WO2024094190A1 - Irak4降解剂及其用途 - Google Patents

Irak4降解剂及其用途 Download PDF

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WO2024094190A1
WO2024094190A1 PCT/CN2023/129712 CN2023129712W WO2024094190A1 WO 2024094190 A1 WO2024094190 A1 WO 2024094190A1 CN 2023129712 W CN2023129712 W CN 2023129712W WO 2024094190 A1 WO2024094190 A1 WO 2024094190A1
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methyl
mmol
alkyl
difluoromethyl
pyrazol
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PCT/CN2023/129712
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English (en)
French (fr)
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冯焱
叶正清
李世强
丁陈利
陈海继
徐娟
王俏
韦红
谢舒晨
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上海领泰生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/20—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D239/22—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems

Definitions

  • the present invention relates to compounds that regulate one or more interleukin-1 receptor associated kinases 4 (IRAK4) by ubiquitination and/or degradation, and their use as drugs for immune and/or inflammatory diseases.
  • IRAK4 interleukin-1 receptor associated kinases 4
  • Interleukin-1 receptor kinase 4 is a serine/threonine-specific protein kinase with biologically important kinase activity, and plays an important role in activating the immune system. Studies have shown that IRAK4 is a key factor downstream of the interleukin (IL)-1 ⁇ family receptors (including IL-1R, IL-18R, IL-33R, IL-36R) and Toll-like receptor (TLR) signaling pathways.
  • IL-1 receptor interleukin-1 receptor kinase 4
  • TLR Toll-like receptor
  • IRAK4-deficient mice and IRAK4-deficient patients do not respond to stimulation of TLRs (except TLR3) and the IL-1 ⁇ family (Suzuki, Suzuki et al., Nature, 2002; Davidson, Currie et al., The Journal of Immunology, 2006; Kuvon Bernuth et al., JEM, 2007; Kim, Staschke et al., JEM, 2007).
  • TLR/IL-1 ⁇ -mediated signaling pathways can be divided into MyD88-dependent signaling pathways and MyD88-independent pathways.
  • the signal transduction pathways mediated by IL-1R and TLR2, TLR4, TLR7/8, and TLR9 all rely on MyD88 as a regulatory factor to activate downstream inflammatory signaling pathways. After TLR/IL-1 ⁇ binds to the ligand, MyD88 molecules are recruited.
  • MyD88 further recruits IRAK4 to the TLR/IL-1 ⁇ complex through its N-terminal death domain, and interacts with and activates IRAK1 or IRAK2 (Kollewe, Mackensen et al., Journal of Biological Chemistry, 2004; Precious et al., J. Biol. Chem., 2009), thereby transmitting signals downstream to the E3 ubiquitin ligase TNF receptor-associated factor (TRAF6), activating the serine/threonine kinase TAK1, and then activating the NF- ⁇ B and MAPK signaling pathways (Wang, Deng et al., Nature, 2001), causing the release of a variety of inflammatory cytokines and anti-apoptotic molecules.
  • TRAF6 E3 ubiquitin ligase TNF receptor-associated factor
  • IRAK4-dependent TLR/IL-1 ⁇ signaling has been shown to be associated with a variety of diseases, such as multiple sclerosis, atherosclerosis, myocardial infarction, myocarditis (Valaperti, Nishii et al., Circulation, 2013), Vogt-Koyanagi-Harada syndrome, systemic lupus erythematosus (SLE), obesity (Ahmad, R., P.
  • gynecological diseases such as endometriosis, dysmenorrhea, dyspareunia and endometriosis, especially pain associated with endometriosis and other symptoms associated with endometriosis such as dysmenorrhea, dyspareunia, dysuria and dysfecia (Akoum, Lawson et al., Human Reproduction, 2007; Allhorn, Boing et al., Reproductive Biology and Endocrinology, 2008; Lawson, Bourcier et al., Journal of Reproductive Immunology, 2008; Sikora, Mielczarek-Palacz et al., American Journal of Reproductive Immunology, 2012; Khan, Kitajima et al., Journal of Obstetrics and Gynaecology Research, 2013; Santulli, Borghese et al., Human Reproduction, 2013); ocular diseases such as retinal ischemia,
  • liver diseases such as fatty liver hepatitis, especially non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH)
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • Velayudham et al. Am J Physiol Gastrointest Liver Physiol, 2011; Miura, Kodama et al., Gastroenterology, 2010; Kamari, Shaish et al., J Hepatol, 2011; Ye, Li et al., Gut, 2012; Roh, Seki, J Gastroenterol Hepatol, 2013; Ceccarelli, S., V.
  • IRAK4-mediated signal transduction pathways The regulation of IRAK4-mediated signal transduction pathways is mainly related to its kinase function, however, there are also some reports indicating that in some cell types, the signal regulation of IRAK4 on downstream processes is related to the non-kinase function of IRAK4. Cushing et al. stated that although the phosphorylation level of IRAK4 was reduced in human skin fibroblasts stimulated by IL-1 ⁇ , the pharmacological inhibition of IRAK4 did not lead to the inhibition of IL-6 and TNF- ⁇ . Supporting these results, the scaffolding function of IRAK4 in IRAK4-deficient fibroblasts is important for IL1 signaling compared with wild-type cells.
  • IRAK4 kinase activity is not necessary in human B cells and T cells, dendritic cells and monocytes, and siRNA gene ablation also showed that IRAK4 has a scaffolding function in these cells.
  • a variety of potent and selective inhibitors against IRAK4 have been reported, such as CA-4948, BAY-1834845, BMS-986126, and PF-06650833, etc. These inhibitors can selectively inhibit the kinase activity of IRAK4 and are mainly used for the prevention and treatment of autoimmune diseases, inflammatory diseases, and tumor diseases.
  • IRAK4 has the functions of scaffold protein and active kinase, and on the other hand, traditional small molecule inhibitors are prone to drug resistance. Therefore, inhibiting the kinase activity of IRAK4 alone may not be sufficient to produce a therapeutic effect.
  • PROTAC Proteolysis Targeting Chimeria
  • This bifunctional molecule recognizes the target protein and E3 ubiquitin ligase in vivo, brings the target protein and E3 ubiquitin ligase closer to form a ternary complex, ubiquitinates the target protein, and then degrades the target protein through the ubiquitin-proteasome pathway in vivo.
  • PROTAC only needs to bring the target protein closer to the E3 ubiquitin ligase to degrade the substrate.
  • This mode of action allows this technology to be applied to some undruggable targets; on the other hand, since the target protein can be released after being degraded to continue to participate in the degradation process of the next protein, this catalytic degradation effect allows a smaller PROTAC drug dose to achieve efficient degradation; on the other hand, traditional small molecule inhibitors are prone to drug resistance, often because of point mutations, which cause the small molecule inhibitors to lose their inhibitory effect on the target, while PROTAC can directly degrade the target protein, which can avoid the drug resistance caused by point mutations to a certain extent. Therefore, compared with traditional small molecule inhibitors, the use of PROTAC technology for the development of new drug small molecules has high advantages and feasibility, and is expected to become the next generation of promising new drugs.
  • PROTAC technology has also been used in the transformation of drugs for a variety of targets, such as androgen receptors, estrogen protein receptors, BTK, etc. US2019/0151295, Several types of degraders targeting IRAK4 are disclosed in US2019/0192688, WO2019/160915 and WO2020/113233, and more degraders targeting IRAK4 are yet to be developed.
  • IRAK4 degraders are suitable for treating and preventing animal immune diseases characterized by an overreactive immune system, and particularly have a good therapeutic or preventive effect on the following diseases: rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and/or psoriasis.
  • rheumatoid arthritis atopic dermatitis
  • HS hidradenitis suppurativa
  • vitiligo vitiligo
  • dermatomyositis dermatomyositis
  • alopecia areata
  • urticaria polymyositis
  • interstitial lung disease systemic lupus erythematosus
  • the present invention provides IRAK4, and/or its stereoisomers, enantiomers, diastereomers, deuterated substances, hydrates, solvates,
  • the invention relates to a pharmaceutical composition, a prodrug and/or a pharmaceutically acceptable salt thereof for use in treating and/or preventing immune and/or inflammatory diseases.
  • the present invention provides IRAK4, and/or its stereoisomers, enantiomers, diastereomers, deuterated compounds, hydrates, solvates, prodrugs and/or pharmaceutically acceptable salts thereof, for use in treating and/or preventing diseases caused by abnormal expression of IRAK4 and IRAK4-related proteins in the IL-1R/TLR pathway and/or abnormal secretion or proliferation of chemical factors, cytokines or immune cells mediated by IRAK4.
  • the IRAK4 degrading agent is a compound represented by Formula I, Formula II or Formula III or a pharmaceutically acceptable salt thereof:
  • Ring A is phenyl or pyridyl
  • Ring B is C 6 -C 10 cycloalkyl or 6-10 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, and the C 6 -C 10 cycloalkyl and 6-10 membered heterocycloalkyl are optionally substituted by a substituent selected from halogen, oxo, cyano, amino, hydroxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl or -O-(C 1 -C 6 alkyl);
  • Ring C is C 6 -C 12 cycloalkyl or 6-12 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, and the C 6 -C 12 cycloalkyl and 6-12 membered heterocycloalkyl are optionally substituted by a substituent selected from halogen, cyano, amino, hydroxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl or -O-(C 1 -C 6 alkyl);
  • Ring D is a C 6 -C 10 aryl group, and the C 6 -C 10 aryl group is optionally substituted with a halogen, cyano, amino, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl or -O-(C 1 -C 6 alkyl) substituent;
  • Ring Y is a 5-6 membered heteroaryl group
  • X is a bond, -O-, -NH-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-;
  • L is -( CH2 ) j- , wherein one or more methylene groups in said - ( CH2 ) j- are optionally replaced by a group selected from -NR3'- , -O-, -S-, -S(O ) -, -S(O) 2- , -S(O)2NR3'-, -CR1'R2'-, -C(O)-, -C (O)O-, -OC(O)-, -NR3'C(O)O-, -OC(O) NR3'-, -C (O) NR3'-, -NR3'C (O)-, -NR4'C (O ) NR3'- , vinylene or ethynylene;
  • R 1' and R 2' are each independently halogen, -OH, -NH 2 , C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 hydroxyalkyl, -O(C 1 -C 4 -NH(C 1 -C 4 alkyl);
  • R 3' and R 4' are each independently hydrogen or C 1 -C 6 alkyl
  • R d are each independently hydrogen, deuterium, halogen, cyano, C 1 -C 6 alkyl, wherein the alkyl is optionally substituted by one or more groups selected from halogen, hydroxyl, and amino;
  • R c is -O(C 1 -C 3 alkyl), -N(C 1 -C 3 alkyl) 1-2 or C 1 -C 6 alkyl, wherein the alkyl is optionally substituted by one or more groups independently selected from hydroxy, amino, halogen, cyano or -O-(C 1 -C 3 alkyl);
  • R b is hydrogen or C 1 -C 6 alkyl, the alkyl group is optionally substituted by one or more groups independently selected from hydroxyl, amino, halogen, cyano;
  • Ra is hydrogen, halogen, C1 - C6 alkyl or -O-( C1 - C6 alkyl), wherein the alkyl is optionally substituted by halogen or hydroxy;
  • Each R 1 is independently selected from: C 1 -C 4 alkyl, -O(C 1 -C 4 alkyl), -N(C 1 -C 4 alkyl) 1-2 , CN, halogen, -OH, -NH 2 , wherein the alkyl group is optionally substituted by a group selected from halogen, cyano, -OH, C 1 -C 4 alkyl, -O(C 1 -C 4 alkyl)
  • each R 2 is independently hydrogen, C 1 -C 4 alkyl, -O(C 1 -C 4 alkyl), C 3 -C 8 cycloalkyl, 3-8 membered heterocycloalkyl, 6-10 membered aryl, 5-6 membered heteroaryl, CN, halogen, -OH, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally selected from halogen, cyano, -OH, -NH 2 , C 1 -C 4 alkyl and -O(C 1 -C 4 alkyl);
  • X' is CH or N
  • n 0, 1, 2, 3, or 4;
  • n 0, 1, 2, 3, or 4;
  • p 1 or 2;
  • j 0, 1, 2, 3, 4 or 5.
  • the IRAK4 degrading agent is a compound represented by Formula I-1, I-2 or Formula II-1 or a pharmaceutically acceptable salt thereof:
  • R 3 is H, halogen, C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl);
  • R c , R d , Ring B, L, Ring C, X, X′, p, R 2 and m are as defined herein.
  • the disease is characterized by the use of an IRAK4 degrader for treating and/or preventing an immune disease.
  • the IRAK4 degrader is used for an immune disease, and the immune disease is selected from: adult Still's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet's disease, benign mucous membrane pemphigoid (mucous membrane pemphigoid), bullous pemphigoid, Castleman's disease (CD), celiac disease, Coxsackie myocarditis, Crohn's disease, dermatitis herpetiformis, atopic dermatitis, dermatomyositis, discoid lupus, endometriosis, eosinophilic fasciitis, erythema nodosum, fibrosing alveolitis, primary glomerulonephritis, Goodpasture syndrome,
  • the IRAK4 degrader is used for an immune disease selected from: rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and/or psoriasis.
  • an immune disease selected from: rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and/or psoriasis.
  • the disease is characterized by IRAK4 degrading agents for treating and/or preventing inflammatory diseases the use of.
  • the IRAK4 degrader is used for an immune disease, and the inflammatory disease is selected from: familial Mediterranean fever, tumor necrosis factor-associated periodic syndrome, mevalonate kinase deficiency, pyridine-associated autoinflammatory with neutrophilic dermatosis (PAAND), suppurative sterile arthritis, pyoderma gangrenosum and acne (PAPA), familial cold autoinflammatory syndrome (FCAS), familial chronic lichenoid keratosis (FKLC), NLRP1-associated autoinflammatory with arthritis and dyskeratosis (NAIAD), IL-1 receptor antagonist (DIRA) deficiency, IL-36 receptor antagonist (DITRA) deficiency, allergic contact dermatitis, CAR-T cell-induced cytokine release syndrome, Crohn's disease, chronic bronchitis, COPD, active ankylosing spondylitis, axial spondyloarthritis, pityria
  • the IRAK4 degrader is used to treat and/or prevent diseases mediated by IL-2R alpha, IL-6, IFA-alpha2, IFN-gamma, IL-1ra, MCP-3, IL-16, IL-12(p40), LIF, IL-5, GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, Eotaxin, Basic FGF, beta-NGF, PDGF-BB, IL-4, MCP-1, IL-8, IL-10, GRO-alpha, HGF, IL-1alpha, IL-1beta, IL-3, SCF, TRAIL, M-CSF, CTACK, IL-15, IL-12(P70), IL-17, IL-23, IL-33 and/or IL-36 cytokines.
  • the IRAK4 degrader is used to treat and/or prevent diseases mediated by IL-4, IL-6, IL-12 (p40), GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, IL-8, IL-10, IL-17, IL-23, IL-33 and/or IL-36 cytokines.
  • the sample is a spleen, skin and/or blood sample.
  • the blood sample is normal human whole blood and/or patient whole blood.
  • the method for treating the disease comprises administering an effective amount of an IRAK4 degrading agent to the subject.
  • the method for treating the disease comprises the IRAK4 degrader as the single compound or in combination with other drugs.
  • the present invention proves through IRAK4 kinase activity test experiment that the A1-A52, B1-B5 or C1-C36 compound of the present invention can effectively bind to the IRAK4 target protein to produce degradation and/or inhibition effect, and proves through Western-Blot that the A1-A52, B1-B5 or C1-C36 compound of the present invention can effectively and specifically degrade the IRAK4 protein in THP-1 cells.
  • the A1-A52, B1-B5 or C1-C36 compound of the present invention and/or its stereoisomers, enantiomers, diastereomers, deuterated compounds, hydrates, solvates, metabolites, prodrugs and/or pharmaceutically acceptable salts can effectively degrade the IRAK4 protein, thereby achieving the effect of preventing or treating diseases or conditions related to IRAK4.
  • the present invention has demonstrated through experiments that the compounds described herein can significantly reduce the expression of IRAK4 in whole blood, skin or liver, indicating that IRAK4 degraders can be used to treat immune diseases.
  • the drug comprises an IRAK4 degrader as an active ingredient; the drug of the present invention may also optionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the drug IRAK4 degrader can be used alone or in combination with other drugs to achieve a better therapeutic effect on inflammatory or immune diseases.
  • the dosage form of the drug can be tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, Solutions, syrups, elixirs, ointments, drops, suppositories, inhalants, sprays.
  • the drug with IRAK4 degrader as the active ingredient can be prepared into any of the above-mentioned pharmaceutical dosage forms according to actual needs, and the drugs in each dosage form can be prepared according to conventional methods in the pharmaceutical field.
  • the route of administration of the drug can be selected according to actual needs, including oral administration, sublingual administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal administration, transdermal administration, parenteral administration, inhalation administration, intratracheal administration, intrapulmonary administration, and bronchial administration.
  • the present invention also provides pharmaceutically acceptable salts of A1-A52, B1-B5 or C1-C36 compounds.
  • pharmaceutically acceptable salt refers to a relatively non-toxic acid addition salt or base addition salt of the compound of the present invention.
  • the acid addition salt is a salt formed by the A1-A52, B1-B5 or C1-C36 compound of the present invention and a suitable inorganic acid or organic acid, which can be prepared during the final separation and purification of the compound, or can be prepared by reacting the purified A1-A52, B1-B5 or C1-C36 compound in its free base form with a suitable organic acid or inorganic acid.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluene, citrate, maleate, fumarate, succinate, tartrate, benzoate, methanesulfonate, p-toluenesulfonate, gluconate, lactobionate and lauryl sulfonate, etc.
  • the base addition salt is a salt formed by the A1-A52, B1-B5 or C1-C36 compound with a suitable inorganic base or organic base, including, for example, salts formed with alkali metals, alkaline earth metals, quaternary ammonium cations, such as sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, tetramethyl quaternary ammonium salts, tetraethyl quaternary ammonium salts, etc.; amine salts include salts formed with ammonia (NH3), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.
  • a suitable inorganic base or organic base including, for example, salts formed with alkali metals, alkaline earth metals, quaternary ammonium cations, such as
  • the compounds of the present invention or their pharmaceutically acceptable salts can be administered to mammals, including humans, orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments or drops), or intratumorally.
  • the compound of the present invention may be administered at a dosage of about 0.05-300 mg/kg body weight/day, preferably 10-300 mg/kg body weight/day, more preferably 10-150 mg/kg body weight/day.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into solid dosage forms for oral administration, including but not limited to capsules, tablets, pills, powders and granules.
  • the compounds A1-A52, B1-B5 or C1-C36 of the present invention are mixed as active ingredients with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (2) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (3) Humectants, such as glycerol, etc.; (4) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, etc.; (5) slow dissolving agents
  • Capsules, tablets and pills may also contain buffers.
  • the solid dosage forms such as tablets, pills, capsules, pills and granules may be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such a composition may be delayed in a certain part of the digestive tract.
  • coating and shell materials such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such a composition may be delayed in a certain part of the digestive tract.
  • Examples of embedding components that can be used are polymeric substances and waxes. If desired, the active ingredient can also be in microencapsulated form with one or more of the above-mentioned excipients.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into liquid dosage forms for oral administration, including but not limited to pharmaceutically acceptable emulsions, solutions, suspensions, syrups and tinctures.
  • the liquid dosage form may contain inert diluents such as water and other solvents, solubilizers, Inert diluents include ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, etc., or mixtures of these substances.
  • the liquid dosage form of the present invention may also contain conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices, etc.
  • the suspending agent includes ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol, microcrystalline cellulose, aluminum methylate and agar, etc., or mixtures of these substances.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including but not limited to physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for re-dissolving into sterile injectable solutions or dispersions.
  • Suitable carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the compounds of the present invention or their pharmaceutically acceptable salts can also be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays and inhalants, etc.
  • dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays and inhalants, etc.
  • active ingredients the A1-A52, B1-B5 or C1-C36 compounds of the present invention or their pharmaceutically acceptable salts are mixed under sterile conditions with a physiologically acceptable carrier and optional preservatives, buffers, or propellants that may be required if necessary.
  • the present invention also provides a pharmaceutical composition, which contains the present invention A1-A52, B1-B5 or C1-C36 compound or its pharmaceutically acceptable salt as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutically acceptable carrier excipient or diluent.
