6.64(dd,J＝8.3,2.1Hz,1H),5.35–5.00(m,1H),4.13(t,J＝8.1Hz,2H),3.70(dd,J＝8.4,5.4Hz,2H),3.17–2.83(m,8H),2.71–2.62(m,2H),2.61–2.54(m,3H),2.43–2.39(m,2H),1.72–1.59(m,6H).
Example 40
With 2, 7-diazaspiro [3.5 ]]T-butyl nonane-7-carboxylate replaces t-butyl 2, 6-diazaspiro [3.4 ] as in example 26, first step]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can be obtained as compound 40 by a similar method and reaction procedure. ESI-MS (m/z): 732.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.62–12.38(m,1H),12.09(s,1H),11.07(s,1H),10.52–10.39(m,1H),7.95(s,1H),7.60–7.52(m,2H),7.50–7.38(m,1H),7.34–7.02(m,2H),6.83–6.55(m,2H),6.35(dd,J＝8.3,2.1Hz,1H),5.05(dd,J＝12.7,5.5Hz,1H),4.30(s,2H),3.83(d,J＝8.5Hz,2H),3.54(s,4H),3.48(s,1H),2.93–2.83(m,2H),2.58(dd,J＝14.0,5.1Hz,4H),2.41–2.26(m,3H),2.05–1.92(m,1H),1.81–1.67(m,4H).
Example 41
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With 2, 7-diazaspiro [3.5 ]]T-butyl nonane-7-carboxylate replaces t-butyl 2, 6-diazaspiro [3.4 ] as in example 26, first step]Octane-6-carboxylate, while substituting 37b for 12b of the fifth step of example 26, can afford compound 41 in a similar manner and reaction procedure. ESI-MS (m/z): 732.6[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.52(d,J＝26.0Hz,1H),12.10(s,1H),11.07(s,1H),10.79–10.56(m,1H),7.96(s,1H),7.85–7.52(m,2H),7.56–7.33(m,1H),7.17(d,J＝5.7Hz,1H),6.83–6.55(m,3H),6.39–6.29(m,1H),5.05(dd,J＝12.8,5.5Hz,1H),4.13(t,J＝8.1Hz,2H),3.69(dd,J＝8.3,5.4Hz,3H),3.54(s,4H),3.06–2.80(m,3H),2.62–2.55(m,3H),2.42–2.32(m,3H),2.06–1.95(m,1H),1.76(t,J＝5.3Hz,4H).
Example 43
With 2, 6-diazaspiro [3.3 ]]Heptane-2-carboxylic acid tert-butyl ester replaces the tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, while substituting 15b for 12b of the fifth step of example 26, can afford compound 43 in a similar manner and reaction procedure. ESI-MS (m/z): 704.7[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.64–12.43(m,1H),12.07(s,1H),11.07(s,1H),10.70–10.48(m,1H),7.95(s,1H),7.61–7.50(m,2H),7.47–7.34(m,1H),7.21–7.05(m,2H),6.79–6.58(m,2H),6.36–6.31(m,1H),5.04(dd,J＝12.7,5.5Hz,1H),4.25(s,2H),3.85(d,J＝4.4Hz,6H),3.51–3.41(m,4H),2.90–2.83(m,1H),2.69–2.52(m,5H),2.04–1.92(m,1H).
Example 44
With 2, 6-diazaspiro [3.3 ]]Heptane-2-carboxylic acid tert-butyl ester replaces the tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, while substituting 37b for 12b of the fifth step of example 26, can afford compound 44 in a similar manner and reaction procedure. ESI-MS (m/z): 704.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.64–12.42(m,1H),12.08(s,1H),11.07(s,1H),10.72–10.38(m,1H),7.96(s,1H),7.66–7.55(m,2H),7.50–7.30(m,1H),7.17(dd,J＝5.8,1.7Hz,1H),6.79–6.59(m,3H),6.39–6.29(m,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.08(t,J＝8.0Hz,2H),3.85(d,J＝3.5Hz,4H),3.72–3.61(m,3H),3.56–3.39(m,3H),2.92–2.85(m,1H),2.76–2.53(m,5H),2.05–1.94(m,1H).