  • the present invention A1-A52, B1-B5 or C1-C36 compound or its pharmaceutically acceptable salt is usually mixed with a pharmaceutically acceptable carrier, excipient or diluent.
  • the composition of the present invention can be formulated into a conventional pharmaceutical preparation according to a conventional preparation method.
  • the compounds of the present invention or their pharmaceutically acceptable salts can be administered alone or (if necessary) in combination with other pharmaceutically acceptable therapeutic agents, such as in combination with other anti-tumor drugs, anti-inflammatory drugs or autoimmune drugs.
  • the components to be combined can be administered simultaneously or sequentially, in the form of a single preparation or in the form of different preparations.
  • the combination can include not only a combination of the compounds of the present invention and one other active agent, but also a combination of the compounds of the present invention and two or more other active agents.
  • compositions are pharmaceutically acceptable as used herein means that the substance or composition must be compatible with other ingredients of the formulation and not harmful to patients.
  • the treatment described in the present invention includes preventive and palliative treatment.
  • the sample is a blood sample from a patient or a normal person
  • the sample is a plasma sample from a patient or a normal person
  • the sample is a peripheral blood mononuclear cell sample from a patient or a normal person.
  • measuring the level of immune biomarkers in a sample comprises using the AlphaLISA method. In some embodiments, measuring the level of immune biomarkers in a sample comprises using a method selected from the examples.
  • Alkyl refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched alkyl groups; C1 - C8 alkyl refers to an alkyl group containing 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl
  • Cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent; "C 3 -C 11 cycloalkyl” refers to a cycloalkyl group including 3 to 11 carbon atoms; “C 3 -C 8 -membered cycloalkyl” refers to a cycloalkyl group including 3 to 8 carbon atoms; “C 5 -C 10 -membered cycloalkyl” refers to a cycloalkyl group including 5 to 10 carbon atoms;
  • Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; preferably C 3 -C 8 -membered cycloalkyl; more preferably C 3 -C 6 -membered cycloalkyl.
  • Polycyclic cycloalkyl includes cycloalkyl of spiro ring, condensed ring and bridged ring.
  • “Spiro cycloalkyl” refers to a polycyclic group in which a carbon atom (called spiro atom) is shared between monocyclic rings. They may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system. According to the number of spiro atoms shared between rings, spiro cycloalkyl is divided into monospiro cycloalkyl, bispiro cycloalkyl or polyspiro cycloalkyl, preferably 7-12 bispiro cycloalkyl.
  • Non-limiting examples of spiro cycloalkyl include:
  • fused cycloalkyl refers to a full-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic fused cycloalkyl.
  • fused cycloalkyl include:
  • Bridged cycloalkyl refers to a full-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected. They may contain one or more double bonds, but none of the rings has a completely conjugated ⁇ electron system. They can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups according to the number of constituent rings. Non-limiting examples of bridged cycloalkyl groups include:
  • the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc.
  • the cycloalkyl may be optionally substituted or unsubstituted.
  • the cycloalkyl group is a C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 monocyclic or polycyclic (eg, spiro, fused or bridged) cycloalkyl group.
  • Heterocycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein one or more (e.g., 2, 3, 4, or 5) ring atoms are selected from nitrogen, oxygen, or S(O) r (wherein r is an integer of 0, 1, or 2), but does not include the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon.
  • 3-11-membered heterocycloalkyl refers to a ring group containing 3 to 11 ring atoms
  • 5-10-membered heterocycloalkyl refers to a ring group containing 5 to 10 ring atoms
  • 3-8-membered heterocycloalkyl refers to a ring group containing 3 to 8 ring atoms, preferably "3-11-membered heterocycloalkyl” containing 1-2 heteroatoms selected from N, O, or S, and more preferably 3-11-membered heterocycloalkyl containing 1 or 2 N atoms.
  • the monocyclic heterocycloalkyl is preferably a 3-8 membered monocyclic heterocycloalkyl containing 1-2 N heteroatoms; non-limiting examples of the monocyclic heterocycloalkyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and piperazinyl.
  • Polycyclic heterocycloalkyl includes spiro ring, condensed ring and bridged heterocycloalkyl.
  • “Spiro heterocycloalkyl” refers to a polycyclic heterocycloalkyl group in which one atom (called spiro atom) is shared between monocyclic rings, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) r (wherein r is an integer 0, 1, 2), and the remaining ring atoms are carbon. They can contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system.
  • spiro cycloalkyl is divided into monospiro heterocycloalkyl, bispiro heterocycloalkyl or polyspiro heterocycloalkyl, preferably containing 1-2 saturated "3-11-membered bispiro heterocycloalkyl" selected from N, O or S heteroatoms; more preferably containing 1 or 2 N atoms. Saturated "7-11-membered bispiro heterocycloalkyl".
  • Non-limiting examples of spiro heterocycloalkyl include: wait.
  • “Fused heterocycloalkyl” refers to a polycyclic heterocycloalkyl group in which each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) r (wherein r is an integer of 0, 1, 2), and the remaining ring atoms are carbon.
  • the number of constituent rings it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocycloalkyl, preferably containing 1-3 "3-11-membered bicyclic fused heterocycloalkyl" selected from N, O or S heteroatoms; more preferably containing 1 or 2 N atoms of saturated "3-11-membered bicyclic fused heterocycloalkyl".
  • fused heterocycloalkyl include: wait.
  • Bridged heterocycloalkyl refers to a polycyclic heterocycloalkyl group in which any two rings share two atoms that are not directly connected, they may contain one or more double bonds, but none of the rings has a completely conjugated ⁇ electron system, and one or more of the ring atoms is selected from nitrogen, oxygen or S(O) r (wherein Where r is an integer 0, 1, 2), and the remaining ring atoms are carbon. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, and non-limiting examples of bridged heterocycloalkyl include:
  • heterocycloalkyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples include:
  • the heterocycloalkyl group may be optionally substituted or unsubstituted.
  • the heterocycloalkyl group is a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered monocyclic or polycyclic (e.g., spirocyclic, fused, or bridged) heterocycloalkyl group, wherein the number of heteroatoms may be 1, 2, 3, 4, or 5, and each heteroatom is independently nitrogen, oxygen, or S(O) r (wherein r is an integer of 0, 1, or 2).
  • Aryl refers to an all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring that shares adjacent pairs of carbon atoms) and a polycyclic group with a conjugated ⁇ electron system
  • C 6 -C 10 aryl refers to an all-carbon aromatic group containing 6-10 carbons, such as phenyl and naphthyl; preferably phenyl.
  • the aryl ring can be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:
  • the aryl group may be optionally substituted or unsubstituted.
  • the aryl group is a 6-10 membered aryl group.
  • Heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms, wherein the heteroatoms include nitrogen, oxygen or S(O) r (wherein r is an integer of 0, 1, 2), a 5-6 membered heteroaryl refers to a heteroaromatic system containing 5-6 ring atoms, and a 5-10 membered heteroaryl refers to a heteroaromatic system containing 5-10 ring atoms, preferably a 5-6 membered heteroaryl; more preferably a 5-6 membered heteroaryl containing 1 or 2 N atoms; non-limiting examples include furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, pyrazole, imidazolyl, triazolyl, tetrazolyl, etc.; preferably pyridyl.
  • the heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples include:
  • the heteroaryl group may be optionally substituted or unsubstituted.
  • the heteroaryl group is a 5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl group, wherein the number of heteroatoms may be 1, 2, 3, 4 or 5, each heteroatom is independently nitrogen, oxygen or S.
  • structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric and geometric (or conformational) forms of the structure; R and S configurations of each asymmetric center, Z and E double bond isomers and Z and E conformational isomers. Therefore, single-layer chemical isomers as well as enantiomeric, diastereomeric and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the present invention. Unless otherwise stated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.
  • structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structure include replacement of hydrogen by deuterium or tritium, or replacement of carbon 13 by a C- or 14 C oxygen-rich carbons are within the scope of the present invention. These compounds are useful as analytical tools, as probes in biological assays or as therapeutic agents according to the present invention.
  • “Stereoisomers” include all isomers of a single compound that differ only in the orientation of their atoms in space.
  • stereoisomer includes mirror image isomers of a compound (including enantiomers of the (R-) or (S-) configuration of a compound), mixtures of mirror image isomers (physical mixtures of enantiomers, as well as racemates or racemic mixtures), geometric (cis/trans or E/Z, R/S) isomers of a compound and isomers of a compound that have multiple chiral centers but are not mirror images of each other (diastereomers).
  • the chiral centers of a compound may undergo epimerization in vivo; therefore, for these compounds, administration of the compound in the (R-) form is considered equivalent to administration of the compound in the (S-) form.
  • the compounds of the present invention may be administered in individual isomeric forms and substantially free of other isomers.
  • the bifunctional compound of the present invention is an isotopic derivative because it has at least one desired isotopic substitution of an atom in an amount greater than the natural abundance of the isotope, i.e., enriched.
  • the compound includes deuterium or multiple deuterium atoms. Substitution with heavier isotopes such as deuterium, i.e., 2H, can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be advantageous in certain circumstances.
  • the bifunctional compounds of the present invention include N-oxides, crystalline forms (also known as polymorphs), active metabolites of compounds having the same type of activity, tautomers, and non-solvated as well as solvated and hydrated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. in the compound.
  • the solvated forms of the conjugates presented herein are also considered to be disclosed herein.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • control sample refers to an individual or sample from a group of individuals who do not have a disease or condition (e.g., arthritis, rheumatoid arthritis, myositis, lupus erythematosus and/or systemic sclerosis), or an internal control, as determined by techniques known in the art.
  • a control or baseline level is first determined, or measured prior to measurement in a sample, or obtained from a database of such control samples.
  • Control compound (I) and “control (I)” are used interchangeably in the present invention.
  • Control compound (II) and “control (II)” are used interchangeably in the present invention.
  • an “effective amount” refers to an amount that, when administered to a subject, produces beneficial or desired results, including clinical results, such as degradation, inhibition or alleviation of symptoms of the disorder being treated in the subject as compared to a control.
  • “Pharmaceutically acceptable carrier, adjuvant or carrier” refers to a non-toxic carrier, adjuvant or carrier that does not destroy the pharmacological activity of the compound in which it is formulated.
  • Pharmaceutically acceptable carriers, adjuvants or carriers that can be used in the composition of the present invention include, but are not limited to, ion exchangers, alumina, stearates, egg Phospholipids, serum proteins, e.g. human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, salts or partial glycerol mixtures of electrolytes, e.g.
  • presulfated guanidine disodium phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium gluconate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • Patient refers to an animal, preferably a mammal, most preferably a human.
  • Cytokine refers to IL-2Ralpha, MIG, MIP-1beta, IL-6, IFN-al pha2, IFN-gamma, SDF-1alpha, IL-1ra, MCP-3, IL-16, IL-12 (p40), LIF, TNF-beta, IL-5, GM-CSF, MIF, TNF-alpha, RANTES, IL-2, IL-1beta, IL-18, Eotaxin, BasicFGF , VEGF, beta-NGF, PDGF-BB, IP-10, IL-13, IL-4, MCP-1, IL-8, MIP-1alpha, IL-10, G-CSF, GRO-alpha, HGF, IL-1alpha, IL-3, SCF, TRAIL, M-CSF, CTACK, IL-15, IL-7, IL-12 (p70), IL-17, IL-9, SCGF-beta, KC, IL-17A and MCP-17A
  • measuring the cytokine levels in normal human or mouse samples includes using cultured peripheral blood mononuclear cells (PBMCs) for determination. In some embodiments, measuring the cytokine levels in normal human or mouse samples includes using Luminex methods for determination.
  • PBMCs peripheral blood mononuclear cells
  • the present invention will be further described in detail and completely below in conjunction with the examples, but the present invention is by no means limited to the contents of the examples.
  • the raw materials in the examples of the present invention are known and can be bought on the market, or can be synthesized by methods known in the art.
  • the experimental methods of the examples of the present invention that do not specify specific conditions are usually according to conventional conditions, or according to the conditions recommended by the raw materials or commodity manufacturers.
  • DMSO N,N-dimethylformamide
  • Glucose refers to glucose
  • Solutol refers to polyethylene glycol-15 hydroxystearate.
  • LPS lipopolysaccharide
  • R848 refers to Reximod.
  • PBMC peripheral blood mononuclear cells
  • IRAK4 refers to interleukin 1 receptor-associated kinase 4.
  • IL refers to interleukin.
  • IMQ refers to imiquimod.
  • Topical refers to local administration.
  • FIG. 1 shows the degradation effect of compound A on IRAK4 in FIG. 1A and FIG. 1B , respectively.
  • Fig. 2 Intracellular IRAK4 protein expression levels in PBMCs of various patients.
  • FIG3 shows the inhibitory effect of compound A on LPS-induced IL-6 in normal human PMBCs.
  • FIG4 shows the inhibitory effect of compound A on cytokine secretion by human peripheral blood mononuclear cells stimulated by LPS.
  • FIG5 shows the inhibitory effect of compound A on cytokine secretion by human peripheral blood mononuclear cells stimulated by R848.
  • FIG6 shows the inhibitory effects of compound A on IL-6 in normal human whole blood and patient whole blood in FIG6A, FIG6B, FIG6C and FIG6D respectively.
  • FIG. 7 shows the degradation of IRAK4 in PBMC, spleen and skin of C57 mice by compound A in FIG. 7A , FIG. 7B and FIG. 7C , respectively.
  • FIG8 shows the efficacy of compound A in the imiquimod-induced psoriasis model in C57 mice in FIG8A , FIG8B , and FIG8C , respectively.
  • Example 1 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecane-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 2 5-((1R, 4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-((1r, 4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazole -4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • the preparation method is as in Example 1.
  • Step 1 Preparation of tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidine-1-carboxylate
  • HATU 900 mg, 2.4 mmol
  • DMF 20 ml
  • 5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxylic acid 470 mg, 1.9 mmol
  • DIEA 1 mL, 4.7 mmol
  • the reaction solution was stirred at 25°C overnight.
  • Step 2 Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 3 Preparation of tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 4 Preparation of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • the reaction mixture was directly concentrated under reduced pressure to give N-(1-(1-((3-azaspiro[5.5]undecane-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • the obtained product was directly used for the next step.
  • Step 5 Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
  • Step 3 Preparation of benzyl 4-(3-((1-(1-(tert-butyloxycarbonyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
  • HATU (380 mg, 0.50 mmol) was added to a solution of 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylic acid tert-butyl ester (0.21 g, 0.3 mmol) (0.5 ml, 1.0 mmol) in DMF (5 ml), and then stirred at room temperature for 2 h and then tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.21 g, 0.3 mmol) was added, and the reaction solution was stirred at 25°C overnight.
  • Step 4 Preparation of benzyl 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
  • reaction solution was directly concentrated under reduced pressure to give 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylic acid benzyl ester.
  • Step 5 Preparation of tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • tert-Butyl 9-formyl-3-azaspiro[5.5]undecane-3-carboxylate 144 mg, 0.45 mmol
  • NaBH 3 CN 138 mg, 1.2 mmol
  • benzyl 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate 230 mg, 0.4 mmol
  • DIEA 0.35 mL, 2.0 mmol
  • Step 6 4-(3-((1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl) Preparation of Benzyl Pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
  • Step 7 Preparation of benzyl 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
  • reaction mixture was then stirred at 25°C for 16 h.
  • Water (30 mL) was added to the reaction mixture, and the mixture was extracted with EA (50 mL ⁇ 3).
  • the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 8 Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
  • Step 2 Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
  • the crude product was purified by Prep-HPLC (acetonitrile/0.05% NH 4 OH aqueous solution, 5% to 95%) to give 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.
  • Step 3 4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethane
  • Step 4 Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Step 5 Preparation of tert-butyl 9-((4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 6 Preparation of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was then directly concentrated under reduced pressure to obtain N-(1-(1-((3-azaspiro[5.5]undecane-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Step 7 Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate
  • Step 2 Preparation of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-(4-(piperidin-4-yl)butyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-(4-(piperidin-4-yl)butyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Step 3 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)butyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
  • Example 8 N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecane-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (crude product), which was directly used in the next step.
  • Step 3 Preparation of N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate
  • N-(3-(Difluoromethyl)-1-((1R,4R)-4-(aldehyde)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.23 mmol) was dissolved in THF (10 mL), tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (56 mg, 0.23 mmol) and STAB (146 mg, 0.69 mmol) were added, and the mixture was reacted at room temperature for 2 h. Water (50 mL) was added.
  • Step 2 Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 3 Preparation of N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • the crude product is used to prepare N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide using reverse phase.
  • Step 1 Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 2 Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 3 Preparation of tert-butyl 4-(2-((((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 5 Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 3 Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • the crude product was prepared by reverse phase to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Example 12 N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 13 N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 14 N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction mixture was dissolved in 4% paraformaldehyde and trifluoroacetic acid (1 mL) was added and stirred at room temperature for 2 h.
  • the reaction solution was directly concentrated under reduced pressure to give N-(3-(difluoromethyl)-1-((1R,4S)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was directly used in the next step without purification.
  • Step 3 Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 15 5-((S)-3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate
  • Step 2 Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate
  • Step 5 Preparation of 1-(5-(4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • N-Benzyl-N-methyl-2-(piperidin-4-yl)ethane-1-amine 250 mg, 1.04 mmol was dissolved in DMSO (10 mL), and DIEA (671 mg, 5.2 mmol) and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid pentafluorophenyl ester (451 mg, 1.04 mmol) were added, and the mixture was reacted at room temperature for 2 h.
  • Step 6 Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 7 Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL x 3), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 8 Preparation of 5-((S)-3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 16 N-(3-(difluoromethyl)-1-(1-(2-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 17 N-(3-(difluoromethyl)-1-((1R,4R)-4-((2-(1-(3-(2,6-dioxopyridin-3-yl)-4-fluorobenzoyl)piperidin-4-yl)ethyl)(methyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid methyl ester
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (40 mL x 3), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid
  • Step 3 Preparation of tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxamido)ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step without purification.
  • Step 5 Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-((2-(1-(3-(2,6-dioxopyridin-3-yl)-4-fluorobenzoyl)piperidin-4-yl)ethyl)(methyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was diluted with water (20 mL), extracted with ethyl acetate (20 mL x 3), and the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Example 18 5-((S)-3-aminopiperidin-1-yl)-N-(1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate
  • Step 3 Preparation of 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was purified by reverse phase chromatography (45% ACN/0.1% NH 4 HCO 3 aqueous solution) to obtain 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.
  • Step 4 Preparation of tert-butyl ((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate
  • Step 5 Preparation of 5-((S)-3-aminopiperidin-1-yl)-N-(1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure, and then a saturated solution of NaHCO 3 was added to adjust the pH to 8-9, extracted with MeOH/DCM (10%, 20 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated.
  • Step 1 Preparation of tert-butyl 9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was directly concentrated under reduced pressure to obtain 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was directly used in the next step without purification.
  • Step 3 Preparation of N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 20 N-(1-(1-(2-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was directly used in the next step.
  • Step 3 Preparation of 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 22 Preparation of N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazole-4-)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL x 3), the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 1 Preparation of (S)-(1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylic acid tert-butyl ester
  • Step 2 Preparation of (S)-5-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 24 N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate
  • Step 2 Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 3 N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
  • amines N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-
  • Example 25 (S)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 27 N-(3-(difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate
  • Step 2 Preparation of N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was directly concentrated under reduced pressure to obtain N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was directly used in the next reaction without purification.
  • Step 3 Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 28 N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate
  • reaction solution was diluted with water (40 mL), extracted with ethyl acetate (50 mL x 3), the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of 1-(2-methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was directly concentrated under reduced pressure to obtain 1-(2-methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was directly used in the next step without purification.
  • Step 3 Preparation of 1-(5-(9-(2-(1,3-dioxolan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was diluted with water (80 mL), extracted with ethyl acetate (50 mL x 3), the organic phase was washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 4 Preparation of 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)propanal
  • Step 5 Preparation of N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide
  • the reaction solution was diluted with water (50 mL), extracted with dichloromethane (60 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • the crude product was purified by pre-HPLC (CH 3 CN/0.08% NH 4 HCO 3 aqueous solution, 5% to 95%) to obtain N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Example 29 N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • N-(3-(Difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide 80 mg, 0.16 mmol
  • 3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecane-9-carbaldehyde 70 mg, 0.16 mmol
  • THF 3 mL
  • sodium triacetoxyborohydride 102 mg, 0.48 mmol
  • Step 1 (S)-(1-(3-((1-(1-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylic acid tert-butyl Preparation of esters
  • reaction solution was stirred at room temperature for 2 h.
  • the reaction solution was diluted with water (20 mL) and ethyl acetate (20 mL x 3).
  • the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • Step 2 Preparation of (S)-5-(3-aminopiperidin-1-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of 4-(3-hydroxypropoxy)piperidine-1-carboxylic acid benzyl ester
  • Step 3 Preparation of 1-(2-chloro-5-(4-(3-hydroxypropoxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • Step 4 Preparation of 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propanal
  • Step 5 Preparation of 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Example 32 N-(3-(Difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • Step 1 Preparation of tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate
  • Step 3 Preparation of benzyl (2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate
  • reaction solution was diluted with water (50 mL), extracted with ethyl acetate (70 mL x 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Step 4 Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione
  • reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the compound 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.
  • Step 5 Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
  • reaction solution was diluted with water (20 mL), extracted with ethyl acetate (25 mL x 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the crude product was prepared by reverse phase to obtain compound N-(3-(difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.
  • Step 1 Preparation of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate
  • Step 2 Preparation of tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate
  • Step 3 Preparation of tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate
  • Step 4 Preparation of tert-butyl (4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)carbamate
  • Step 5 Preparation of benzyl 4-(((4-((tert-butyloxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate
  • Step 6 Preparation of benzyl 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate
  • Step 7 Preparation of benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate
  • Step 8 Preparation of tert-butyl 4-((((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate
  • Step 9 Preparation of benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate
  • 6-(Trifluoromethyl)picolinic acid 90 mg, 0.47 mmol
  • HATU 178 mg, 0.47 mmol
  • DIEA 140 mg, 1.08 mmol
  • tert-butyl 4-((((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate 190 mg, 0.36 mmol
  • DMF 5 mL
  • the reaction solution was then stirred at room temperature for 2 h. Water (30 mL) was added to the reaction solution, and EA (20 mL ⁇ 3) was used.
  • Step 10 Preparation of N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • reaction solution was filtered through celite, and the filtrate was directly concentrated under reduced pressure to give N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide.
  • Step 11 N-(2-((1S,4S)-4-(((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy) Preparation of 6-(trifluoromethyl)-2-(4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • reaction solution was diluted with water (50 mL) and washed with DCM (20 mL ⁇ 3).
  • the organic phase was washed with saturated brine (60 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure.
  • Step 1 Preparation of benzyl 4-(2-((4-((tert-butyloxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of benzyl 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 3 Preparation of benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of benzyl 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 5 Preparation of benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • 6-(Trifluoromethyl)picolinic acid 101 mg, 0.53 mmol
  • HATU 217 mg, 0.57 mmol
  • DIEA 170 mg, 1.32 mmol
  • benzyl 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (236 mg, 0.44 mmol) in DMF (5 mL) while stirring.
  • the reaction mixture was then stirred at room temperature for 2 h. Water (50 mL) was added to the reaction mixture and the mixture was extracted with EA (20 mL ⁇ 3).
  • Step 6 Preparation of N-(2-((1S,4S)-4-hydroxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 7 Preparation of N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 1 Preparation of tert-butyl (4-hydroxy-4-(methylamino)methyl)cyclohexyl)carbamate
  • Step 2 Preparation of benzyl ((4-((tert-butyloxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate
  • Step 3 Preparation of benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate
  • Step 4 Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate
  • Step 5 Preparation of benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate
  • Step 6 Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate
  • 6-(Trifluoromethyl)picolinic acid (538 mg, 2.78 mmol), HATU (1.06 g, 2.78 mmol) and DIEA (828 mg, 6.42 mmol) were added to a solution of benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate (940 mg, 2.14 mmol) in DMF (10 mL) with stirring. The reaction solution was then stirred at room temperature overnight. The reaction solution was diluted with water (100 mL) and extracted with EA (30 mL ⁇ 3).
  • Step 7 Preparation of N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 8 Preparation of tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate
  • Step 9 Preparation of N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 10 Preparation of N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • reaction solution was diluted with water (50 mL), then extracted with DCM (20 mL ⁇ 3), and the organic phase was washed with saturated brine (60 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure.
  • Example 36 N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-methoxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 1 Preparation of benzyl 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 2 Preparation of benzyl 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 3 Preparation of benzyl 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate
  • Step 4 Preparation of N-(6-methoxy-2-((1S,4S)-4-methoxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-2H-indazol-5-yl)-6-(trifluoromethyl)picolinamide
  • Step 5 Preparation of N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-methoxycyclohexyl)-6-methoxy-2H-indazol-5-yl)-6-(trifluoromethyl)picolinamide
  • Test Example 1 IRAK4 kinase activity test
  • KinEASE-STK S1 serine/threonine kinase kit (Cisbio) was used to detect the inhibitory effect of the compound on IRAK4 kinase activity.
  • the specific method was as follows: the compound was dissolved in dimethyl sulfoxide, and then diluted in an isocratic manner with the buffer of the kit so that the final concentration of the test compound in the reaction system ranged from 10000nM to 0.038nM. Then, 2.5nM IRAK4 kinase (Carna), 1 ⁇ M biotinylated peptide substrate (Cisbio) and 7 ⁇ M adenosine triphosphate (Sigma-Aldrich) were added in sequence and incubated at 37°C for 120min.
  • the inhibition rate of the compound at each concentration was calculated by comparing the fluorescence intensity ratio with the control group, and then the nonlinear curve fitting of the logarithmic concentration-inhibition rate was performed by GraphPad Prism 7 to obtain the IC 50 value of the compound.
  • the compounds of the present invention can effectively bind to the target protein and inhibit the kinase activity of IRAK4.
  • Test Example 2 Degradation of IRAK4 in THP-1 cells by compounds
  • THP-1 cells (Stem Cell Bank of the Chinese Academy of Sciences) were inoculated into each well of a 24-well cell culture plate, with a cell density of 5 ⁇ 10 5 cells/well; the cell plate was placed in a 5% carbon dioxide incubator and cultured overnight at 37°C, and then 50 ⁇ L of compound dimethyl sulfoxide solution was added, with the final concentration of the compound ranging from 0.03 to 3000 nM. After further culture for 24 hours, the cells were collected into a 1.5 mL centrifuge tube and centrifuged at 1000 rpm and 4°C for 5 minutes.
  • cell pellet was washed twice with 1 ⁇ DPBS, and the resuspended cells were lysed with 200 ⁇ L lysis buffer (cell lysis buffer was Western and IP cell lysis buffer (Biyuntian), supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Biyuntian)), and then placed on ice for 30 minutes and centrifuged at 14000g and 4°C for 10 minutes. The supernatant was taken and the IRAK4 protein level was detected by Western Blot.
  • cell lysis buffer was Western and IP cell lysis buffer (Biyuntian)
  • 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail Biyuntian
  • the above cell lysate was also used to measure the IRAK4 degradation effect using the human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C).
  • the specific method was to take 2 ⁇ L of the cell lysate supernatant sample to be tested and add it to a white 384-well plate (PerkinElmer, 6007299), and then add 4 ⁇ L of 5 ⁇ anti-IRAK4 receptor beads (PerkinElmer, final concentration of 10 ⁇ g/mL), and incubate at 23°C for 30 minutes. Then, 4 ⁇ L of 5 ⁇ biotin-labeled anti-IRAK4 antibody (PerkinElmer, final concentration of 1 nM) was added and incubated at 23°C for 60 minutes.
  • the compounds of the present invention can effectively degrade IRAK4 kinase protein in cells and have excellent degradation activity.
  • Test Example 3 Determination of cytokine concentrations induced by LPS in normal human whole blood and PBMC secretion
  • the cell plate was affixed with a sealing film and placed in a 5% carbon dioxide incubator at 37°C for 5 hours. After centrifugation at 2000 rpm for 10 minutes, 30 ⁇ L of supernatant plasma was taken from each well and transferred to a new 96-well transparent cell plate for cytokine concentration testing and frozen at -80°C for testing.
  • the interleukin-6 (IL-6) AlphaLISA detection kit (PerkinElmer, AL223C) was used to measure the IL-6 concentration in the supernatant plasma samples of LPS-induced human whole blood (blood collected from healthy people aged 22-50 years).
  • IL-6 standard solution Prepare different concentrations of IL-6 standard solution according to the product instructions, with a concentration range of 0-100000 pg/mL. Take 2 ⁇ L of each concentration of IL-6 standard solution and the cell supernatant sample to be tested and add them to a white 384-well plate (PerkinElmer, 6007299), then add 8 ⁇ L of 5 ⁇ anti-IL-6 receptor microbeads (PerminElmer, final concentration of 10 ⁇ g/mL) and biotin-labeled anti-IL-6 antibody (PerminElmer, final concentration of 1 nM) mixed solution to each well, and incubate at 23°C for 60 minutes.
  • PerkinElmer 6007299
  • PBMC PB010C
  • RPMI 1640 medium Gibco, A1049101
  • fetal bovine serum Gibco, 10099141
  • penicillin 100U/mL
  • streptomycin 100 ⁇ g/mL streptomycin
  • the cell plate was again placed in a 5% CO 2 incubator at 37°C for 5 hours, centrifuged at 2000 rpm for 10 minutes, and 100 ⁇ L of cell supernatant was transferred from each well to a new 96-well transparent cell plate for cytokine concentration testing and frozen at -80°C for testing.
  • the interleukin-6 (IL-6) AlphaLISA detection kit (PerkinElmer, AL223C) was used to measure the IL-6 concentration in the supernatant of LPS-induced human PBMC cells.
  • IL-6 standard solution Prepare different concentrations of IL-6 standard solution according to the product instructions, with a concentration range of 0-100000 pg/mL. Take 2 ⁇ L of each concentration of IL-6 standard solution and the cell supernatant sample to be tested and add them to a white 384-well plate (PerkinElmer, 6007299), then add 8 ⁇ L of 5 ⁇ anti-IL-6 receptor microbeads (final concentration of 10 ⁇ g/mL) and biotin-labeled anti-IL-6 antibody (final concentration of 1 nM) mixed solution to each well, and incubate at 23°C for 60 minutes.
  • Cryopreserved normal human PBMC (Miaoshun, PB010C) were resuspended in RPMI 1640 medium (Gibco, A1049101), supplemented with 10% fetal bovine serum (Gibco, 10099141), 100U/mL penicillin and 100 ⁇ g/mL streptomycin (Gibco, 15140122). 0.95mL of cells were plated in each well of a 24-well cell culture plate, and then 50 ⁇ L of the diluted compound solution was added to the corresponding wells with cells, so that the final concentration of the compound was in the range of 0.01-3000nM. The final concentration of DMSO was 0.25%.
  • the cell plate was placed in a 5% CO2 incubator at 37°C for 24 hours, and the cells were collected into a 1.5mL centrifuge tube and centrifuged at 1000rpm and 4°C for 5 minutes.
  • the cell pellet was washed twice with 1 ⁇ DPBS, and the resuspended cells were lysed with 100 ⁇ L lysis buffer, placed on ice for 30 minutes, and then centrifuged at 14000g and 4°C for 10 minutes.
  • the cell lysis supernatant samples after centrifugation were frozen at -80°C for testing.
  • the cell lysis buffer was Western and IP cell lysis buffer (Biyuntian, P0013), supplemented with 1mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Biyuntian, P1008).
  • the total protein concentration in the cell lysate samples was determined using the BCA protein quantification kit (Tiangen, PA115-02).
  • the prepared PBMC lysate samples were used to measure the IRAK4 concentration using the human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C) to determine the degradation effect of the compound on IRAK4.
  • the method is as follows:
  • the AlphaLISA signal value was measured on the microplate reader EnVision (PerkinElmer, 2105) in the AlphaScreen standard setting mode.
  • the standard curve was drawn by the AlphaLISA signal value of each concentration of IRAK4 standard solution.
  • the total protein concentration of each sample obtained by BCA quantification was used for corresponding correction to determine the IRAK4 concentration of each cell lysate supernatant.
  • the IRAK4 concentration of the control group was compared to calculate the degradation rate of the compound at each concentration, and then the DC 50 and D max of the compound were obtained by nonlinear curve fitting using the logarithmic concentration-inhibition rate using GraphPad Prism 8. value.
  • Fresh whole blood was collected from normal volunteers or patients (rheumatoid arthritis, dermatomyositis) and anticoagulated with sodium heparin. 0.95 mL of whole blood was placed in each well of a 24-well cell culture plate, and 50 ⁇ L of the diluted compound solution was added to the corresponding wells with whole blood, so that the final concentration of the compound was in the range of 10 to 10,000 nM. The final concentration of DMSO was 0.1%. After the cell plate was placed in a 5% CO 2 incubator and cultured at 37°C for 24 hours after administration, the whole blood samples in each well were collected, and PBMCs were separated from the whole blood using FICOLL separation solution (GE, 17-1440-02) according to the steps in the product manual.
  • FICOLL separation solution GE, 17-1440-02
  • the separated cells were washed once with 1 ⁇ DPBS, and then the red blood cells were lysed with red blood cell lysis solution (Nanjing Senbeijia, BL-O51-100ml) for 5 minutes, and the cells were washed once with 1 ⁇ DPBS. Finally, the white precipitated cells collected after centrifugation at 3000 rpm for 5 minutes were PBMCs.
  • red blood cell lysis solution Najing Senbeijia, BL-O51-100ml
  • the cell lysis buffer was Western and IP cell lysis buffer (Biyuntian, P0013), supplemented with 1mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Biyuntian, P1008). After centrifugation, the cell lysis supernatant protein sample was frozen at -80°C for testing.
  • the total protein concentration in the whole blood cell lysate samples prepared from normal subjects or patients was determined using the BCA protein quantification kit (Tian Gen, PA115-02).
  • the whole blood cell lysate samples of normal people or patients were prepared and the IRAK4 concentration was measured using the human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C) to determine the degradation effect of the compound on IRAK4.
  • the method is as follows:
  • the AlphaLISA signal value was measured on the microplate reader EnVision (PerkinElmer, 2105) in the AlphaScreen standard setting mode.
  • the standard curve was drawn by the AlphaLISA signal value of each concentration of IRAK4 standard solution.
  • the total protein concentration of each sample obtained by BCA quantification was used for corresponding correction to determine the IRAK4 concentration of each cell lysate supernatant.
  • the IRAK4 concentration of the control group was compared to calculate the degradation rate of the compound at each concentration, and then the DC 50 and D max of the compound were obtained by nonlinear curve fitting using the logarithmic concentration-inhibition rate using GraphPad Prism 8. value.
  • FIG. 1A and FIG. 1B show that compound A of the present invention has a good degradation effect on IRAK4 of normal human peripheral blood mononuclear cells, dermatomyositis patients, and rheumatoid arthritis patients.
  • Test Example 5 Determination of IRAK4 protein expression levels in PBMCs of various patients
  • This method is suitable for isolating PBMC from whole blood of patients with osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, etc., as well as whole blood of normal people.
  • the cell lysis buffer was Western and IP cell lysis buffer (Biyuntian, P0013), supplemented with 1mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Biyuntian, P1008). After centrifugation, the cell lysis supernatant protein sample was frozen at -80°C for testing.
  • the total protein concentration in the cell protein samples was determined using the BCA protein quantification kit (Tiangen, PA115-02).
  • the prepared cell protein samples were used to measure the expression level of IRAK4 protein in PBMC of various patients or normal people using the human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C).
  • the method is as follows:
  • FIG. 2 shows that the intracellular IRAK4 protein in PBMCs of patients with rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus and systemic sclerosis is highly expressed.
  • Test Example 6 Inhibitory effect of compounds on LPS-induced IL-6 in normal human PMBC
  • PBMC normal human PBMC
  • RPMI 1640 medium Gibco, A1049101
  • fetal bovine serum Gibco, 10099141
  • penicillin 100U/mL
  • streptomycin 100 ⁇ g/mL
  • 150 ⁇ L of PBMC was plated in each well of a 96-well transparent cell plate (Labserv, 310109008) to a cell density of 2 ⁇ 105 cells/well. Then 50 ⁇ L of the diluted compound solution was added to the corresponding wells with cells to make the final concentration of the compound in the range of 0.026-10000nM.
  • the final concentration of DMSO was 0.25%.
  • 10 ⁇ L of LPS (0111:B4) (Sigma, L4391) was added.
  • the cell plate was again placed in a 5% CO 2 incubator and cultured at 37°C for 5 hours, then centrifuged at 2000 rpm for 10 minutes, and 100 ⁇ L of cell supernatant was transferred from each well to a new 96-well transparent cell plate for cytokine concentration testing and frozen at -80°C for testing.
  • the IL-6 concentration in the supernatant of normal human PBMC cells was determined using the human IL-6 Alpha LISA detection kit (PerkinElmer, AL223C). The method is as follows:
  • IL-6 standard solution Prepare different concentrations of IL-6 standard solution according to the product instructions, with a concentration range of 0-100000 pg/mL. Take 2 ⁇ L of each concentration of IL-6 standard solution and the cell supernatant sample to be tested and add them to a white 384-well plate (PerkinElmer, 6007299), then add 8 ⁇ L of 5 ⁇ anti-IL-6 receptor microbeads (final concentration of 10 ⁇ g/mL) and biotin-labeled anti-IL-6 antibody (final concentration of 1 nM) mixed solution to each well, and incubate at 23°C for 60 minutes.
  • FIG3 illustrates that compound A of the present invention has a good inhibitory effect on IL-6 in human peripheral blood mononuclear cells stimulated by lipopolysaccharide.
  • Test Example 7 Human PBMC cytokine test
  • PBMC peripheral blood mononuclear cells
  • Lipopolysaccharide (0111:B4) (Sigma-Aldrich, L4391)
  • Vacuum blood collection tube lithium heparin anticoagulation (BD, 367880)
  • PBMCs After the cryopreserved normal human PBMCs are revived, they are resuspended in RPMI 1640 medium (supplemented with 10% fetal bovine blood, 100U/mL penicillin and 100 ⁇ g/mL streptomycin). PBMCs are plated in a 96-well transparent cell plate to a cell density of 2 ⁇ 10 5 cells/well. Then, the test compound diluted in the culture medium is added to the compound wells to make the final concentration of the compound 500nM (containing 0.25% DMSO). At the same time, culture medium containing 0.25% DMSO is added to the positive and negative wells, respectively.
  • RPMI 1640 medium supplied with 10% fetal bovine blood, 100U/mL penicillin and 100 ⁇ g/mL streptomycin.
  • PBMCs are plated in a 96-well transparent cell plate to a cell density of 2 ⁇ 10 5 cells/well. Then, the test compound diluted in the culture medium is added to the compound well
  • the cell plate After the sample is added, the cell plate is placed in a 5% CO 2 incubator and incubated at 37°C for 20 hours, and LPS or R848 is added to the compound wells and the positive control wells. After the sample is added, the cell plate is again placed in a 5% CO 2 incubator and incubated at 37°C for 5 hours. Afterwards, centrifuge at 2000 rpm for 10 minutes. Finally, take the cell supernatant from each well and freeze it at -80°C for later use.
  • Figures 4 and 5 show that at a concentration of 500 nM, the degradation agent compound A of the present invention generally has an inhibitory effect on the cytokine secretion of human peripheral blood mononuclear cells stimulated by lipopolysaccharide or reximod, and is superior to the inhibitor compounds of the same concentration (control I and control II).
  • Test Example 8 Inhibitory effect of compounds on whole blood IL-6 in normal subjects or patients
  • Fresh whole blood was collected from normal volunteers or patients (rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis) and anticoagulated with sodium heparin. 190 ⁇ L of whole blood was spread in a 96-well clear cell plate (Labserv, 310109008), and 10 ⁇ L of diluted compound solution was added to the corresponding wells with whole blood, so that the final concentration of the compound was in the range of 10-10000 nM. The final concentration of DMSO was 0.25%. After the drug-treated cell plate was placed in a 5% CO 2 incubator at 37°C for 20 hours, 10 ⁇ L of LPS was added.
  • the cell plate was placed in a 5% CO 2 incubator again at 37°C for 5 hours, centrifuged at 2000 rpm for 10 minutes, and 50 ⁇ L of supernatant was transferred from each well to a new 96-well clear cell plate for cytokine concentration testing, and frozen at -80°C for testing.