Example 45
With 2, 8-diaza-spiro [4.5 ]]Tert-butyl decane-2-carboxylate substitution of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 14b for 12b of the fifth step of example 26, can afford compound 45 using similar methods and reaction steps. ESI-MS (m/z): 760.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.58(d,J＝9.5Hz,1H),12.08(s,1H),11.07(s,1H),10.84–10.52(m,1H),8.30–7.94(m,1H),7.68–7.56(m,2H),7.51–7.40(m,1H),7.21–7.10(m,2H),6.89(dd,J＝9.1,2.4Hz,2H),6.81(dd,J＝8.6,2.2Hz,1H),5.05(dd,J＝12.9,5.4Hz,1H),3.61–3.38(m,4H),3.19–2.99(m,6H),2.92–2.84(m,1H),2.65–2.54(m,4H),2.47–2.39(m,4H),2.17–2.12(m,1H),2.04–1.96(m,2H),1.82–1.65(m,5H).
Example 46
With 2, 6-diazaspiro [3.3 ]]Heptane-2-carboxylic acid tert-butyl ester replaces the tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, while substituting 14b for 12b of the fifth step of example 26, can afford compound 46 in a similar manner and reaction procedure. ESI-MS (m/z): 718.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.52(s,1H),12.08(s,1H),11.07(s,1H),10.74–10.56(m,1H),7.96(d,J＝15.9Hz,1H),7.63(d,J＝8.4Hz,1H),7.60–7.57(m,1H),7.52–7.29(m,1H),7.21–7.14(m,1H),6.91–6.77(m,2H),6.75–6.50(m,1H),6.37–6.29(m,1H),5.05(dd,J＝12.9,5.3Hz,1H),3.86(d,J＝4.5Hz,4H),3.56–3.44(m,4H),3.41–3.37(m,4H),3.13–3.07(m,1H),2.93–2.81(m,1H),2.63–2.54(m,2H),2.46–2.44(m,1H),2.38–2.29(m,1H),2.16–2.07(m,1H),2.04–1.95(m,1H),1.77–1.65(m,1H).
Example 47
The first step: 2- (2, 6-dioxo-piperidin-3-yl) -5-methyl-isoindole-1, 3-dione (300 mg,1.10 mmol) and N-bromosuccinimide (294.18 mg,1.65 mmol) were dissolved in acetonitrile (20 mL) and azobisisobutyronitrile (18.09 mg,0.11 mmol) was added. The reaction system was purged with nitrogen three times with a nitrogen ball and reacted at 75℃for 8 hours under the protection of the nitrogen ball. The reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed with water and then with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give compound 47a (300 mg, yield 78%) as ESI-MS (m/z): 351.2[ M+H) ] + 。
And a second step of: 1d (200 mg,0.55 mmol) and 1-Boc-3-azetidinone (93.44 mg,0.55 mmol) were dissolved in a mixed solvent of MeOH (5 mL) and DCE (5 mL), sodium acetate (74.27 mg,0.55 mmol) was added, and stirred at room temperature for 30min. Then, sodium cyanoborohydride (68.60 mg,1.09 mmol) was added to the reaction solution, and stirring was continued at room temperature for 6 hours. The reaction solution was diluted with methylene chloride, washed with saturated sodium hydrogencarbonate and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give compound 47b (52 mg, yield 18%) as ESI-MS (m/z): 522.8[ M+H)] +
And a third step of: 47b (52 mg,0.1 mmol) was dissolved in dichloromethane (4 mL), TFA (1 mL) was added, and the mixture was stirred at room temperature for 30min. The reaction system was concentrated to give compound 47c (40 mg, yield 95%). ESI-MS (m/z): 422.6[ M+H ]] +
Fourth step: 47c (40 mg,0.1 mmol) and 47a (83.31 mg,0.14 mmol) were dissolved in DMF (1 mL), DIPEA (36.79 mg,0.28 mmol) was added and the temperature was raised to 90℃for 16h. The reaction mixture was purified by HPLC to give Compound 47 (12 mg, yield 18%) ESI-MS (m/z): 692.7[ M+H)] + ； 1 H NMR(500MHz,DMSO-d 6 ))δ12.61(s,1H),12.11(s,1H),11.13(s,1H),10.70–10.59(m,1H),7.99(s,1H),7.88(d,J＝7.6Hz,1H),7.84–7.76(m,2H),7.59(d,J＝5.7Hz,1H),7.52–7.30(m,1H),7.21–7.09(m,2H),6.91–6.85(m,1H),5.15(dd,J＝12.8,5.4Hz,1H),3.79(s,2H),3.47–3.43(m,3H),3.15–3.06(m,5H),2.91–2.85(m,1H),2.74–2.52(m,3H),2.46–2.40(m,4H),2.17–1.92(m,1H).