  • the IL-6 concentration in the whole blood supernatant of normal subjects or patients was determined using the human IL-6 AlphaLISA detection kit (PerkinElmer, AL223C). The method is as follows:
  • IL-6 standard solution Prepare different concentrations of IL-6 standard solution according to the product instructions, with a concentration range of 0-100000 pg/mL. Take 2 ⁇ L of each concentration of IL-6 standard solution and the cell supernatant sample to be tested and add them to a white 384-well plate (PerkinElmer, 6007299), then add 8 ⁇ L of 5 ⁇ anti-IL-6 receptor microbeads (final concentration of 10 ⁇ g/mL) and biotin-labeled anti-IL-6 antibody (final concentration of 1 nM) mixed solution to each well, and incubate at 23°C for 60 minutes.
  • the inhibition rate of the compound at each concentration was calculated by comparing it with the IL-6 concentration of the control group, and then the nonlinear curve fitting of the logarithmic concentration-inhibition rate was performed by GraphPad Prism 7 to calculate the IC 50 value of the compound.
  • Figures 6A, 6B, 6C and 6D show that compound A of the present invention has a good inhibitory effect on IL-6 in the whole blood of normal people induced by lipopolysaccharide, IL-6 in the whole blood of dermatomyositis patients, IL-6 in the whole blood of rheumatoid arthritis patients and IL-6 in the whole blood of systemic lupus erythematosus patients.
  • Test Example 9 Effect of Compound A on Tissue IRAK4 Protein Expression after Multiple Oral Administration in Male BALB/C Mice
  • a certain amount of compound A was weighed, and 5% DMSO was first added to dissolve the compound into a clear solution, and then 15% Solutol was added and shaken to mix, and finally 80% physiological saline was added and mixed.
  • the dose of compound A was set to 30 mpk, 60 mpk, and 90 mpk; oral administration, twice a day; the vehicle was 5% DMSO + 15% Solutol + 80% saline, and the grouping information is shown in Table 5:
  • the animals were grouped according to their body weight, and the grouping information is shown in Table 5. Then, the drugs were administered. The drugs were administered intragastrically multiple times, and the animals were euthanized by CO 2 3 days later. After CO 2 euthanasia , blood was collected from the heart, anticoagulated with heparin, and PBMCs were isolated and prepared to detect the degradation of IRAK4; spleen and skin tissues were also obtained to detect the protein level of IRAK4.
  • Protein levels of IRAK4 in PBMC, spleen, and skin Protein levels of IRAK4 in PBMC, spleen, and skin.
  • Figures 7A, 7B and 7C show that IARK4 in animal PBMC, spleen and skin was well degraded after oral administration of compound A. As the administration time increased, the degradation rate of IRAK4 in PBMC and spleen tissues also increased, reaching more than 80%.
  • Test Example 10 Pharmacodynamic effect of compound A on imiquimod-induced C57 mouse psoriasis model
  • VecticalTM (calcitriol) Ointment, Galderma, LOT: 321449
  • mice Female C57BL/6 mice (weight 19-21 g), 9 weeks old, 10 mice per group.
  • Imiquimod cream weigh 62.5 mg of imiquimod cream for each mouse and apply it directly to the back, and weigh 20 mg of imiquimod cream and apply it directly to the right ear;
  • Calcitriol ointment 75 mg of calcitriol ointment was weighed for each mouse and applied directly to the back, and 24 mg of calcitriol ointment was weighed for each mouse and applied directly to the right ear;
  • test compound Dex was formulated into corresponding concentrations for oral administration
  • 5mg/kg Take 5 Dex tablets, add 7.5ml normal saline, vortex and sonicate until evenly suspended, and store at 4°C for later use. Before administration, be sure to vortex and sonicate until evenly suspended, and prepare once every three days;
  • Test compound A was formulated into corresponding concentrations for oral administration.
  • Vehicle 1% DMSO + 10% Solutol + 89% (5% Dextrose), prepared and used immediately before each administration.
  • test compound A Prepare in EP tubes. Weigh compound A, add 1% of the total volume of DMSO, vortex and sonicate until completely dissolved, then add 10% of the total volume of Solutol, vortex and sonicate until clear, and add 89% of the total volume of 5% glucose solution step by step, while vortexing, and keep stirring with a magnetic stirrer before administration. The compound was prepared and used immediately each time.
  • Day-1 Shave the back of the mice and use a mold to ensure a uniform area of about 2cm*3cm. Select 60 mice;
  • mice were randomly divided into 6 groups according to body weight and right ear thickness, with 10 mice in each group;
  • imiquimod was applied on the back skin and right ears of mice in groups G2 to G6 at noon starting from Day 0 for sensitization and continued for 7 days.
  • mice iv. Starting from Day 0, the back skin of mice was clinically scored before administration every morning, and the ear thickness was measured every other day. Compound A was administered at 9:00 a.m. and 17:00 p.m.; imiquimod was applied at 13:30 p.m. for sensitization. The detailed grouping and dosing schedule is shown in Table 6.
  • This experiment evaluated the effect of the compound on improving clinical scores in the imiquimod-induced mouse psoriasis model.
  • the average clinical score of the solvent control group gradually increased, reaching 8.00 points on the 5th day, indicating that the imiquimod-induced mouse psoriasis model was successfully established.
  • Compound A at both 10 mg/kg and 100 mg/kg doses had an inhibitory effect on the clinical score of psoriasis in mice in a dose-dependent manner.
  • Compound A showed significant difference compared with the solvent control group at both 10 mg/kg and 100 mg/kg doses starting from Day 3 and lasted until the end of the experiment (Day 5).
  • Figure 8C shows that on day 5, compound A at doses of 10 mg/kg and 100 mg/kg had an inhibitory effect on the ear thickness difference ⁇ ear thickness (ear thickness value on the day of administration - ear thickness value before group administration), and compound A at 100 mg/kg had a significant statistical difference in the inhibitory effect on the ear thickness difference ⁇ ear thickness (p ⁇ 0.0001).
  • the compound of the present invention can be used as an IRAK4 degrader to treat rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus, systemic sclerosis and/or psoriasis.

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Abstract

涉及式Ⅰ、式Ⅱ化合物的用途，具体涉及化合物A1-A52、B1-B5 或C1-C36 在制备用于治疗和/或预防炎症性或免疫性疾病药物中的用途；涉及化合物A1-A52、B1-B5 或C1-C36 在制备治疗和/或预防类风湿性关节炎、特应性皮炎、化脓性汗腺炎、白癜风、皮肌炎、斑秃、荨麻疹、多发性肌炎、间质性肺疾病、系统性红斑狼疮、系统性硬化症、银屑病等疾病药物的用途。

Description

IRAK4降解剂及其用途
技术领域:本发明涉及通过泛素化和/或降解调节一种或多种白细胞介素-1受体相关激酶4(IRAK4)的化合物，并将其用于免疫性和/或炎症性疾病的药物的用途。
背景技术：白介素-1受体激酶4(IRAK4)是一种丝氨酸/苏氨酸特异性蛋白激酶，具有生物学上重要的激酶活性，在激活免疫系统中起重要作用。研究表明，IRAK4是白细胞介素(IL)-1β家族受体(包括IL-1R、IL-18R、IL-33R、IL-36R)和Toll样受体(TLR)信号传导通路下游的关键因子,IRAK4缺失的小鼠和IRAK4缺失的患者均对TLR(TLR3除外)和IL-1β家族的刺激不起反应(Suzuki,Suzuki等人，Nature,2002；Davidson,Currie等人，The Journal of Immunology,2006；Kuvon Bernuth等人，JEM,2007；Kim,Staschke等人，JEM,2007)。
根据有无MyD88的参与，TLR/IL-1β介导的信号通路可分为MyD88依赖型信号通路和MyD88非依赖性通路，其中IL-1R和TLR2、TLR4、TLR7/8、TLR9所介导的信号转导通路都依赖MyD88作为调节因子激活下游的炎症信号通路。TLR/IL-1β与配体结合后，募集MyD88分子，MyD88通过其N末端死亡结构域进一步募集IRAK4到TLR/IL-1β复合物中，并与IRAK1或IRAK2相互作用且将其激活(Kollewe,Mackensen等人，Journal of Biological Chemistry,2004；Precious等人，J.Biol.Chem.,2009)，从而向下游传递信号至E3泛素连接酶TNF受体相关因子(TRAF6)，活化丝氨酸/苏氨酸激酶TAK1，进而激活NF-κB及MAPK信号通路(Wang,Deng等人，Nature,2001)，引起多种炎症细胞因子和抗凋亡分子的释放。IRAK4依赖性的TLR/IL-1β信号通路已经被证明与多种疾病相关：如多发性硬化、动脉粥样硬化、心肌梗死、心肌炎(Valaperti,Nishii等人，Circulation,2013)伏格特-小柳-原田综合征(Vogt-Koyanagi-Harada syndrome)、系统性红斑狼疮(SLE)、肥胖(Ahmad,R.,P.Shihab等人，Diabetology&Metabolic Syndrome,2015)、1型糖尿病、类风湿性关节炎、脊椎关节炎(特别是牛皮癣性脊椎关节炎和别克捷列夫氏病(Bekhterev's disease))、红斑狼疮、银屑病、白癜风、巨细胞动脉炎、慢性炎性肠道疾病和病毒性疾病，例如HIV(人免疫缺陷病毒)、肝炎病毒(Staschke等人，The Journal of Immunology,2009；Marquez等人，Ann Rheum Dis,2014；Zambrano-Zaragoza等人，International Journal of Inflammation,2014；Wang等人，Experimental and Therapeutic Medicine,2015；Ciccia等人，Rheumatology,2015)；皮肤病如银屑病、特应性皮炎、金德勒综合征(Kindler's syndrome)、大疱性类天疱疮、变应性接触性皮炎、斑秃、反常性痤疮(acneinversa)和寻常痤疮；其他炎症性疾病如过敏症、贝切特氏病、痛风、成人斯蒂尔氏病(adult-onset Still's disease)、心包炎和慢性炎性肠道疾病例如溃疡性结肠炎和克罗恩氏病(Crohn's disease)、移植排斥反应和移植物抗宿主反应；妇科疾病例如子宫内膜异位症(adenomyosis)、痛经、交媾困难和子宫内膜组织异位症(endometriosis)，特别是与子宫内膜异位症相关的疼痛和其他与子宫内膜异位症相关的症状例如痛经、交媾困难、排尿困难和大便困难(Akoum,Lawson等人，Human Reproduction,2007；Allhorn,Boing等人，Reproductive Biology and Endocrinology,2008；Lawson,Bourcier等人，Journal of Reproductive Immunology,2008；Sikora, Mielczarek-Palacz等人，American Journal of Reproductive Immunology,2012；Khan,Kitajima等人，Journal of Obstetrics and Gynaecology Research,2013；Santulli,Borghese等人，Human Reproduction,2013)；眼部疾病如视网膜缺血、角膜炎、过敏性结膜炎、干燥性角结膜炎、黄斑变性和眼色素层炎(Kaarniranta和Salminen,J Mol Med(Berl),2009；Sun和Pearlman,Investigative Ophthalmology&Visual Science,2009；Redfern和McDermott,Experimental EyeResearch,2010；Kezic,Taylor等人，J Leukoc Biol,2011；Chang,McCluskey等人，Clinical&Experimental Ophthalmology,2012；Guo,Gao等人，Immunol Cell Biol,2012；Lee,Hattori等人，Investigative Ophthalmology&Visual Science,2012；Qi,Zhao等人，Investigative Ophthalmology&Visual Science,2014)；纤维化疾病如肝纤维化、心肌炎、原发性胆汁性肝硬化、囊性纤维化(Zhao,Zhao等人，Scand J Gastroenterol,2011；Benias,Gopal等人，Clin Res Hepatol Gastroenterol,2012；Yang,L.和E.Seki,Front Physiol,2012；Liu,Hu等人，Biochim Biophys Acta.,2015)；慢性肝脏疾病，例如脂肪肝肝炎，特别是非酒精性脂肪性肝病(NAFLD)和/或非酒精性脂肪性肝炎(NASH)、酒精性脂肪性肝炎(ASH)(Nozaki,Saibara等人，Alcohol Clin Exp Res,2004；Csak,T.,A.Velayudham等人，Am J Physiol Gastrointest Liver Physiol,2011；Miura,Kodama等人，Gastroenterology,2010；Kamari,Shaish等人，J Hepatol,2011；Ye,Li等人，Gut,2012；Roh,Seki,J Gastroenterol Hepatol,2013；Ceccarelli,S.,V.Nobili等人，World J Gastroenterol,2014；Miura,Ohnishi,World J Gastroenterol,2014；Stojsavljevic,Palcic等人，World J Gastroenterol,2014)；心血管疾病和神经障碍，例如心肌再灌注损伤、心肌梗死、高血压(Oyama,Blais等人，Circulation,2004；Timmers,Sluijter等人，Circulation Research,2008；Fang和Hu,MedSci Monit,2011；Bijani,International Reviews of Immunology,2012；Bomfim,DosSantos等人，Clin Sci(Lond),2012；Christia和Frangogiannis,European Journal of Clinical Investigation,2013；Thompson和Webb,Clin Sci(Lond),2013；Hernanz,Martínez-Revelles等人，British Journal of Pharmacology,2015；Frangogiannis,Curr Opin Cardiol,2015；Bomfim,Echem等人，Life Sciences,2015),以及阿尔茨海默氏病、中风、颅脑创伤、肌萎缩性侧索硬化(ALS)和帕金森氏症(Brough,Tyrrell等人，Trends in Pharmacological Sciences,2011；Carty和Bowie,Biochemical Pharmacology,2011；Denes,Kitazawa,Cheng等人，The Journal of Immunology,2011；Lim,Kou等人，TheAmerican Journal of Pathology,2011；Béraud和Maguire-Zeiss,Parkinsonism&Related Disorders,2012；Denes,Wilkinson等人，Disease Models&Mechanisms,2013；Noelker,Morel等人，Sci.Rep.,2013；Wang,Wang等人，Stroke,2013；Xiang,Chao等人，Rev Neurosci,2015；Lee,Lee等人，J Neuroinflammation,2015)；瘙痒和疼痛(包括急性、慢性、炎性和神经性疼痛)如痛觉过敏、异常性疼痛、经前痛、与子宫内膜异位症相关的疼痛、手术后疼痛、间质性膀胱炎、CRPS(复杂性局部疼痛综合征)、三叉神经痛、前列腺炎、脊髓损伤引起的疼痛、炎症引发的疼痛、下腰痛、癌痛、化疗相关的疼痛、HIV治疗诱发的神经病、烧伤引起的疼痛和慢性疼痛(Wolf,Livshits等人，Brain,Behavior and Immunity,2008；Kim,Lee等人，Toll-like Receptors:Roles in Infection and Neuropathology,2009；del Rey,Apkarian等人，Annals of the New York Academy of Sciences,2012；Guerrero,Cunha等人，European Journal of Pharmacology,2012；Kwok,Hutchinson等人，PLoS ONE,2012；Nicotra,Loram等人，Experimental Neurology,2012；Chopra和Cooper,J Neuroimmune Pharmacol,2013；David,Ratnayake等人，Neurobiology of Disease,2013；Han,Zhao等 人，Neuroscience,2013；Liu和Ji,Pflugers Arch.,2013；Stokes,Cheung等人，Journal of Neuroinflammation,2013；Zhao,Zhang等人，Neuroscience,2013；Liu,Zhang等人，Cell Research,2014；Park,Stokes等人，Cancer Chemother Pharmacol,2014；Van der Watt,Wilkinson等人，BMC Infect Dis,2014；Won,K.A.,M.J.Kim等人，J Pain,2014；Min,Ahmad等人，Photochem Photobiol.,2015；Schrepf,Bradley等人，Brain Behav Immun,2015；Wong,L.,J.D.Done等人，Prostate,2015)；肿瘤疾病如某些淋巴瘤：ABC-DLBCL(活化B细胞弥漫性大细胞B细胞淋巴瘤)、套细胞淋巴瘤和沃尔丹斯特伦疾病(disease)，以及慢性淋巴细胞性白血病、黑色素瘤、胰腺肿瘤和肝细胞癌(Ngo,Young等人，Nature,2011；Puente,Pinyol等人，Nature,2011；Ochi,Nguyen等人，J Exp Med,2012；Srivastava,Geng等人，Cancer Research,2012；Treon,Xu等人，New England Journal of Medicine,2012；Choi,Kim等人，Human Pathology,2013；Liang,Chen等人，Clinical Cancer Research,2013)、ras-依赖性肿瘤、乳腺癌、卵巢癌、结肠直肠癌、头颈部癌、肺癌、前列腺癌。
IRAK4介导的信号传导途径的调节主要与其激酶功能有关，但是，也有一些报道表明在某些细胞类型中，IRAK4对下游过程的信号调节与IRAK4的非激酶功能有关。Cushing等人就表示，尽管在IL-1β刺激的人皮肤成纤维细胞中IRAK4磷酸化水平降低，但IRAK4的药理抑制作用并不会导致IL-6和TNF-α的抑制。支持这些结果的是，IRAK4缺乏的成纤维细胞与野生型细胞相比，IRAK4的支架功能对于IL1信号传导很重要。同时，Chiang和他的同事也表示，IRAK4激酶活性在人类B细胞和T细胞、树突状细胞和单核细胞中不是必要的，且siRNA基因切除也显示IRAK4在这些细胞中具有支架功能作用。现已报道了多种针对IRAK4的强效的选择性抑制剂，如CA-4948、BAY-1834845、BMS-986126以及PF-06650833等，这些抑制剂都能选择性的抑制IRAK4的激酶活性，主要用于自身免疫性疾病，炎性疾病和肿瘤性疾病的预防和治疗。然而，一方面由于IRAK4具有支架蛋白和活性激酶的作用，且另一方面传统的小分子抑制剂易产生耐药性，因此，仅抑制IRAK4激酶活性可能不足以产生治疗效果。
蛋白降解靶向嵌合体(Proteolysis Targeting Chimeria,PROTAC)是一种不同于传统小分子抑制剂的技术，传统小分子抑制剂通常需要作用于靶蛋白的活性位点才能抑制其活性，而PROTAC为一种异质双功能分子，其一端为可识别靶蛋白的小分子抑制剂，通过连接链，另一端为可识别E3泛素连接酶的E3泛素连接酶配体，这种双功能分子在体内识别靶蛋白和E3泛素连接酶，将靶蛋白和E3泛素连接酶拉近，形成三元复合物，将靶蛋白泛素化后，在体内通过泛素－蛋白酶体途径将靶蛋白降解。相较于传统小分子抑制剂，一方面PROTAC只需要将靶蛋白与E3泛素连接酶拉近，使底物降解，这种作用模式使得这种技术可以应用于一些不可成药靶点；另一方面，由于靶蛋白在被降解后，PROTAC分子还可释放出来继续参与下一蛋白的降解过程，因此这种具有催化效果的降解作用，使得较少的PROTAC药物剂量就可以实现高效的降解；再一方面，传统的小分子抑制剂易产生耐药性常常是因为发生了点突变，使得小分子抑制剂失去了对靶点的抑制作用，而PROTAC可以直接将靶蛋白降解，在一定程度上能够避免点突变产生的耐药性。因此，相较于传统小分子抑制剂，运用PROTAC技术进行新药小分子研发具有很高的优势和可行性，有望成为下一代极具前途的新型药物。PROTAC技术也已被应用于多种靶点药物的改造，如雄性激素受体、雌性激素蛋白受体、BTK等等。US2019/0151295、 US2019/0192688、WO2019/160915和WO2020/113233中公开了几类靶向IRAK4的降解剂，更多的靶向IRAK4的降解剂丞待开发。
发明概述
现有技术公开了许多IRAK4抑制剂(参见：Annual Reports in Medical Chemistry(2014),49,117-133)。
WO2016/083433A1实施例公开了如下化合物(本发明对照化合物(Ⅰ))：
WO2015/150995A1实施例公开了如下化合物(本发明对照化合物(Ⅱ))：
如本文所述，本发明人发现IRAK4降解剂适用于治疗和预防以过度反应的免疫系统为特征的动物免疫性疾病，特别对以下疾病：类风湿性关节炎、特应性皮炎、化脓性汗腺炎(HS)、白癜风、皮肌炎、斑秃、荨麻疹、多发性肌炎、间质性肺疾病、系统性红斑狼疮、系统性硬化症和/或银屑病具有很好的治疗或预防作用。
发明内容
化合物A1-A52分别对应于下式：













化合物A1-A52的制备和作为药物的用途已在WO2022/088551A1公布。
化合物B1-B5分别对应于下式：

化合物B1-B5的制备和作为药物的用途已在WO2022/028547A1公布。
化合物C1-C36分别对应于下式：








化合物C1-C36的制备和作为药物的用途见本发明说明部分。
本发明提供了IRAK4，和/或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶 剂化物、前药和/或其药学上可接受的盐，其用于治疗和/或预防免疫性和/或炎症性疾病的用途。
本发明提供了IRAK4，和/或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药和/或其药学上可接受的盐，其用于治疗和/或预防由IL-1R/TLR通路中IRAK4及IRAK4相关蛋白的异常表达和/或IRAK4所介导的化学因子、细胞因子或免疫细胞异常分泌或增生引起的疾病的用途。
在本发明的一些方案中，IRAK4降解剂为式Ⅰ、式Ⅱ或式Ⅲ所示的化合物或其药学上可以接受的盐：
其中：
环A为苯基或吡啶基；
环B为C6-C10环烷基或含有1-2个选自N、O或S杂原子的6-10元杂环烷基，所述C6-C10环烷基和6-10元杂环烷基任选被选自卤素、氧代、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
环C为C6-C12环烷基或含有1-2个选自N、O或S杂原子的6-12元杂环烷基，所述C6-C12环烷基和6-12元杂环烷基任选被选自卤素、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
环D为C6-C10芳基，所述C6-C10芳基任选被卤素、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
环Y为5-6元杂芳基；
X为键、-O-、-NH-、-C(O)-、-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-；
L为-(CH2)j-，所述-(CH2)j-中的1个或多个亚甲基任选的被选自-NR3’-、-O-、-S-、-S(O)-、-S(O)2-、-S(O)2NR3’-、-CR1’R2’-、-C(O)-、-C(O)O-、-OC(O)-、-NR3’C(O)O-、-OC(O)NR3’-、-C(O)NR3’-、-NR3’C(O)-、-NR4’C(O)NR3’-、亚乙烯基或亚乙炔基的基团替代；
R1’、R2’各自独立为卤素、-OH、-NH2、C1-C4烷基、C1-C4卤代烷基、C1-C4羟基烷基、-O(C1-C4 烷基)或-NH(C1-C4烷基)；
R3’、R4’各自独立的为氢或C1-C6烷基；
Rd各自独立的为氢、氘、卤素、氰基、C1-C6烷基，所述烷基任选的被1个或多个选自卤素、羟基、氨基的基团取代；
Rc为-O(C1-C3烷基)、-N(C1-C3烷基)1-2或C1-C6烷基，所述烷基任选被1个或多个独立的选自羟基、氨基、卤素、氰基或-O-(C1-C3烷基)的基团取代；
Rb为氢或C1-C6烷基，所述烷基任选被1个或多个独立的选自羟基、氨基、卤素、氰基的基团取代；
Ra为氢、卤素、C1-C6烷基或-O-(C1-C6烷基)，所述烷基任选被卤素或羟基取代；
各个R1独立地选自：C1-C4烷基、-O(C1-C4烷基)、-N(C1-C4烷基)1-2、CN、卤素、-OH、-NH2，所述烷基任选被选自卤素、氰基、-OH、C1-C4烷基、-O(C1-C4烷基)的基团取代
各个R2独立地为氢、C1-C4烷基、-O(C1-C4烷基)、C3-C8环烷基、3-8元杂环烷基、6-10元芳基、5-6元杂芳基、CN、卤素、-OH，所述烷基、环烷基、杂环烷基、芳基、杂芳基任选被选自卤素、氰基、-OH、-NH2、C1-C4烷基和-O(C1-C4烷基)；
X’为CH或N；
m是0、1、2、3或者4；
n是0、1、2、3或者4；
p为1或者2；
j为0、1、2、3、4或者5。
在本发明的一些优选方案中，IRAK4降解剂为式Ⅰ-1、Ⅰ-2或式Ⅱ-1所示的化合物或其药学上可以接受的盐：

其中：
R3为H、卤素、C1-C6烷基或-O-(C1-C6烷基)；
Rc、Rd、环B、L、环C、X、X’、p、R2和m本发明所定义。
在本发明的一些优选方案中，所述疾病的特征在于IRAK4降解剂用于治疗和/或预防免疫性疾病的用途。
在本发明的一些优选方案中，IRAK4降解剂用于免疫性疾病，所述免疫性疾病选自：成人斯蒂尔病、斑秃、强直性脊柱炎、自身免疫性肝炎、自身免疫性心肌炎、自身免疫性胰腺炎、自身免疫性视网膜病变、自身免疫性荨麻疹、白塞病、良性黏膜类天疱疮(黏膜类天疱疮)、大疱性类天疱疮、卡斯尔曼病(CD)、乳糜泻、柯萨奇心肌炎、克罗恩病、疱疹样皮炎、特应性皮炎、皮肌炎、盘状狼疮、子宫内膜异位症、嗜酸性筋膜炎、结节性红斑、纤维化肺泡炎、原发肾小球肾炎、Goodpasture综合征、自身免疫性溶血性贫血、过敏性紫癜(HSP)、化脓性汗腺炎(HS)、IgG4相关的硬化性疾病、包涵体肌炎(IBM)、间质性膀胱炎(IC)、兰伯特-伊顿综合征、线性IgA疾病(LAD)、系统性红斑狼疮、慢性莱姆病、多发性硬化症、系统性硬化症、特发炎症性肌病(IIM)、眼瘢痕性类天疱疮视神经炎、回纹风湿病(PR)、天疱疮、自身免疫性脑脊髓炎、POEMS综合征、结节性多动脉炎、原发性胆汁性胆管炎、原发性硬化性胆管炎、银屑病、银屑病关节炎、反应性关节炎、复发性多软骨炎、腹膜后纤维化、风湿热、类风湿关节炎、结节病、自身免疫性巩膜炎、硬斑病、干燥综合征、大动脉炎、颞动脉炎、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、甲状腺眼病(Ted)、Tolosa-Hunt综合征(THS)、横贯性脊髓炎、溃疡性结肠炎(UC)、未分化结缔组织病(UCTD)、葡萄膜炎、系统性血管炎、白癜风、Vogt-小柳-原田病、重症肌无力、痛风、脊柱关节病、晶型关节病、骨关节炎、类风湿性关节炎、多发性肌炎、间质性肺疾病、巨细胞动脉炎、风湿性多肌痛、肉芽肿性血管炎、嗜酸性肉芽肿性血管炎、嗜酸性粒细胞血管炎、显微镜下多血管炎、冷球蛋白血症、皮肤白细胞破碎性血管炎、混合性结缔组织病、史蒂芬-约翰逊综合症、肺动脉高压、心内膜炎、动脉粥样硬化、多形性红斑、急性冠脉综合征、特发性肺纤维化、非酒精性脂肪肝、肾纤维化、1型糖尿病、初级干燥、川崎病、脓疱型银屑病、慢性肉芽肿病、视神经脊髓炎荨麻疹、掌跖脓疱病、败血症、大疱性皮肤病、阿茨海默病、慢性自发性荨麻疹、慢性牛皮癣、幼年特受性关节炎、中轴性脊柱关节炎或格雷夫斯病。
在本发明的一些优选方案中，IRAK4降解剂用于免疫性疾病，所述免疫性疾病选自：类风湿性关节炎、特应性皮炎、化脓性汗腺炎(HS)、白癜风、皮肌炎、斑秃、荨麻疹、多发性肌炎、间质性肺疾病、系统性红斑狼疮、系统性硬化症和/或银屑病。
在本发明的一些优选方案中，所述疾病的特征在于IRAK4降解剂用于治疗和/或预防炎症性疾病 的用途。
在本发明的一些优选方案中，IRAK4降解剂用于免疫性疾病，所述炎症性疾病选自：家族性地中海热、肿瘤坏死因子相关周期性综合征、甲羟戊酸激酶缺乏症、吡啶相关自身炎症伴中性粒细胞性皮肤病(PAAND)、化脓性无菌关节炎、坏疽性脓皮病和痤疮(PAPA)、家族性感冒自身炎症综合征(FCAS)、家族性慢性地衣样角化病(FKLC)、NLRP1相关自身炎症伴关节炎和角化不良(NAIAD)、IL-1受体拮抗剂(DIRA)缺乏症、IL-36受体拮抗剂(DITRA)缺乏症、过敏性接触性皮炎、CAR-T细胞诱导的细胞因子释放综合征、克罗恩氏病、慢性支气管炎、COPD、活动性强直性脊柱炎、中轴型脊柱关节、毛发红糠疹、炎性肠病、脊柱关节炎、急性肺损伤、泛发性脓疱型银屑病、寻常痤疮或结肠炎。
在本发明的一些优选方案中，IRAK4降解剂用于治疗和/或预防由IL-2R alpha﹑IL-6﹑IFA-alpha2﹑IFN-gamma﹑IL-1ra﹑MCP-3﹑IL-16﹑IL-12(p40)﹑LIF﹑IL-5﹑GM-CSF﹑TNF-alpha﹑IL-2﹑IL-1alpha、IL-1beta﹑IL-18﹑Eotaxin﹑Basic FGF﹑beta-NGF﹑PDGF-BB﹑IL-4﹑MCP-1﹑IL-8﹑IL-10﹑GRO-alpha﹑HGF﹑IL-1alpha、IL-1beta﹑﹑IL-3﹑SCF﹑TRAIL﹑M-CSF﹑CTACK﹑IL-15﹑IL-12(P70)、IL-17、IL-23、IL-33和/或IL-36细胞因子所介导的疾病的用途。
在本发明的一些优选方案中，IRAK4降解剂用于治疗和/或预防由IL-4、IL-6﹑IL-12(p40)﹑GM-CSF﹑TNF-alpha﹑IL-2﹑IL-1alpha、IL-1beta、IL-18﹑IL-8﹑IL-10﹑IL-17、IL-23、IL-33和/或IL-36细胞因子所介导的疾病中的用途。
在本发明的一些优选方案中，所述样品是脾脏、皮肤和/或血液样品。
在本发明的一些优选方案中，所述血液样品是正常人全血和/或病人全血。
在本发明的一些优选方案中，所述疾病的方法包括将有效量的IRAK4降解剂施用于所述对象。
在本发明的一些优选方案中，所述疾病的治疗方法，包括IRAK4降解剂为所述的单一的化合物或与其它药物的联用。
本发明通过IRAK4激酶活性测试实验证明本发明所述A1-A52、B1-B5或C1-C36化合物能够有效的与IRAK4靶蛋白结合产生降解和/或抑制效果，通过Western-Blot证明本发明所述A1-A52、B1-B5或C1-C36化合物能够有效的特异性的降解THP-1细胞中的IRAK4蛋白。本发明所述A1-A52、B1-B5或C1-C36化合物，和/其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、代谢物、前药和/或其药学上可接受的盐可有效的降解IRAK4蛋白，从而达到预防或治疗与IRAK4相关的疾病或病症的效果。
本发明通过实验证明，本文所述的化合物能够明显减少全血、皮肤或肝脏中IRAK4的表达，表明IRAK4降解剂可用于免疫性疾病的治疗。
本发明中，所述药物包含IRAK4降解剂，以其为活性成分；本发明所述药物还可任选的包含药学上可接受的载体﹑稀释剂或赋形剂。
本发明中，所述药物IRAK4降解剂可以单独使用，也可以与其它药物联用以实现对炎性或免疫性疾病更好的治疗效果。
本发明中，所述药物的剂型可为片剂、丸剂、胶囊剂、散剂、颗粒剂、乳液剂、混浮剂、分散液、 溶液剂、糖浆剂、酏剂、软膏剂、滴剂、栓剂、吸入剂、喷射剂。IRAK4降解剂为活性成分的药物可以根据实际需要被制备成上述任一种药物剂型，各剂型的药物均可以按照药学领域的常规方法制备。
本发明中，所述药物的给药途径可以根据实际需要选择口服给药、舌下给药、静脉注射、腹腔注射、肌肉注射、皮下注射、鼻腔给药、经皮给药、肠胃外给药、吸入给药、气管内给药、肺内给药、支气管给药的任意一种方式。
本发明还提供A1-A52、B1-B5或C1-C36化合物药学上可接受的盐。术语“药学上可接受的盐”是指相对无毒的本发明化合物的酸加成盐或碱加成盐。所述酸加成盐为本发明A1-A52、B1-B5或C1-C36化合物与合适的无机酸或者有机酸形成的盐，这些盐可在化合物最后的分离和提纯过程中制备，或者可用纯化的A1-A52、B1-B5或C1-C36化合物以其游离碱形式与适宜的有机酸或无机酸进行反应来制备。代表性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月硅酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为A1-A52、B1-B5或C1-C36化合物与合适的无机碱或者有机碱形成的盐，包括例如与碱金属、碱土金属、季铵阳离子形成的盐，例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基季铵盐、四乙基季铵盐等；胺盐包括与氨(NH3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。
本发明的化合物或其药学上可接受的盐可给药于哺乳动物包括人，可口服、直肠、肠胃外(静脉内、肌肉内或皮下)、局部给药(粉剂、软膏剂或滴剂)、或瘤内给药。
本发明化合物的给药剂量可以大约为0.05-300mg/kg体重/天，优选10-300mg/kg体重/天，更优选10-150mg/kg体重/天。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的固体剂型，包括但不限于胶囊剂、片剂、丸剂、散剂和颗粒剂等。在这些固体剂型中，本发明A1-A52、B1-B5或C1-C36化合物作为活性成分与至少一种常规惰性赋形剂(或载体)混合，例如与柠檬酸钠或磷酸二钙，或与下述成分混合：(1)填料或增容剂，如淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸等；(2)粘合剂，如羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶等；(3)保湿剂，如甘油等；(4)崩解剂，如琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐和碳酸钠等；(5)缓溶剂，如石蜡等；(6)吸收加速剂，如季铵化合物等；(7)润湿剂如鲸蜡醇和单硬脂酸甘油酯等；(8)吸附剂，如高岭土等；和(9)润滑剂，如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠等，或其混合物。胶囊剂、片剂和丸剂中也可包含缓冲剂。所述固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材料例如肠溶衣和其他本领域公知的材料进行包衣或微囊化。它们可包含不透明剂，并且这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时、活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的液体剂型，包括但不限于药学上可接受的乳液、溶液、悬浮液、糖浆和酊剂等。除了作为活性成分的A1-A52、B1-B5或C1-C36化合物或其药学上可接受的盐外，液体剂型可包含本领域中常规采用的惰性稀释剂如水和其他溶剂、增溶 剂和乳化剂，如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油类，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油等或这些物质的混合物等。除了这些惰性稀释剂外，本发明液体剂型也可包含常规助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料等。所述悬浮剂包括如乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇只、微晶纤维素、甲醇铝和琼脂等或这些物质的混合物。
本发明化合物或其药学上可接受的盐可以配制为用于肠胃外注射的剂型包括但不限于生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，以及用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物或其药学上可接受的盐也可以配制为用于局部给药的剂型包括如软膏剂、散剂、栓剂、滴剂、喷射剂和吸入剂等。作为活性成分的本发明A1-A52、B1-B5或C1-C36化合物或其药学上可接受的盐在无菌条件下和生理上可接受的载体及任选的防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明还提供药物组合物，它含有本发明A1-A52、B1-B5或C1-C36化合物或其药学上可接受的盐作为活性成分，以及药学上可接受载体、赋形剂或稀释剂。在制备药物组合物时，通常是将本发明A1-A52、B1-B5或C1-C36化合物或其药学上可接受的盐与药学上可接受载体、赋形剂或稀释剂混合。可以按常规制备方法将所述本发明组合物配制为常规药物制剂。例如片剂、丸剂、胶囊剂、散剂、颗粒剂、乳液剂、混浮剂、分散液、溶液剂、糖浆剂、酏剂、软膏剂、滴剂、栓剂、吸入剂、喷射剂等。
本发明所述的化合物或其药学上可接受的盐可以单独给药，或者(如果需要)与其他药学上可接受的治疗剂联合给药，如与其他抗肿瘤药物、抗炎症药物或自身免疫性药物组合。待组合的各成分可同时或顺序的给予，以单一制剂形式或以不同制剂的形式给予。所述组合不仅可包括本发明化合物和一种其他活性剂的组合，也可包括本发明化合物和两种或更多种其他活性剂的组合。
本发明所述的药学上可接受是指物质或组合物必须与制剂的其它成分相容，且对患者无害。
本发明所述的治疗包括预防性及缓解性治疗。
本发明的一些实施方案中样品是患者或者正常人的血液样品，一些实施方案中样品是患者或者正常人的血浆样品，一些实施方案中样品是患者或者正常人的外周血单个核细胞样品。
本发明的一些实施方案中,测量样品中的免疫性生物标志物的水平包括使用AlphaLISA方法。在一些实施方案中,测量样品中的免疫性生物标志物的水平包括使用选自实施例中所述的方法。
详细说明：除非有相反陈述、下列用在说明书和权利要求书中的术语具有下述含义。
“烷基”是指饱和的脂肪族烃基团，包括直链或支链烷基；C1-C8烷基是指含有1-8个碳原子的烷基，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基 戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基或其各种支链异构体；优选C1-C6烷基；更优选C1-C4烷基。所述烷基可以是取代的或未取代的。在一些实施方案中，所述烷基为C1、C2、C3、C4、C5、C6、C7或C8烷基。
“环烷基”指饱和或部分不饱和的单环或多环环状烃取代基；“C3-C11环烷基”指包括3至11个碳原子的环烷基；“C3-C8元环烷基”指包括3至8个碳原子的环烷基；“C5-C10元环烷基”指包括5至10个碳原子的环烷基；
单环环烷基的非限制性实施例包含环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选环丙基、环丁基、环戊基、环己基；优选C3-C8元环烷基；更优选C3-C6元环烷基。
多环环烷基包括螺环、稠环和桥环的环烷基。“螺环烷基”指单环之间共用一个碳原子(称螺原子)的多环基团，他们可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基基或多螺环烷基，优选7-12元双螺环烷基。螺环烷基的非限制性实施例包含：
等。
“稠环烷基”指系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选双环稠环烷基。稠环烷基的非限制性实施例包含：
等。
“桥环烷基”指任意两个环共用两个不直接连接的碳原子的全碳多环基团，他们可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，根据组成环的数目可以分为双环、三环、四环或多环桥环烷基。桥环烷基的非限制性实施例包含：
等。
所述环烷基环可以稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性实施例包括茚满基、四氢萘基、苯并环庚烷基等。所述环烷基可以是任选取代的或未取代的。
在一些实施方案中，所述环烷基为C3、C4、C5、C6、C7、C8、C9、C10、C11、C12单环或多环(例如螺环、稠环或桥环)环烷基。
“杂环烷基”指饱和的或部分不饱和的单环或多环环状烃取代基，其中一个或多个(例如2、3、4或5)环原子选自氮、氧或S(O)r(其中r是整数0、1或2)，但不包含-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。“3-11元杂环烷基”指包含3至11个环原子的环基，“5-10元杂环烷基”指包含5至10个环原子的环基，“3-8元杂环烷基”指包含3至8个环原子的环基，优选含有1-2个选自N、O或S杂原子的“3-11元杂环烷基”，更优选含有1个或2个N原子的3-11元杂环烷基。
单环杂环烷基优选为含有1-2个N杂原子的3-8元单环杂环烷基；单环杂环烷基的非限制性实施例包含吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等，优选哌啶基和哌嗪基。
多环杂环烷基包括螺环、稠环和桥环的杂环烷基。“螺杂环烷基”指单环之间共用一个原子(称螺原子)的多环杂环烷基团，其中一个或多个环原子选自氮、氧或S(O)r(其中r是整数0、1、2)，其余环原子为碳。他们可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。根据环与环之间共用螺原子的数目将螺环烷基分为单螺杂环烷基、双螺杂环烷基或多螺杂环烷基，优选含有1-2个选自N、O或S杂原子的饱和的“3-11元双螺杂环烷基”；更优选含有1个或2个N原子的饱和的“7-11元双螺杂环烷基”。螺杂环烷基的非限制性实施例包含：
等。
“稠杂环烷基”指系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环烷基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子选自氮、氧或S(O)r(其中r是整数0、1、2)，其余环原子为碳。根据组成环的数目可以分为双环、三环、四环或多环稠杂环烷基，优选含有1-3个选自N、O或S杂原子的“3-11元双环稠杂环烷基”；更优选含有1个或2个N原子的饱和的“3-11元双环稠杂环烷基”。稠杂环烷基的非限制性实施例包含：
等。
“桥杂环烷基”指任意两个环共用两个不直接连接的原子的多环杂环烷基团，他们可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子选自氮、氧或S(O)r(其 中r是整数0、1、2)，其余环原子为碳。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，桥杂环烷基的非限制性实施例包含：
等。
所述杂环烷基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环烷基，非限制性实施例包含：
所述杂环烷基可以是任选取代的或未取代的。