Example 48
With 2, 6-diazaspiro [3.3 ]]Heptane-2-carboxylic acid tert-butyl ester replaces the tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26 ]Octane-6-carboxylate, compound 48 can be obtained by a similar method and reaction procedure. ESI-MS (m/z): 718.6[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60–12.45(m,1H),12.08(s,1H),11.06(s,1H),10.80–10.52(m,1H),7.96(d,J＝7.1Hz,1H),7.61–7.52(m,2H),7.49–7.38(m,1H),7.21–7.05(m,3H),6.74–6.42(m,1H),6.34(d,J＝8.3Hz,1H),5.27–4.81(m,1H),3.85(d,J＝3.8Hz,4H),3.64–3.46(m,5H),3.45–3.36(m,4H),2.93–2.85(m,1H),2.65–2.55(m,1H),2.48–2.43(m,2H),2.28–2.19(m,1H),2.07–1.95(m,2H),1.69–1.58(m,1H).
Example 49
Replacement of the 1-Boc-3-azetidinone of the second step of example 47 with tert-butyl 3-formylazetidine-1-carboxylate resulted in compound 49 in a similar manner and reaction procedure. ESI-MS (m/z): 706.8[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(d,J＝6.5Hz,1H),12.09(s,1H),11.13(s,1H),10.77–10.54(m,1H),8.19(s,2H),7.98(s,1H),7.87(d,J＝7.6Hz,1H),7.82–7.75(m,2H),7.58(d,J＝5.7Hz,1H),7.52–7.40(m,1H),7.20–7.06(m,2H),6.93–6.83(m,1H),5.15(dd,J＝12.8,5.4Hz,1H),3.75(s,2H),3.43–3.40(m,6H),3.07(t,J＝4.9Hz,4H),2.94–2.86(m,3H),2.71–2.61(m,2H),2.59–2.54(m,3H),2.10–2.03(m,1H).
Example 50
Compound 50 can be obtained by a similar procedure and reaction steps substituting 1-Boc-3-azetidinone for 1-tert-butoxycarbonylpiperidine-4-carbaldehyde in example 47 in the second step. ESI-MS (m/z): 734.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.68–12.53(m,1H),12.10(s,1H),11.13(s,1H),10.67(d,J＝46.1Hz,1H),8.20(s,2H),7.98(s,1H),7.90–7.86(m,1H),7.85–7.78(m,2H),7.58(d,J＝5.7Hz,1H),7.52–7.41(m,1H),7.21–7.07(m,2H),6.93–6.85(m,1H),5.15(dd,J＝12.8,5.4Hz,1H),3.66(s,2H),3.10(t,J＝4.6Hz,4H),2.96–2.77(m,5H),2.65–2.56(m,2H),2.20(d,J＝7.1Hz,2H),2.10–1.94(m,4H),1.71(d,J＝12.6Hz,2H),1.30–1.11(m,4H).