在一些实施方案中，所述杂环烷基为3、4、5、6、7、8、9、10、11、12元单环或多环(例如螺环、稠环或桥环)杂环烷基，其中杂原子的个数可以为1、2、3、4或5个，每个杂原子独立地为氮、氧或S(O)r(其中r是整数0、1或2)。
“芳基”指全碳单环或稠合多环(也就是共享毗邻碳原子对的环)且具有共轭的π电子体系的多环基团，“C6-C10芳基”指含有6-10个碳的全碳芳基，例如苯基和萘基；优选苯基。所述芳基环可以稠合于杂芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为芳基环，非限制性实施例包含：
所述芳基可以是任选取代的或未取代的。在一些实施方案中，所述芳基为6-10元芳基。
“杂芳基”指包含1至4个杂原子的杂芳族体系，所述杂原子包括氮、氧或S(O)r(其中r是整数0、1、2)，5-6元杂芳基指含有5-6个环原子的杂芳族体系，5-10元杂芳基指含有5-10个环原子的杂芳族体系，优选5-6元杂芳基；更优选含有1个或2个N原子的5-6元杂芳基；非限制实施例包括呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、吡唑、咪唑基、三唑基、四唑基等；优选吡啶基。所述杂芳基环可以稠合于芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，非限制性实施例包含：
所述杂芳基可以是任选取代的或未取代的。在一些实施方案中，所述杂芳基为5、6、7、8、9、10元杂芳基，其中杂原子的个数可以为1、2、3、 4或5个，每个杂原子独立地为氮、氧或S。
除非另有说明，否则本文描述的结构还意味着结构的所有异构(例如：对映体，非对映异构和几何(或构象)的形式；每个非对称中心，Z和E双键异构体和Z和E构象异构体的R和S配置。因此，单层化学异构体以及对映体，非对映异构和几何(或构象)混合物的本发明化合物在本发明的范围内。除非另有说明，否则本发明化合物的所有互变异构形式在本发明的范围内。另外，除非另有说明，否则本文描绘的结构还意味着包括仅在一个或多个同位素富集的原子存在下不同的化合物。例如，具有本结构的化合物包括通过氘或氚更换氢，或者通过a替换碳13C-或14富含C氧的碳在本发明的范围内。这些化合物可用于作为分析工具，作为生物测定中的探针或根据本发明的治疗剂。“立体异构体”包括单个化合物的所有异构体,其仅在它们的原子在空间中的取向不同。术语立体异构体包括化合物的镜像异构体(包括化合物的(R-)或(S-)构型的对映异构体),镜像异构体的混合物(对映异构体的物理混合物,以及外消旋体或外消旋混合物),几何(cis/trans或E/Z,R/S)化合物的异构体和具有多个手性中心但彼此不是镜像的化合物的异构体(非对映异构体)。化合物的手性中心可在体内发生差向异构化；因此,对于这些化合物,以(R-)形式施用化合物被认为等同于以(S-)形式施用化合物。因此,本发明的化合物可以以单独的异构体形式和基本上不含其他异构体的形式制备和使用,或者以各种异构体的混合物形式,例如立体异构体的外消旋混合物的形式制备和使用。在一些实施方案中,本发明的双官能化合物是同位素衍生物,因为它具有原子的至少一个所需同位素取代,其量高于同位素的天然丰度,即富集。在一个实施方案中,该化合物包括氘或多个氘原子。用较重的同位素(如氘)取代,即2H,可以提供由更高的代谢稳定性导致的某些治疗优势,例如,增加的体内半衰期或减少的剂量需求,因此在某些情况下可能是有利的。此外,本发明的双官能化合物包括N-氧化物、结晶形式(也称为多晶型物)、具有相同类型活性的化合物的活性代谢物、互变异构体、以及与药学上可接受的溶剂的非溶剂化以及溶剂化和水合形式如化合物中的水、乙醇等。本文提出的缀合物的溶剂化形式也被认为是本文公开的。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物、以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药、利于活性成分的吸收进而发挥生物活性。
“对照样品”或“对照样品”是指分别没有患有疾病或病症的一组个体的个体或样品(例如关节炎，类风湿关节炎，肌炎，红斑狼疮和/或系统性硬化症)或内部控制，通过本领域已知的技术确定。在一些实施例中，先确定控制或基线水平，或在样本中测量之前测量，或者从这种控制样本的数据库获得。
“对照化合物(Ⅰ)”与“对照(Ⅰ)”在本发明中可互换使用。
“对照化合物(Ⅱ)”与“对照(Ⅱ)”在本发明中可互换使用。
“诱导”与“刺激”在本发明中可互换使用。
“有效量”是指当施用于受试者时产生有益的或期望的结果,包括临床结果,例如,与对照相比,降解、抑制或减轻受试者中正在治疗的病症的症状的量。
“药学上可接受的载体,佐剂或载体”是指无毒载体,佐剂或不破坏其配制化合物的药理活性的载体。可用于本发明组合物的药学上可接受的载体,佐剂或载体包括但不限于离子交换剂,氧化铝,硬脂酸盐,卵 磷脂,血清蛋白,例如人血清白蛋白,磷酸盐等缓冲物质,甘氨酸,山梨酸,山梨酸钾,饱和蔬菜脂肪酸,水,盐或电解质的部分甘油混合物,如预硫酸胍,磷酸二钠,磷酸二钠,氯化钠,锌盐,胶体二氧化硅,葡萄铝酸镁,聚乙烯吡咯烷酮,基于纤维素的物质,聚乙二醇,羧甲基纤维素,聚丙烯酸酯,蜡,聚乙烯-聚氧化丙烯-嵌段聚合物,聚乙二醇和羊毛脂肪。
“患者”是指动物，优选哺乳动物，最优选人。
“细胞因子”是指选自IL-2Ralpha,MIG,MIP-1beta,IL-6,IFN-al pha2,IFN-gamma,SDF-1alpha,IL-1ra,MCP-3,IL-16,IL-12(p40),LIF,TNF-beta,IL-5,GM-CSF,MIF,TNF-alpha,RANTES,IL-2,IL-1beta,IL-18,Eotaxin,BasicFGF,VEGF,beta-NGF,PDGF-BB,IP-10,IL-13,IL-4,MCP-1,IL-8,MIP-1alpha,IL-10,G-CSF,GRO-alpha,HGF,IL-1alpha,IL-3,SCF,TRAIL,M-CSF,CTACK,IL-15,IL-7,IL-12(p70),IL-17,IL-9,SCGF-beta,KC,IL-17A和MCP-17A。在一些实施方案中,测量正常人或者小鼠样品中的细胞因子水平包括使用培养的外周血单个核细胞(PBMC)测定。在一些实施方案中,测量正常人或者小鼠样品中的细胞因子水平包括使用Luminex方法测定。
以下例证性实施方案用于说明本发明。本发明并不限于这些实施例。
下面将结合实施例对本发明做进一步详细、完整地说明，但决非限制本发明，本发明也并非仅局限于实施例的内容。本发明实施例中的原料是已知的并且可以在市场上买到、或者可以采用或按照本领域已知的方法来合成。在无特殊说明的情况下，本发明实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。
缩写定义
“DMSO”指N,N-二甲基甲酰胺。
“Glucose”指葡萄糖。
“Solutol”指聚乙二醇-15羟基硬脂酸酯。
“PO”指口服。
“LPS”指脂多糖。
“R848”指雷希莫特。
“PBMC”指人外周血单个核细胞。
“IRAK4”指白细胞介素1受体相关激酶4。
“IL”指白细胞介素。
“IMQ”指咪喹莫特。
“Topical”指局部给药。
“BID”指每日2次。
“QD”指每日1次。
附图说明
图1分别以图1A﹑图1B标示化合物A对IRAK4的降解作用。
图2各类病人PBMC胞内IRAK4蛋白表达水平图。
图3化合物A对LPS诱导的正常人PMBC的IL-6抑制作用。
图4化合物A对LPS刺激人外周血单个核细胞分泌的细胞因子的抑制作用。
图5化合物A对R848刺激人外周血单个核细胞分泌的细胞因子的抑制作用。
图6分别以图6A﹑图6B﹑图6C﹑图6D标示化合物A对正常人全血﹑病人全血IL-6抑制作用。
图7分别以图7A﹑图7B﹑图7C标示化合物A对C57小鼠PBMC﹑脾脏﹑皮肤IRAK4的降解。
图8分别以图8A﹑图8B﹑图8C标示化合物A在咪喹莫特诱导C57小鼠银屑病模型上的药效。
具体实施方法：
Ⅰ化合物制备实施例：
实施例1：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代基四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
将N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(150mg,粗品,0.18mmol)，4-氯-3-(2,4-二氧代基四氢嘧啶-1(2H)-基)苯甲酸五氟苯基酯(78mg,0.18mmol)和N,N-二异丙基乙胺(116mg,0.9mmol)在二甲亚砜(5mL)中的溶液在室温下搅拌过夜。将所得混合物倒入水(50mL)中并搅拌10分钟。过滤，滤饼通过制备型HPLC纯化，得到目标产物5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代基四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝874.5.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.50(d,J＝5.9Hz,1H),8.78(d,J＝7.7Hz,1H),8.39(d,J＝4.0Hz,1H),8.26(d,J＝5.7Hz,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.39(dd,J＝8.3,2.0Hz,1H),7.27-6.96(m,1H),6.90-6.41(m,1H),5.30-5.05(m,1H),4.77(d,J＝17.4Hz,1H),4.26-4.14(m,1H),3.86-3.70(m,3H),3.66-3.41(m,5H),3.30-3.25(m,2H),2.96-2.84(m,2H),2.79-2.70(m,2H),2.22-2.08(m,2H),2.06-1.83(m,8H),1.75-1.65(m,2H),1.63-1.24(m,7H),1.19-0.91(m,4H).
实施例2：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-((1r,4R)-4-((9-(4-氯-3-(2,4-二氧代基四氢嘧啶-1(2H)-基)苯甲酰基)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑 -4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
制备方法参照实施例1。
LC-MS:(ESI,m/z):[M+H]+＝874.4
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.50(d,J＝6.3Hz,1H),8.78(d,J＝7.7Hz,1H),8.38(d,J＝4.4Hz,1H),8.25(d,J＝5.7Hz,1H),7.64(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.39(dd,J＝8.2,1.9Hz,1H),7.26-6.95(m,1H),6.91-6.41(m,1H),5.32-5.03(m,1H),4.77(d,J＝17.4Hz,1H),4.17(t,J＝12.9Hz,1H),3.84-3.71(m,3H),3.67-3.57(m,4H),3.45(d,J＝9.8Hz,1H),3.30-3.25(m,2H),2.79-2.71(m,2H),2.38-2.24(m,4H),2.13-1.77(m,8H),,1.79-1.66(m,2H),1.60-1.35(m,9H),1.12-0.92(m,2H).
实施例3：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
将N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(160mg,0.26mmol)，3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(110mg,0.26mmol)和DIEA(168mg,1.3mmol)在DMSO(5mL)中的混合物在室温搅拌过夜。反应液倒入水中(50mL)并搅拌10min。过滤，滤饼经Prep-HPLC纯化得到5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝870.3.
1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.50(d,J＝5.7Hz,1H),8.79(d,J＝7.7Hz,1H),8.39(d,J＝4.0Hz,1H),8.26(d,J＝5.6Hz,1H),7.41-7.29(m,2H),7.27-6.95(m,2H),6.91-6.40(m,1H),5.31-5.00(m,1H),4.77(d,J＝17.5Hz,1H),4.27-4.15(m,1H),3.86-3.71(m,5H),3.65-3.39(m,8H),2.89(d,J＝10.4Hz,2H),2.68(t,J＝6.5Hz,2H),2.15(d,J＝6.1Hz,2H),2.08-1.87(m,8H),1.76-1.65(m,2H),1.63-1.38(m,5H),1.36-1.21(m,2H),1.19-0.95(m,4H).
实施例4：N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯的制备
25℃时向5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酸(470mg,1.9mmol)和DIEA(1mL,4.7mmol)的DMF(20ml)溶液中加入HATU(900mg,2.4mmol)，混合物在25℃搅拌2h后再加入4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(0.5g,1.5mmol)，反应液再在25℃搅拌过夜。反应液中加水(30mL)，然后用EA(50mL×3)萃取，合并有机相，并用饱和食盐水洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(EA)纯化得到4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝547.4.
步骤2：N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
25℃时向4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(0.2g,0.366mmol)的DCM(3mL)溶液中加TFA(1mL)。反应液在25℃搅拌1h。反应液直接减压浓缩得到N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝447.2.
步骤3：9-((4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
25℃时向N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(200mg,0.45mmol)和DIEA(0.5ml,1.35mmol)的MeOH(5mL)溶液中加入9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(150mg,0.45mmol)和NaBH3CN(100mg,2.25mmol)。反应液在25℃搅拌16h。过滤， 减压浓缩，所得粗品经flash柱(MeOH:DCM＝3:97)纯化得到9-((4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝712.4.
步骤4：N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
25℃时向9-((4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(0.14g,0.2mmol)的DCM(3mL)溶液中加TFA(1mL)。反应液在25℃搅拌1h。反应液直接减压浓缩得到N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。所得产品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝612.5.
步骤5：N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
25℃时向N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(110mg,0.18mmol)和1-(2-氯-5-五氟苯甲酰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(90mg,0.19mmol)的DMSO(2mL)溶液中加DIEA(0.1mL,0.54mmol)。反应液在25℃搅拌16h。反应液倒入水中(10mL)，过滤，滤饼经Prep-HPLC纯化得到.
LC-MS:(ESI,m/z):[M+H]+＝862.4.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.39(dd,J＝8.2,1.8Hz,1H),7.25-6.95(m,1H),6.91(d,J＝8.0Hz,1H),4.26-4.14(m,1H),3.87-3.68(m,9H),3.66-3.50(m,3H),3.31-3.20(m,2H),2.95-2.83(m,2H),2.80-2.68(m,2H),2.21-2.08(m,2H),2.07-1.87(m,6H),1.70(d,J＝8.7Hz,2H),1.63-1.41(m,5H),1.38-1.25(m,2H),1.16-1.02(m,4H).
实施例5：N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
将5-氯吡唑并[1,5-a]嘧啶-3-羧酸乙酯(3.0g,13.3mmol)和benzyl哌嗪-1-羧酸苄酯(2.9g,13.3mmol)溶于ACN(30mL)溶液中，再加DIEA(5.2g,40mmol)，将反应液加热到60℃搅拌2小时，减压浓缩，粗品用乙酸乙酯(100mL)溶解，饱和食盐水洗，减压浓缩，粗品用柱层析(PE:EA＝2:1)纯化得到5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯。
LC-MS:(ESI,m/z):[M+H]+＝410.2.
1H NMR(400MHz,DMSO-d6)δ8.76(d,J＝7.9Hz,1H),8.22(s,1H),7.41-7.33(m,5H),6.86(d,J＝7.9Hz,1H),5.13(s,2H),4.19(q,J＝7.1Hz,2H),3.88-3.74(m,4H),3.62-3.49(m,4H),1.28(t,J＝7.1Hz,3H).
步骤2：5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸的制备
将5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯(2.6g,6.3mmol)溶于甲醇(26mL)中，加入氢氧化锂(1.0g,25.2mmol)，反应液60℃搅拌2小时，反应结束后向反应液加稀盐酸(2mol/L)使反应液pH至2，然后过滤，所得滤饼用水洗并真空干燥得到5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸。
LC-MS:(ESI,m/z):[M+H]+＝382.2.
步骤3：4-(3-((1-(1-(叔丁氧羰基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯的制备
25℃时向5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-羧酸(270mg,0.4mmol和DIEA (0.5ml,1.0mmol)的DMF(5ml)溶液中加HATU(380mg,0.50mmol)，然后在室温搅拌2h后再加4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(0.21g,0.3mmol)，反应液在25℃搅拌过夜。反应加水(30mL)淬灭，用EA(50mL×3)萃取，有机相用饱和食盐水洗涤，无水Na2SO4干燥，过滤，减压浓缩，所得粗品经层析柱(MeOH:DCM＝0～3:97)纯化得到4-(3-((1-(1-(叔丁氧羰基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝680.4.
步骤4：4-(3-((3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯的制备
向4-(3-((1-(1-(叔丁氧羰基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯(0.29g,0.42mmol)的DCM(5mL)溶液中加TFA(1mL)，然后反应液在室温搅拌2h。反应液直接减压浓缩得到4-(3-((3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯.
LC-MS:(ESI,m/z):[M+H]+＝580.5.
步骤5：9-((4-(4-(5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
向4-(3-((3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯(230mg,0.4mmol)和DIEA(0.35mL,2.0mmol)的MeOH(10mL)溶液中9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(144mg,0.45mmol)和NaBH3CN(138mg,1.2mmol)。反应液在25℃搅拌过夜。反应加水(30mL)淬灭，然后用EA(50mL×3)萃取，有机相用饱和食盐水洗涤，无水Na2SO4干燥，过滤，减压浓缩，所得粗品经flash柱(MeOH:DCM＝0～3:97)纯化得到9-((4-(4-(5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝845.5.
步骤6：4-(3-((1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基) 吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯的制备
向9-((4-(4-(5-(4-((苄氧基)羰基)哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(0.29g,0.34mmol)的DCM(3mL)溶液中加TFA(1mL)，然后在25℃搅拌1h。反应液直接减压浓缩得到4-(3-((1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝745.4.
步骤7：4-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯的制备
向4-(3-((1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯(240mg,0.32mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(144mg,0.33mmol)的DMSO(5mL)溶液中加DIEA(0.1mL,0.6mmol)。然后反应液在25℃搅拌16h。反应液中加水(30mL)，用EA(50mL×3)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，过滤，减压浓缩。所得粗品经flash柱(MeOH:DCM＝0～3:97)纯化得到4-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝995.0.
步骤8：N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
0℃时向4-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌嗪-1-羧酸苄酯(290mg, 0.29mmol)的DCM(15mL)溶液中加碘代三甲硅烷(0.85mL,0.6mmol)，然后反应液在25℃搅拌16h。反应液倒入水中，用DCM(20mL×3)萃取，无水Na2SO4干燥，过滤，减压浓缩，所得粗品经Prep-HPLC纯化得到N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(哌嗪-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝861.1.
1H NMR(400MHz,MeOD)δ8.62(d,J＝7.9Hz,1H),8.43(brs,2.67H,FA),8.35(s,1H),7.64(d,J＝8.3Hz,1H),7.54(d,J＝1.6Hz,1H),7.43(d,J＝8.2Hz,1H),7.08 6.77(m,2H),4.57-4.43(m,1H),4.15-4.03(m,4H),3.79(t,J＝6.4Hz,2H),3.75-3.65(m,2H),3.63-3.48(m,2H),3.47-3.37(m,2H),3.37-3.30(m,2H),3.05-2.95(m,2H),2.95-2.77(m,4H),2.43-2.27(m,4H),1.92-1.75(m,3H),1.74-1.65(m,3H),1.65-1.45(m,2H),1.39-1.12(m,5H).
实施例6：5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
向5-氯吡唑并[1,5-a]嘧啶-3-羧酸乙酯(2.0g,8.9mmol)的ACN(20mL)溶液中加8-氧杂-3-氮杂双环[3.2.1]辛烷(1.3g,8.9mmol)和DIEA(3.4g,26mmol)，然后反应液加热到60℃反应2h。反应加水(200mL)淬灭，用EA(200mL×2)萃取，有机相用无水Na2SO4干燥，过滤，减压浓缩。所得粗产物经层析柱(PE:EA＝1～5:1)纯化得到5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯。
LC-MS:(ESI,m/z):[M+H]+＝303.2.