Example 51
By 2, 6-diaza-spiro [3.4 ]]octane-2-Carbonic acid tert-butyl ester instead of tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while substituting 18b for 12b of the fifth step of example 26, can give compound 51 by a similar method and reaction procedure. ESI-MS (m/z): 746.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.47–12.34(m,1H),11.99(s,1H),11.00(s,1H),10.77–10.47(m,1H),8.12–7.75(m,1H),7.58(d,J＝8.6Hz,1H),7.54–7.48(m,1H),7.43–7.30(m,1H),7.23(d,J＝2.4Hz,1H),7.15(dd,J＝8.8,2.4Hz,1H),7.11(dd,J＝5.7,1.6Hz,1H),6.73–6.60(m,1H),6.51–6.38(m,1H),4.99(dd,J＝12.8,5.5Hz,1H),3.95(d,J＝13.0Hz,2H),3.24–3.16(m,4H),3.15–3.05(m,4H),2.90–2.77(m,3H),2.56–2.46(m,2H),2.25(d,J＝6.8Hz,2H),2.08(t,J＝6.8Hz,2H),1.99–1.91(m,1H),1.71(d,J＝12.8Hz,2H),1.59–1.46(m,1H),1.15–1.06(m,2H).
Example 52
Substitution with tert-butyl 1, 4-diazacycloheptane-1-carboxylateTertiary butyl 2, 6-diazaspiro [3.4 ] as first step in example 37]Octane-6-carboxylate, while substituting 18b for 12b of the fifth step of example 26, can afford compound 52 using similar methods and reaction steps. ESI-MS (m/z): 734.5[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.49(s,1H),12.07(s,1H),11.07(s,1H),10.63(s,1H),7.91(s,1H),7.64–7.56(m,2H),7.45–7.36(m,1H),7.31–7.14(m,3H),7.05–6.76(m,1H),6.67(d,J＝8.7Hz,1H),5.06(dd,J＝12.7,5.4Hz,1H),4.00(d,J＝12.3Hz,2H),3.57–3.49(m,4H),2.97–2.73(m,6H),2.61–2.52(m,4H),2.31–2.29(m,1H),1.99(s,1H),1.93–1.88(m,2H),1.80–1.70(m,3H),1.16–1.05(m,2H).
Example 53
With 3, 9-diazaspiro [5.5 ]]Undecane-3-carboxylic acid tert-butyl ester replacing tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, while substituting 18b for 12b of the fifth step of example 26, can afford compound 53 using similar methods and reaction steps. ESI-MS (m/z): 788.7[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.94(s,1H),11.07(s,1H),10.51(s,1H),7.65(d,J＝8.7Hz,1H),7.53(d,J＝5.7Hz,1H),7.48–7.38(m,1H),7.30(d,J＝2.3Hz,1H),7.27–7.06(m,3H),6.87(d,J＝8.7Hz,1H),5.06(dd,J＝12.8,5.4Hz,1H),4.03(d,J＝12.8Hz,2H),3.13–3.05(m,4H),3.03–2.82(m,4H),2.65–2.53(m,3H),2.39–2.31(m,4H),2.19–2.12(m,2H),2.04–1.99(m,1H),1.83–1.74(m,3H),1.63–1.47(m,8H).
Example 54
With 2-Boc-octahydropyrrolo [3,4-c ]]Pyrrole replacement tert-butyl 2, 6-diazaspiro [3.4 ] as first step in example 26]Octane-6-carboxylate, while 18b was used instead of 12b in the fifth step of example 26, can be obtained by similar method and reaction procedureAnd compound 54.ESI-MS (m/z): 746.9[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.60(s,1H),12.09(s,1H),11.06(s,1H),10.78–10.58(m,1H),7.94(s,1H),7.78–7.52(m,2H),7.50–7.35(m,1H),7.31–7.13(m,3H),6.94–6.82(m,1H),6.70–6.59(m,1H),5.05(dd,J＝12.7,5.4Hz,1H),4.02(d,J＝13.1Hz,2H),3.47–3.41(m,2H),3.03–2.84(m,7H),2.62–2.54(m,4H),2.48–2.43(m,2H),2.29–2.18(m,2H),2.05–1.94(m,1H),1.84–1.71(m,3H),1.18–1.09(m,2H).