步骤2：5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸的制备
向5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯(2.6g,8.6mmol)的H2O/MeOH(3ml/26mL)混合液中加LiOH(1.4g,34mmol)，然后反应液加热到60℃反应12h。反应液经硅藻土过滤，滤液减压浓缩。所得粗产物经Prep-HPLC(乙腈/0.05％NH4OH水溶液,5％～95％)纯化得到5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸。
LC-MS:(ESI,m/z):[M+H]+＝275.3.
步骤3：4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲 基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯的制备
向5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸(274mg,1mmol)的DMF(5mL)溶液中加4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(316mg,1mmol)，HATU(456mg,1.2mmol)和DIEA(387mg,3mmol)，然后反应液在25℃反应12h。反应液倒入水中，然后用EA(50mL×2)萃取，有机相用无水Na2SO4干燥，过滤，减压浓缩。所得粗产物经层析柱(MeOH:DCM＝0～1:9)纯化得到4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝573.4.
步骤4：5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(240mg,0.42mmol)的HCl/1.4-二氧六环(5mL)溶液在室温搅拌1h。反应液直接减压浓缩得到5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝473.4.
步骤5：9-((4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
向5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(130mg,0.27mmol)的THF(10mL)溶液中加9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(77mg,0.27mmol和STAB(175mg,0.8mmol)。然后反应液在25℃搅拌12h。反应液经硅藻土 过滤，滤液减压浓缩。所得粗产物经柱层析(MeOH:DCM＝0～1:9)纯化得到9-((4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝738.3.
步骤6：N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将9-((4-(4-(5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(100mg,0.14mmol)的HCl/1.4-二氧六环(4mL)溶液在25℃搅拌1h。然后反应液直接减压浓缩得到N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝638.3.
步骤7：5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
向N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(150.0mg,0.24mmol)的DMSO(2mL)溶液中加3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(123mg,0.28mmol)和DIEA(93mg,0.72mmol)，然后反应液在室温搅拌12h。反应液倒入水(20mL)中，用EA(30mL×2)萃取，有机相用无水Na2SO4干燥，减压浓缩。所得粗品经Prep-HPLC(乙腈/0.05％NH4HCO3水溶液,5％～95％)纯化得到5-(8-氧杂-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝884.5.
1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.41(s,1H),8.81(d,J＝7.8Hz,1H),8.39(s,1H),8.28 (d,J＝1.3Hz,1H),7.37(d,J＝8.4Hz,1H),7.32(s,1H),7.27-6.95(m,2H),6.82(d,J＝7.9Hz,1H),4.50-4.37(m,2H),4.30-4.00(m,2H),3.84(s,3H),3.68-3.34(m,6H),3.28-3.11(m,2H),2.98-2.78(m,2H),2.73-2.60(m,2H),2.22-1.63(m,14H),1.61-1.17(m,8H),1.18-0.93(m,4H).
实施例7：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)丁基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(4-(4-(4-(5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)丁基)哌啶-1-羧酸叔丁酯的制备
向5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(110mg,0.24mmol)的THF(5mL)溶液中加4-(4-氧代丁基)哌啶-1-羧酸叔丁酯(100mg,0.36mmol)和STAB(170mg,0.71mmol)。反应液在25℃搅拌2h。反应加水(30mL)淬灭，用DCM(30mL×3)萃取，有机相用无水Na2SO4干燥，过滤，减压浓缩。所得粗产物经flash柱(MeOH:DCM＝0～3:97)纯化得到4-(4-(4-(4-(5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)丁基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝698.0.
步骤2：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-(4-(哌啶-4-基)丁基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
向4-(4-(4-(4-(5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)丁基)哌啶-1-羧酸叔丁酯(0.2g,0.26mmol)的DCM(5mL)溶液中加TFA(1mL)。反应液在25℃搅拌2h。反应液直接减压浓缩得到5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-(4-(哌啶-4-基)丁基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝598.1.
步骤3：5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)丁基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰 胺的制备
向5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-(4-(哌啶-4-基)丁基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(160mg,0.26mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(130mg,0.26mmol)的DMSO(4mL)溶液中加DIEA(0.14mL,0.78mmol)。反应液在25℃搅拌2h。反应加水(10mL)淬灭，用DCM(20mL×3)萃取，用无水Na2SO4干燥，过滤，减压浓缩，所得粗品经Prep-HPLC纯化得到5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(4-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)丁基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝848.3.
1H NMR(400MHz,DMSO-d6)δ10.49(s,1H),9.50(d,J＝5.3Hz,1H),8.78(d,J＝7.7Hz,1H),8.40(s,1H),8.25(d,J＝5.0Hz,1H),7.63(d,J＝8.3Hz,1H),7.54(s,1H),7.38(d,J＝8.1Hz,1H),7.20-7.00(m,1H),6.98-6.45(m,1H),5.40-5.05(m,1H),4.77(d,J＝17.3Hz,1H),4.54-4.34(m,1H),4.28-4.12(m,1H),3.83-3.72(m,3H),3.67-3.57(m,3H),3.45(d,J＝9.6Hz,1H),3.02-2.85(m,3H),2.80-2.70(m,2H),2.36-2.26(m,2H),2.08-1.88(m,8H),1.76-1.47(m,4H),1.47-1.22(m,6H),1.18-1.02(m,2H).
实施例8：N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-(((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将N-(3-(二氟甲基)-1-((1R,4R)-4-(醛基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(50mg,0.10mmol)和3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(24mg,0.10mmol)溶于THF(3mL)中，在搅拌下向反应液中加入三乙酰氧基硼氢化钠(67mg,0.32mmol)，反应液室温搅拌2h。向反应液中加入水(50mL)，乙酸乙酯萃取(30mL x 3)，合并有机相，无水硫酸钠干燥，减压浓缩，粗品经正向柱层析纯化得到9-(((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝712.3.
步骤2：N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将9-(((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(95mg,0.13mmol)溶于DCM(3mL)，加入TFA(1mL)，反应液室温搅拌1h。反应液直接减压浓缩得到N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(粗品)，直接用于下步反应。
LC-MS:(ESI,m/z):[M+H]+＝612.3.
步骤3：N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(130mg,0.13mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(57mg,0.13mmol)溶于DMSO(5mL)中，在搅拌下向反应液中加入DIEA(84mg,0.65mmol)后，使反应液在室温下搅拌反应1h。反应结束后将反应液倒入水(50mL)中，用EA萃取(30mL x 3)，合并有机相并用饱和食盐水(80mL)洗涤，经无水硫酸钠干燥，过滤，滤液减压浓缩后经prep-HPLC分离纯化得到N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝862.3.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),9.39(s,1H),8.82(d,J＝6.3Hz,1H),8.37(s,1H),8.28(s,1H),7.63(d,J＝7.9Hz,1H),7.55(s,1H),7.39(d,J＝7.9Hz,1H),7.25-6.95(m,1H),6.90(d,J＝7.6Hz,1H),4.27-4.07(m,1H),3.90-3.50(m,12H),3.40-3.30(m,2H),2.79-2.70(m,2H),2.43-2.23(m,4H),2.20-1.95(m,4H),1.93-1.83(m,2H),1.82-1.30(m,11H),1.09-0.94(m,2H).
实施例9：N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(2-((((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯的制备
将N-(3-(二氟甲基)-1-((1R,4R)-4-(醛基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(110mg,0.23mmol)用THF(10mL)溶解，加入4-(2-(甲基氨基)乙基)哌啶-1-羧酸叔丁酯(56mg,0.23mmol)和STAB(146mg,0.69mmol)，室温反应2h，加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品用柱层析(MeOH:DCM＝1:11)纯化得到4-(2-((((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝700.4.
步骤2：N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将4-(2-((((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(95mg，0.14mmol)溶于DCM(3mL)中，加入TFA(1mL)，室温反应1h。反应额有直接减压浓缩得到N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝600.3.
步骤3：N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的准备
将N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(95mg,0.14mmol)溶于DMSO(5mL)，加入DIEA(77mg,0.60mmol) 和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(61mg,0.14mmol)，室温反应2h。加水(50mL)并搅拌5分钟，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用反相制备得到N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝850.4.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),9.39(s,1H),8.83(d,J＝7.9Hz,1H),8.38(s,1H),8.29(s,1H),7.63(d,J＝8.2Hz,1H),7.54(d,J＝1.8Hz,1H),7.38(dd,J＝8.2,1.8Hz,1H),7.24-6.97(m,1H),6.91(d,J＝8.0Hz,1H),4.49-4.38(m,1H),4.23-4.13(m,1H),3.83-3.76(m,4H),3.75-3.70(m,4H),3.66-3.52(m,2H),3.10-2.95(m,1H),2.84-2.67(m,3H),2.29(t,J＝7.0Hz,2H),2.14-2.01(m,7H),1.93-1.84(m,2H),1.82-1.46(m,7H),1.41-1.32(m,2H),1.17-0.95(m,4H).
实施例10：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-(羟甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将((1R,4R)-4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲醇(410mg,1.67mmol)溶于DMF(10mL)，加入5-(8-氧-3-氮杂螺双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-羧酸(459mg,1.67mmol)，HATU(825mg,2.17mmol)和DIEA(646mg,5.01mmol)，室温反应2h。加水(50mL)并搅拌5分钟，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析纯化得到5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-(羟甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝502.3.
步骤2：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-甲醛环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-(羟甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(290mg，0.58mmol)，溶于ACN(10mL)，加入IBX(324mg,1.16mmol)，85℃反应1.5h，减压浓缩除去ACN，加水(30mL)，用乙酸乙酯(30mL x 3)萃取，饱和食盐水(50mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析纯化得到5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-甲醛环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝500.3.
步骤3：4-(2-((((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯的制备
将5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-甲醛环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.20mmol)用THF(10mL)溶解，加入4-(2-(甲基氨基)乙基)哌啶-1-羧酸叔丁酯(48mg,0.20mmol)和STAB(127mg,0.6mmol)，室温反应2h，加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(MeOH:DCM＝1:11)纯化得到4-(2-((((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝726.4.
步骤4：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将4-(2-((((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(110mg,0.15mmol)溶于DCM(3 mL)中，加入TFA(1mL)，室温反应1h，减压浓缩，得到5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝626.3.
步骤5：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(110mg,0.15mmol)溶于DMSO(5mL)，加入DIEA(97mg,0.75mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(65mg,0.15mmol)，室温反应2h。加水(50mL)并搅拌5分钟，用乙酸乙酯(50mL x 3)提取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用反相制备得到5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝876.2.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),9.41(s,1H),8.82(d,J＝7.9Hz,1H),8.38(s,1H),8.28(s,1H),7.63(d,J＝8.2Hz,1H),7.54(d,J＝1.9Hz,1H),7.38(dd,J＝8.2,2.0Hz,1H),7.30-6.95(m,1H),6.82(d,J＝8.0Hz,1H),4.48-4.42(m,2H),4.26-4.09(m,2H),3.79-3.50(m,4H),3.27-3.17(m,2H),3.10-2.90(m,1H),2.85-2.70(m,3H),2.29(t,J＝6.8Hz,2H),2.15-2.07(m,5H),2.07-2.01(m,2H),1.95-1.83(m,4H),1.82-1.47(m,9H),1.41-1.31(m,2H),1.17-0.96(m,4H).
实施例11：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-(((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(3-(二氟甲基)-1-((1R,4R)-4-甲醛环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.20mmol)用THF(10mL)溶解，加入3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(48mg,0.20mmol)和STAB(127mg,0.6mmol)，室温反应2h，加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(MeOH:DCM＝1:11)纯化得到9-(((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝738.5.
步骤2：N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将9-(((1R,4R)-4-(4-(5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(105mg,0.14mmol)溶于DCM(3mL)中，加入TFA(1mL)，室温反应1h。减压浓缩，得到N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺，直接用于下一步。
步骤3：5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将N-(1-((1R,4R)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(110mg,0.14mmol)溶于DMSO(5mL)，加 入DIEA(90mg,0.70mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(61mg,0.14mmol)，室温反应2h。加水(50mL)并搅拌5分钟，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用反相制备得到5-(8-氧-3-氮杂双环[3.2.1]辛烷-3-基)-N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝888.2.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.41(s,1H),8.81(d,J＝7.9Hz,1H),8.37(s,1H),8.28(s,1H),7.64(d,J＝8.2Hz,1H),7.55(d,J＝1.8Hz,1H),7.39(dd,J＝8.2,1.9Hz,1H),7.24-6.97(m,1H),6.82(d,J＝8.0Hz,1H),4.47-4.41(m,2H),4.31-3.93(m,3H),3.81-3.72(m,1H),3.67-3.51(m,3H),3.25-3.15(m,2H),2.77-2.72(m,2H),2.35-2.25(m,4H),2.15-2.07(m,2H),2.16-2.98(m,2H),1.96-1.29(m,19H),1.10-0.92(m,2H).
实施例12：N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-氟苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.14mmol)用二甲亚砜(3mL)溶解，搅拌下加入3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-氟苯甲酸五氟苯酯(58mg,0.14mmol)和N,N-二异丙基乙胺(54mg,0.42mmol)。室温下搅拌2小时后，加水(50mL)，过滤，滤饼经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-氟苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝846.4.
1H NMR(400MHz,DMSO-d6)δ10.53(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.50(d,J＝7.7Hz,1H),7.36(d,J＝4.0Hz,2H),7.23-6.97(m,1H),6.91(d,J＝8.0Hz,1H),4.25-4.15(m,1H),3.84-3.69(m,10H),3.65-3.49(m,2H),3.31-3.24(m,2H),2.89(d,J＝10.3Hz,2H),2.73(t,J＝6.6Hz,2H),2.20-2.04(m,2H),2.04-1.80(m,6H),1.70-1.43(m,7H),1.43-1.20(m,2H),1.20-0.96(m,4H).
实施例13：N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.12mmol)用二甲亚砜(3mL)溶解，搅拌下加入3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酸五氟苯酯(58mg,0.14mmol)和N,N-二异丙基乙胺(46mg,0.36mmol)。室温下搅拌2小时后，加水(50mL)，过滤，滤饼经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝842.4.
1H NMR(400MHz,DMSO-d6)δ10.37(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.37-7.29(m,2H),7.28-6.96(m,2H),6.91(d,J＝7.9Hz,1H),4.25-4.15(m,1H),3.83-3.63(m,9H),3.61-3.47(m,3H),3.32-3.24(m,2H),2.95-2.65(m,4H),2.25-1.85(m,11H),1.70(d,J＝9.3Hz,2H),1.58-1.15(m,7H),1.12-0.91(m,4H).
实施例14：N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(2-((((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯的制备
N-(3-(二氟甲基)-1-((1R,4S)-4-甲醛环己基)-1H-吡唑-4-基)-5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(120mg,0.21mmol)和4-(2-(甲基氨基)乙基)哌啶-1-羧酸叔丁酯(51mg,0.21mmol)用四氢呋喃(10mL)溶解，搅拌下加入三乙酰氧基硼氢化钠(222mg,1.05mmol)。室温搅拌过夜，加水(50mL)，用二氯甲烷(20mL x 3)萃取，饱和食盐水(60mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(甲醇:二氯甲烷＝1:9)纯化得到4-(2-((((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝798.5.
步骤2：N-(3-(二氟甲基)-1-((1R,4S)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
4-(2-((((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺)-1H-吡唑-1-基)环己基)甲基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(80mg,0.10mmol)用二氯甲烷(3mL)溶解，三氟乙酸(1mL)加入并室温搅拌2小时，反应液直接减压浓缩得到N-(3-(二氟甲基)-1-((1R,4S)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝614.3.
步骤3：N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
N-(3-(二氟甲基)-1-((1R,4S)-4-((甲基(2-(哌啶-4-基)乙基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(80mg,0.10mmol)用二甲亚砜(3mL)溶解，搅拌下加入3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(47mg,0.11mmol)和N,N-二异丙基乙胺(38mg,0.30mmol)。室温下搅拌过夜，加水(50mL)，过滤，滤饼经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝860.4.
1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.37(s,1H),8.72(d,J＝8.0Hz,1H),8.37(s,1H),8.24(d,J＝5.1Hz,1H),7.36(dd,J＝8.5,2.1Hz,1H),7.32(d,J＝2.1Hz,1H),7.24-6.94(m,2H),6.86(d,J＝8.1Hz,1H),4.89(d,J＝4.1Hz,1H),4.50-4.15(m,2H),4.03-3.80(m,5H),3.72-3.53(m,4H),3.51-3.40(m,1H),3.10-2.65(m,4H),2.30(t,J＝7.0Hz,2H),2.15-1.95(m,7H),1.92-1.25(m,15H),1.17-0.95(m,4H).
实施例15：5-((S)-3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(羟甲基)环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯的制备
将(S)-5-(3-((叔丁氧羰基)氨基)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-羧酸(150mg,0.42mmol)溶于DMF(10mL)，加入((1R,4R)-4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)环己基)甲醇(112mg,0.46mmol)，DIEA(163mg,1.26mmol)和py-BOP(284mg,0.55mmol)，室温反应2h。加水(50mL)并搅拌5分钟，用乙酸乙酯(50mL x 3)提取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(EA:PE＝0～1)纯化得到((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(羟甲基)环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝589.3.
步骤2：((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-甲醛环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯的制备
将((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(羟甲基)环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯(190mg,0.32mmol)，溶于ACN(10mL)，加入IBX(181mg,0.65mmol)，85℃反应1.5h。向反应液中加水(30mL)，用乙酸乙酯(30mL x 3)萃取，饱和食盐水(50mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(EA:PE＝0～1)纯化得到((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-甲醛环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝586.6.
步骤3：N-苄基-N-甲基-2-(哌啶-4-基)乙烷-1-胺的制备
将4-(2-(苄基(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(345mg,1.04mmol)溶于DCM(5mL)中，加入TFA(1mL)，室温反应1h。反应液直接减压浓缩得到N-苄基-N-甲基-2-(哌啶-4-基)乙烷-1-胺，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝233.2.
步骤5：1-(5-(4-(2-(苄基(甲基)氨基)乙基)哌啶-1-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将N-苄基-N-甲基-2-(哌啶-4-基)乙烷-1-胺(250mg,1.04mmol)溶于DMSO(10mL)，加入DIEA(671mg,5.2mmol)和3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(451mg,1.04mmol)，室温反应2h。反应液中加水(100mL)并搅拌5分钟，用乙酸乙酯(100mL x 3)萃取，饱和食盐水(150mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(MeOH:DCM＝0～1:9)纯化得到1-(5-(4-(2-(苄基(甲基)氨基)乙基)哌啶-1-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝479.2.
步骤6：1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将1-(5-(4-(2-(苄基(甲基)氨基)乙基)哌啶-1-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮(240mg，0.5mmol)溶于MeOH(10mL)中，加入Pd/C(72mg,30％)，室温反应2h。过滤后减压浓缩，得到1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，直接用于下一步。
LC-MS:(ESI,m/z):[M+Na]+＝411.1.
步骤7：((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯的制备
将1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(95mg,0.24mmol)用THF(10mL)溶解，加入((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-甲醛环己基)-1H-吡唑-4-基)氨基甲酰)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯(120mg,0.20mmol)和STAB(127mg,0.60mmol)，室温反应2h。反应液中加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，饱和食盐水(100mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(MeOH:DCM＝0～1；9)纯化得到((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝959.4.
步骤8：5-((S)-3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)羰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯(100mg,0.10mmol)溶于DCM(5mL)中，加入TFA(1mL)，室温反应1h。反应液直接减压浓缩，粗品使用反相制备纯化得到5-((S)-3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-((1R,4S)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝859.4.
1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.36(s,1H),8.74(d,J＝8.0Hz,1H),8.38(s,1H),8.24(s,1H),7.36(dd,J＝8.4,2.1Hz,1H),7.32(d,J＝2.1Hz,1H),7.27-6.97(m,2H),6.88(d,J＝8.1Hz,1H),4.52-4.15(m,4H),3.84(s,3H),3.59(t,J＝6.6Hz,2H),3.24-2.93(m,3H),2.80-2.60(m,4H),2.40-2.25(m,2H),2.16-1.99(m,8H),1.95-1.80(m,3H),1.80-1.29(m,13H),1.17-0.96(m,4H).
实施例16：N-(3-(二氟甲基)-1-(1-(2-(3-(3-(2,4-二氧代基四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
将N-(1-(1-(2-(3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡 唑并[1,5-a]嘧啶-3-甲酰胺(103mg,0.165mmol)和3-(2,4-二氧代基四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(85mg,0.198mmol)加入到DMSO(3mL)中，然后加入DIEA(63.8mg,0.495mmol)，室温搅拌16h,然后加入水(20mL)，用乙酸乙酯(25mL x 4)萃取，饱和食盐水洗，无水硫酸钠干燥，减压浓缩，粗品经Prep-HPLC制备纯化(乙腈/0.5％FA水溶液,5％～95％)得到产物N-(3-(二氟甲基)-1-(1-(2-(3-(3-(2,4-二氧代基四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝872.5.