Example 55
With 2, 6-diazaspiro [3.3 ]]Heptane-2-carboxylic acid tert-butyl ester replaces the tert-butyl 2, 6-diazaspiro [3.4 ] as the first step in example 26]Octane-6-carboxylate, while substituting 18b for 12b of the fifth step of example 26, can afford compound 55 using similar methods and reaction steps. ESI-MS (m/z): 732.2[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ12.52(s,1H),12.10(s,1H),11.07(s,1H),10.76–10.57(m,1H),7.96(s,1H),7.67–7.55(m,2H),7.48–7.36(m,1H),7.33–6.77(m,3H),6.74–6.52(m,1H),6.35(d,J＝8.6Hz,1H),5.06(dt,J＝13.0,4.4Hz,1H),4.02(d,J＝12.9Hz,2H),3.85(s,4H),3.61–3.56(m,4H),3.05–2.81(m,5H),2.64–2.54(m,3H),2.47–2.43(m,1H),2.36–2.26(m,1H),2.04–1.98(m,1H),1.79–1.65(m,1H),1.46–1.43(m,1H).
Example 56
The first step: 2- (2, 6-dioxo-piperidin-3-yl) -5-fluoro-isoindole-1, 3-dione (1.0 g,3.62 mmol) and 1- (tert-butoxycarbonyl) piperazine (846.60 mg,3.80 mmol) were dissolved in DMF (5 mL), DIPEA (1.40 g,10.86 mmol) was added and the temperature was raised to 90℃for 16h. The reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. The filtrate is concentrated, and the mixture is filtered, The residue was purified by silica gel column chromatography (dichloromethane/methanol) to give compound 56a (1.6 g, yield 99%). ESI-MS (m/z): 443.5[ M+H ]] + 。
And a second step of: 56a (1.6 g,3.62 mmol) was dissolved in dioxane (10 mL), 4M HCl dioxane solution (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction system was concentrated to give 56b (1.2 g, yield 96%). ESI-MS (m/z): 343.6[ M+H ]] + 。
And a third step of: 1d (500 mg,1.32 mmol) was dissolved in DMF (10 mL) and DIPEA (852.94 mg,6.60 mmol) was added. Bromoacetaldehyde diethyl acetal (390 mg,1.98 mmol) was added after stirring and the reaction was allowed to proceed at 80℃for 16h. The reaction solution was cooled to room temperature, diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol) to give 56c (128 mg, yield 21.2%). ESI-MS (m/z): 497.2[ M+H ]] + 。
Fourth step: to 56c (128 mg,0.28 mmol) was added a solution of 4M HCl dioxane (2 mL) and reacted at 50℃for 16h. The reaction solution was concentrated to give 56d (100 mg, yield 93%). ESI-MS (m/z): 423.8[ M+H ]] + 。
Fifth step: 56d (50 mg,0.13 mmol) and 56b (59.58 mg,0.13 mmol) were dissolved in 1, 2-dichloroethane (2 mL) and methanol (2 mL) under ice-bath, sodium acetate (17.7 mg,0.13 mmol) was added to the reaction system, after which the reaction system was stirred for 30min, then sodium cyanoborohydride (16.35 mg,0.26 mmol) was added, and the system was stirred at room temperature for another 6h. The reaction solution was diluted with methylene chloride, washed with saturated sodium hydrogencarbonate and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was dissolved in DMF and purified by HPLC to give compound 56 (5 mg, yield 5.2%). ESI-MS (m/z): 735.8[ M+H ] ] + ； 1 HNMR(500MHz,DMSO-d 6 )δ12.79(s,1H),12.07(s,1H),11.08(s,1H),10.57(s,1H),7.88(s,1H),7.68(d,J＝8.5Hz,1H),7.56(d,J＝5.7Hz,1H),7.51–7.39(m,1H),7.35(d,J＝2.3Hz,1H),7.26(dd,J＝8.7,2.4Hz,1H),7.21–7.06(m,2H),6.91–6.85(m,1H),5.07(dd,J＝12.8,5.4Hz,1H),3.45(t,J＝5.0Hz,4H),3.13–3.08(m,4H),2.92–2.79(m,1H),2.63–2.56(m,10H),2.55–2.53(m,4H),2.04–2.00(m,1H).
Test example 1: detection of HPK1 protein degradation Capacity by Compounds
The reagents required are shown in the following table:
experimental procedure
The specific operation is as follows: jurkat E6-1 (hereinafter referred to as Jurkat) cells were grown in cell culture medium [ RPMI 1640 (BI, 01-100-1 ACS), 10% heat-inactivated FBS (BI, 04-002-1A), 1% Penicillin-Streptomycin (BI, 03-031-1B) in Corning 24 well plates (catalog No. 3524)]At a volume of 1 mL/well at 2,500,000 cells/well. Jurkat cells were treated with compounds diluted in 0.2% dmso, diluted according to the following protocol: (1) Transfer 3. Mu.L of the compound stock solution (concentration: 10 mM) to 1497. Mu.L of the medium to prepare a 20. Mu.M compound solution; (2) Starting at 20 μm, 10-fold dilutions were made with 0.2% dmso for a total of 6 doses; (3) 1mL of each concentration of compound solution was added to the cells (final concentration of compound 0,1, 10, 100, 1000, 10000 nM) and incubated for 20h. Collecting cells in 15mL centrifuge tube, centrifuging 300g for five minutes, discarding culture medium, adding 3mL PBS pre-cooled at 4deg.C, washing, discarding washing solution, adding appropriate amount of RIPA lysate (containing Protease and Phosphatase Inhibitor Cocktail) according to cell amount, mixing, standing on ice for 30min, shaking and mixing once every 10min, centrifuging at 4deg.C for 30min at 20,000rpm, transferring the supernatant into new centrifuge tube, and adding Pierce TM Rapid Gold BCA Protein Assay Kit (A53225) protein concentration was quantified. An appropriate amount of SDS-PAGE loading buffer was added to the protein sample and heated in a boiling water bath for 5 minutes to substantially denature the protein and stored at-20℃until use. Electrophoresis was performed using a pre-cast gel (Genscript, M00655 or M00657) at 120V voltage, and film transfer was performed at 4deg.C for 100min at 300mA voltageAfter completion, the protein film was immediately placed in 5% skim milk diluted with TBST, slowly shaken on a shaker, and blocked at room temperature for 1h. Referring to the instructions of HPK1 Rabbit mAb (CST, 53453S), GAPDH (D16H 11) Rabbit Monoclonal antibody (CST, 5174S), the primary antibody was diluted in the recommended ratio, incubated overnight with slow shaking at 4℃and washed 3 times with TBST for 10 min/time; selection of the Goat Anti-Rabbit IgG (H)&L) -HRP Conjugated (Thermofiser, G-21234) with reference to the second antibody instructions, the second antibody was diluted according to the recommended ratio, incubated for 1h with slow shaking at room temperature, washed 3 times with TBST, 10 min/time; ECL luminescence was added, the results were observed by a developing instrument, western results were quantified using Image J software, and HPK1 degradation rate (degradation rate=1-dosing/control) and half-maximal degradation concentration DC were calculated by comparison with the control group without compound 50 . The experimental results are shown in the following table:
from the results of the table above, it can be seen that the compounds of the present invention are capable of significantly degrading the HPK1 protein in Jurkat cells.
Test example 2: detection of the ability of Compounds to secrete the cytokine Interleukin-2 (IL-2) by human PBMC cells and the Effect of Compounds on the viability of human PBMC cells
The required reagents are as follows
Experimental cell source information:
manufacturer(s) Batch of Product specification
Shanghai Xuanfeng Biotechnology Co.,Ltd. SC12132W/XCWBC6037W 10/25million/vial
Experimental procedure
The specific operation is as follows: human PBMC were removed from liquid nitrogen according to standard procedures, thawed and resuscitated in a 37℃water bath, and cells were resuspended in RPMI 1640 medium (10% FBS in each case), washed twice by centrifugation; human PBMC cells were then resuspended in RPMI 1640 medium for use. The compound powder was dissolved to 10mM with DMSO, 2. Mu.L of the compound was added to 998. Mu.L of RPMI 1640 medium, and the mixture was vortexed to the highest concentration point. The compound solution was gradually diluted 5-fold with 0.2% dmso medium for a total of 8 concentration points. As a control, RPMI 1640 medium solution containing dmso at a concentration of 0.1% was used. mu.L of the medium containing 0.5X10 s per well was added to Corning 96-well cell culture plate (cat# 3599) 5 Culture medium of human PBMC cells was then added with an equal volume of compound dilution, control was added with RPMI 1640 medium containing 0.2% DMSO, and incubated in a 37℃cell incubator (Thermo Fisher Scientific, model: 3111) for 1h. Then adding Anti-human CD3 Antibody with the final concentration of 0.37-1.1 mug/ml (adjusted according to different batches of human PBMC) and 1 mug/ml Anti-human CD28Antibody, and placing the mixture in a cell culture incubator at 37 ℃ for incubation for 20 hours. IL-2 content in cell supernatants was detected using a Human IL-2DuoSet ELISA KIT, which was performed according to the instructions of the KIT. Data highest fold ratio of stimulation signal of compound to signal of 0.1% dmso and half maximal effect concentration EC 50 Description. Cell collection using CellTiter-Luminescent Cell Viability Assay kit for detecting cell viability, and cell viability data adopts half inhibition concentration IC of compound 50 Description.
The results of the test for the ability of compounds to secrete the cytokine interleukin-2 (IL-2) towards human PBMC cells and the effect of compounds on human PBMC cell viability are shown in the following table:
from the results of the table above, it can be seen that the compounds of the present invention are capable of significantly stimulating human PBMC cells to secrete the cytokine interleukin-2 (IL-2) with less impact on human PBMC cell viability.
Test example 3: mouse pharmacokinetic testing of Compounds
Test animals: ICR mice (Male, 25-35g,6-9 weeks old, style)
Test procedure: ICR mice were randomly grouped by body weight and Compound 7 of the present invention was administered by gavage at a dose of 10mg/kg in a vehicle of 10% DMSO+10% PEG-400+10% Solutol+70% H 2 O. Fasted for 1h before gastric lavage administration, and fed after 1h blood sampling, and free drinking water. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 24h post-dose. All samples were subjected to quantitative analysis by LC-MS/MS at-80 ℃.
The pharmacokinetic parameters of compound 7 in mice are shown in the following table:
As can be seen from the table, the compound of the invention has high exposure in mice, high maximum blood concentration and good oral absorption.

Claims (6)

1. A compound or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, wherein said compound is selected from the group consisting of any one of the compounds shown in the following table:
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2. a pharmaceutical composition comprising a compound according to claim 1, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
3. Use of a compound according to claim 1, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, or a pharmaceutical composition according to claim 2, for the manufacture of a medicament for the prophylaxis and/or treatment of a disease associated with HPK1 activity.
4. A method of preventing and/or treating a disease associated with HPK1 activity comprising administering to a patient in need thereof a therapeutically effective amount of a compound of claim 1, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide, or prodrug thereof, or a pharmaceutical composition of claim 2.
5. The use according to claim 3 or the method according to claim 4, wherein the disease associated with HPK1 activity comprises cancer or an immune disease; the cancer is preferably lung cancer, thyroid cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, oral cancer, thyroid cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, prostate cancer, cervical cancer, renal cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, cell tumor, glioma, hematological tumor, and lymphoma; the immune diseases are preferably lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis.
6. The use according to claim 3 or the method according to claim 4, wherein the compound according to claim 1, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide or prodrug thereof, or the pharmaceutical composition according to claim 2, is used alone or in combination with other classes of pharmaceutical preparations and/or therapeutic methods.
CN202311524559.XA 2022-11-17 2023-11-15 Efficient HPK1 degradation agent compound and preparation method and application thereof Pending CN117624187A (en)

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