1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),8.27(s,0.62H,HCOOH),7.42-7.28(m,2H),7.26-6.94(m,2H),6.91(d,J＝7.9Hz,1H),4.30-4.10(m,1H),3.84(s,3H),3.79-3.61(m,8H),3.62-3.54(m,6H),2.97-2.91(m,2H),2.71-2.65(m,2H),2.37-2.17(m,2H),2.05-1.85(m,6H),1.75-1.62(m,2H),1.55-1.17(m,9H),1.17-0.93(m,4H).
实施例17：N-(3-(二氟甲基)-1-((1R,4R)-4-((2-(1-(3-(2,6-二氧代吡啶-3-基)-4-氟苯甲酰l)哌啶-4-基)乙基)(甲基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸甲酯的制备
将5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酸(200mg,0.73mmol)溶于DMF(4mL)中，(1R,4R)-4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)环己烷-1-羧酸甲酯(182mg,0.73mmol)，HATU(139mg,0.95mmol)和DIEA(377mg,2.92mmol)加入，反应液室温搅拌过夜。反应液加水(50mL)稀释，乙酸乙酯(40mL x3)萃取，有机相用饱和食盐水洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(EA:PE＝9:1)纯化得到(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸甲酯。
LC-MS:(ESI,m/z):[M+H]+＝504.1.
步骤2：(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸的制备
将(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸甲酯(230mg,0.46mmol)用混合溶液甲醇/水(5mL,V/V＝5/1)溶解，加入氢氧化锂(44mg,1.84mmol)，反应液加热至60℃搅拌2小时。反应液用稀盐酸(2mol/L)调节至pH＝4，乙酸乙酯(40mL x 3)萃取，有机相用饱和食盐水洗涤，经无水硫酸钠干燥，过滤，减压浓缩得到(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝490.6.
步骤3：4-(2-((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)-N-甲基环己烷-1-羧酰氨基)乙基)哌啶-1-羧酸叔丁酯的制备
将(1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己烷-1-羧酸(140mg,0.29mmol)和4-(2-(甲基氨基)乙基)哌啶-1-羧酸叔丁酯(70mg,0.29mmol)溶于DMF(4mL)中，加入HATU(144mg,0.38mmol)和DIEA(155mg,1.2mmol)，反应液室温搅拌过夜。反应液加水(40mL)稀释，用乙酸乙酯(40mL x 4)萃取，有机相经饱和食盐水洗，无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(EA)纯化得到4-(2-((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)-N-甲基环己烷-1-羧酰氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝714.8.
步骤4：N-(3-(二氟甲基)-1-((1R,4R)-4-(甲基(2-(哌啶-4-基)乙基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将4-(2-((1R,4R)-4-(3-(二氟甲基)-4-(5-吗啡啉吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)-N-甲基 环己烷-1-羧酰氨基)乙基)哌啶-1-羧酸叔丁酯(130mg,0.18mmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(0.3mL)并室温搅拌1小时。反应液直接减压浓缩得到N-(3-(二氟甲基)-1-((1R,4R)-4-(甲基(2-(哌啶-4-基)乙基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺，无需纯化，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝614.2.
步骤5：N-(3-(二氟甲基)-1-((1R,4R)-4-((2-(1-(3-(2,6-二氧代吡啶-3-基)-4-氟苯甲酰l)哌啶-4-基)乙基)(甲基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将N-(3-(二氟甲基)-1-((1R,4R)-4-(甲基(2-(哌啶-4-基)乙基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(130mg,0.18mmol)和3-(2,6-二氧代基哌啶-3-基)-4-氟苯甲酸五氟苯基酯(75.3mg,0.18mmol)溶于DMSO(2mL)中，加入DIEA(93mg,0.72mmol)，反应液室温搅拌3小时。反应液中加水(20mL)稀释，乙酸乙酯(20mL x 3)萃取，有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-((1R,4R)-4-((2-(1-(3-(2,6-二氧代吡啶-3-基)-4-氟苯甲酰l)哌啶-4-基)乙基)(甲基)氨甲酰基)环己基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝847.4.
1H NMR(400MHz,DMSO-d6)δ10.90(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.40(d,J＝5.5Hz,1H),8.29(s,1H),7.40-7.33(m,2H),7.31-7.23(m,1H),7.23-6.96(m,1H),6.91(d,J＝8.0Hz,1H),4.52-4.20(m,2H),4.12(dd,J＝12.7,4.9Hz,1H),3.88-3.66(m,8H),3.62-3.48(m,1H),3.433.34(m,1H),3.01(s,3H),2.82-2.62(m,4H),2.60-2.54(m,1H),2.29-2.17(m,1H),2.08-1.98(m,3H),1.95-1.67(m,6H),1.66-1.35(m,6H),1.21-1.02(m,2H).
实施例18：5-((S)-3-氨基哌啶-1-基)-N-(1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(2-((((9H-芴-9-基)甲氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯的制备
将4-(2-(甲基氨基)乙基)哌啶-1-羧酸叔丁酯(240mg,1.0mmol)用DCM(20mL)溶解，加入FmocCl(311mg,1.2mmol)和吡啶(237mg,3.0mmol)，室温反应过夜。反应液加水(50mL)稀释，用DCM(30mL x 3)萃取，有机相经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(EA:PE＝0～4:1)纯化得到4-(2-((((9H-芴-9-基)甲氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+Na]+＝487.3.
步骤2：(9H-芴-9-基)甲基甲基(2-(哌啶-4-基)乙基)氨基甲酸的制备
将4-(2-((((9H-芴-9-基)甲氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(210mg,0.45mmol)溶于DCM(4mL)中，加入TFA(1mL)，室温反应1h。反应液直接减压浓缩得到(9H-芴-9-基)甲基甲基(2-(哌啶-4-基)乙基)氨基甲酸，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝365.2.
步骤3：1-(2-氯-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将(9H-芴-9-基)甲基甲基(2-(哌啶-4-基)乙基)氨基甲酸(210mg,0.45mmol)溶于DMSO(5mL)，加入DIEA(264mg,2.05mmol)和4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(178mg,0.41mmol)，室温反应过夜。反应溶液使用反相色谱纯化(45％ACN/0.1％NH4HCO3水溶液)纯化得到1-(2-氯-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝393.1.
步骤4：((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯的制备
将1-(2-氯-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(50mg,0.13mmol)用THF(10mL)溶解，加入((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-甲酰基环己基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯(30mg,0.05mmol)和STAB(83mg,0.39mmol)，反应0.5小时后，((S)-1-(3-((3-(二氟甲基)-1-((1R,4S)-4-甲酰基环己基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯(60mg,0.10mmol)1小时内分两次加入，然后室温反应2小时。反应液中加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，有机相经饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(MeOH:DCM＝0～1:9)纯化得到 ((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝963.3.
步骤5：5-((S)-3-氨基哌啶-1-基)-N-(1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯(100mg,0.10mmol)溶于DCM(5mL)中，加入TFA(1mL)，室温反应1h。反应液直接减压浓缩，然后加入NaHCO3饱和溶液调节pH至8～9，用MeOH/DCM(10％,20mL x 3)萃取，有机相用无水硫酸钠干燥，过滤，浓缩，粗品使用反相制备纯化得到5-((S)-3-氨基哌啶-1-基)-N-(1-((1R,4S)-4-(((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝863.1.
1H NMR(400MHz,DMSO-d6)δ11.18-9.87(s,1H),9.36(s,1H),8.74(d,J＝8.0Hz,1H),8.37(s,1H),8.24(s,1H),7.63(d,J＝8.2Hz,1H),7.54(d,J＝2.0Hz,1H),7.38(dd,J＝8.2,2.0Hz,1H),7.26-6.97(m,1H),6.87(d,J＝8.0Hz,1H),4.50-4.25(m,2H),4.18(t,J＝11.9Hz,2H),3.80-3.70(m,1H),3.70-3.51(m,2H),3.26-3.18(m,1H),3.08-2.97(m,2H),2.85-2.64(m,5H),2.30(t,J＝7.2Hz,2H),2.14-2.07(m,5H),2.07-1.97(m,2H),1.95-1.83(m,3H),1.80-1.30(m,12H),1.18-0.95(m,4H).
实施例19：N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(100mg,0.39mmol)用二甲亚砜(3mL)溶解，搅拌下加入 4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(205mg,0.47mmol)和N,N-二异丙基乙胺(153mg,1.18mmol)，室温搅拌过夜。反应液中加水(50mL)，用乙酸乙酯(30mL x 3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经柱层析(乙酸乙酯)纯化得到9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M-Boc]+＝405.1.
步骤2：1-(2-氯-5-(3,9-二氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(170mg,0.34mmol)用二氯甲烷(2.5mL)溶解，三氟乙酸(0.5mL)加入并室温搅拌2小时。反应液直接减压浓缩得到1-(2-氯-5-(3,9-二氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝405.1.
步骤3：N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
N-(3-(二氟甲基)-1-((1R,4R)-4-醛基环己基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(50mg,0.10mmol)和1-(2-氯-5-(3,9-二氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(42mg,0.10mmol)用二氯乙烷(10mL)溶解，加入三乙酰氧基硼氢化钠(44mg,0.20mmol)，升温至70℃搅拌过夜。反应液中加水(50mL)，用二氯甲烷(20mL x 3)萃取，经无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(1-((1R,4R)-4-((9-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝874.2.
1H NMR(400MHz,CDCl3)δ9.76(s,1H),8.44(s,1H),8.39(d,J＝5.3Hz,2H),8.30(d,J＝7.6Hz,1H),7.58-7.48(m,2H),7.43(d,J＝1.8Hz,1H),7.38-7.34(m,1H),6.97-6.65(m,1H),6.02(d,J＝7.5Hz,1H),4.98-4.85(m,4H),4.47-4.37(m,4H),4.13-4.06(m,1H),3.86-3.74(m,4H),3.46-3.38(m,2H), 3.00-2.74(m,3H),2.39-2.29(m,3H),2.29-2.10(m,3H),2.10-2.00(m,2H),1.92-1.76(m,3H),1.40-1.10(m,10H),0.95-0.85(m,1H).
实施例20：N-(1-(1-(2-(3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
N-(1-(1-(2-(3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺(23mg,0.03mmol)用二甲亚砜(3mL)溶解，搅拌下加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(14mg,0.03mmol)和N,N-二异丙基乙胺(38mg,0.30mmol)，室温下搅拌2小时。反应液中加水(20mL)，过滤，滤饼经p-TLC(甲醇:二氯甲烷＝1:9)纯化得到N-(1-(1-(2-(3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)乙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝876.3.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.42-7.35(m,1H),7.26-6.97(m,1H),6.91(d,J＝8.0Hz,1H),4.31-4.11(m,1H),3.86-3.66(m,9H),3.61-3.50(m,3H),3.29-3.24(m,1H),2.99-2.90(m,2H),2.80-2.71(m,2H),2.43-2.30(m,2H),2.15-1.84(m,6H),1.73-1.60(m,2H),1.55-1.23(m,10H),1.16-1.01(m,4H).
实施例21：5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-((4-(4-(5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将5-((1R,4R)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,粗品)用THF(10mL)溶解，加入9-甲酰基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(50mg,0.18mmol)和STAB(115mg,0.54mmol)，室温反应2h。反应液中加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤， 减压浓缩，粗品使用柱层析(MeOH:DCM＝0～1:9)纯化得到9-((4-(4-(5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝724.3.
步骤2：N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将9-((4-(4-(5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(100mg,0.14mmol)溶于DCM(4mL)中，加入TFA(1mL)，室温反应1h。反应液直接减压浓缩得到N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺，直接用于下一步。
LC-MS:(ESI,m/z):[M+H]+＝624.4.
步骤3：5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.14mmol)溶于DMSO(10mL)，加入DIEA(90mg,0.70mmol)和3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酸五氟苯酯(58mg,0.14mmol)，室温反应2h。反应液中加水(100mL)并搅拌5分钟，用乙酸乙酯(100mL x 3)萃取，有机相用饱和食盐水(150mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用反相制备纯化得到5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝854.4.
1H NMR(400MHz,DMSO-d6)δ10.37(s,1H),9.50(d,J＝5.7Hz,1H),8.78(d,J＝7.6Hz,1H),8.39(d,J＝3.6Hz,1H),8.26(d,J＝5.6Hz,1H),7.36-7.29(m,2H),7.26-6.96(m,2H),6.88-6.44(m,1H),5.28-5.07(m,1H),4.77(d,J＝17.6Hz,1H),4.31-4.11(m,1H),3.88-3.71(m,3H),3.63-3.45(m,5H),2.95-2.85(m,2H),2.81-2.65(m,2H),2.25-2.10(m,5H),2.07-1.87(m,8H).
实施例22：N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-)-5-(1,1-二氧代硫代吗啡啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-(1,1-二氧代硫代吗啡啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺(60mg,0.1mmol)和3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-甲醛(41mg,0.1mmol)用四氢呋喃(2mL)溶解，搅拌下加入三乙酰氧基硼氢化钠(84mg,0.4mmol)并室温搅拌3小时。反应液中加水(50mL)稀释，用乙酸乙酯(50mL x 3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-)-5-(1,1-二氧代硫代吗啡啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝890.1
1H NMR(400MHz,CD3OD-d4)δ8.64(d,J＝7.9Hz,1H),8.37(d,J＝5.8Hz,2H),7.40(d,J＝7.8Hz,1H),7.36-7.29(m,2H),7.06-6.77(m,2H),4.60-4.52(m,2H),4.42-4.34(m,4H),4.25-4.14(m,1H),3.92-3.82(m,1H),3.77-3.62(m,3H),3.49-3.40(m,2H),3.30-3.22(m,4H),3.06-3.00(m,2H),2.92-2.82(m,2H),2.31(s,3H),2.28-2.22(m,2H),2.20-2.06(m,6H),1.82-1.72(m,2H),1.70-1.63(m,2H),1.55-1.39(m,2H),1.33-1.25(m,2H),1.24-1.12(m,3H).
实施例23：(S)-5-(3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：(S)-(1-(3-((3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯的制备
将(S)-(1-(3-((3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯(45mg,0.08mmol)和3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-甲醛(33mg,0.08mmol)溶于DMF(2mL)中，NaBH(OAc)3(51mg,0.24mmol)加入，室温搅拌2小时。反应液中加入水(50mL)，用乙酸乙酯(30mL x 3)萃取，合并上层有机相并用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经正相柱层析纯化得到(S)-(1-(3-((3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝955.5.
步骤2：(S)-5-(3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将(S)-(1-(3-((3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯(30mg,0.03mmol)溶于DCM(0.5mL)中，然后加入TFA(0.1mL)，室温搅拌1小时。饱和NaHCO3水溶液加入到反应液中直至溶液pH值至弱碱性，用混合有机溶剂(MeOH:DCM＝1:10,30mL x 3)萃取，无水硫酸钠干燥，过滤，减压浓缩，粗品经p-HPLC纯化得到(S)-5-(3-氨基哌啶-1-基)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝855.7.
1H NMR(400MHz,MeOD)δ8.49(d,J＝8.0Hz,1H),8.35(s,1H),8.29(s,1H),7.40(d,J＝7.9Hz,1H),7.35-7.29(m,2H),7.04-6.74(m,2H),4.60-4.50(m,1H),4.50-4.25(m,2H),4.25-4.15(m,1H),3.97-3.79(m,1H),3.80-3.63(m,3H),3.50-3.38(m,3H),3.15-3.08(m,1H),3.06-3.01(m,2H),2.94-2.79(m,3H),2.31(s,3H),2.27-2.21(m,2H),2.21-2.04(m,7H),1.93-1.83(m,1H),1.83-1.76(m,2H),1.70-1.44(m,8H),1.22-1.02(m,4H).
实施例24：N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(4-(5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯的制备
将5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-羧酸(130mg,0.5mmol)，4-(4-氨基-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(158mg,0.5mmol)和Py-BOP(780mg,1.5mmol)用DMF(6mL)溶解，然后加入DIEA(780mg,1.5mmol)，室温搅拌16小时。反应液中加入水(50mL)，乙酸乙酯(30mL x 3)萃取，合并上层有机相并用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经正相柱层析纯化得到4-(4-(5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝559.2.
步骤2：N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将4-(4-(5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-3-(二氟甲基)-1H-吡唑-1-基)哌啶-1-羧酸叔丁酯(250mg,0.45mmol)用DCM(4mL)溶解，然后加入TFA(1mL)，室温搅拌1小时。反应液直接减压浓缩得到粗品N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝459.3.
步骤3：N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰 胺的制备
将N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(114mg,0.25mmol)和3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-甲醛(102mg,0.25mmol)用THF(5mL)溶解，三乙酰氧基硼氢化钠(160mg,0.75mmol)加入，室温搅拌16小时。反应液中加入水(50mL)，乙酸乙酯(30mL x 3)萃取，合并上层有机相并用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经prep-HPLC纯化得到白色固体N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝854.6.
1H NMR(400MHz,MeOD)δ8.47(d,J＝7.8Hz,1H),8.35(s,1H),8.27(s,1H),7.40(d,J＝7.4Hz,1H),7.36-7.27(m,2H),7.05-6.95(m,1H),6.31(d,J＝8.0Hz,1H),4.62-4.50(m,2H),4.50-4.39(m,4H),4.26-4.11(m,1H),3.95-3.82(m,1H),3.77-3.60(m,3H),3.49-3.40(m,2H),3.05-2.97(m,2H),2.89-2.79(m,2H),2.37-2.20(m,5H),2.20-2.09(m,6H),1.83-1.75(m,2H),1.71-1.41(m,7H),1.37-1.27(m,2H),1.24-1.08(m,4H).
实施例25：(S)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
将(S)-N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(80mg,粗品,0.11mmol)用二甲亚砜(3mL)溶解，搅拌下加入3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酸五氟苯酯(46mg,0.11mmol)和N,N-二异丙基乙胺(142mg,1.1mmol)，室温搅拌过夜。反应液中加水(50mL)，过滤，滤饼经Pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到(S)-N-(3-(二氟甲基)-1-(1-((3-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝856.3.
1H NMR(400MHz,DMSO-d6)δ10.38(s,1H),9.38(s,1H),8.72(d,J＝8.0Hz,1H),8.38(s,1H),8.24 (s,1H),7.37-7.29(m,2H),7.26-6.96(m,2H),6.86(d,J＝8.0Hz,1H),4.89(d,J＝2.0Hz,1H),4.26-4.14(m,1H),4.00-3.75(m,3H),3.70-3.39(m,6H),3.30-3.24(m,2H),2.93-2.64(m,4H),2.21(s,3H),2.18-2.10(m,2H),2.06-1.65(m,10H),1.61-1.22(m,9H),1.16-0.95(m,4H).
实施例26：(S)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
将(S)-N-(1-(1-((3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(80mg,粗品,0.11mmol)用二甲亚砜(3mL)溶解，搅拌下加入perfluorophenyl 4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(48mg,0.11mmol)和N,N-二异丙基乙胺(142mg,1.1mmol)，室温下搅拌过夜。反应液中加水(30mL)，过滤，滤饼经Pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到(S)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝876.3.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.38(s,1H),8.72(d,J＝7.9Hz,1H),8.38(s,1H),8.24(s,1H),7.63(d,J＝8.2Hz,1H),7.55(s,1H),7.39(d,J＝8.5Hz,1H),7.26-6.97(m,1H),6.86(d,J＝8.0Hz,1H),4.88(d,J＝2.0Hz,1H),4.25-4.15(m,1H),3.97-3.40(m,9H),3.30-3.18(m,2H),2.93-2.85(m,2H),2.78-2.72(m,2H),2.20-2.10(m,2H),2.05-1.65(m,10H),1.60-1.31(m,9H),1.20-0.94(m,4H).
实施例27：N-(3-(二氟甲基)-1-((1R,4S)-4-((9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-(((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
N-(3-(二氟甲基)-1-((1R,4S)-4-甲醛环己基)-1H-吡唑-4-基)-5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(90mg,0.15mmol)和3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(40mg, 0.15mmol)用四氢呋喃(10mL)溶解，加入三乙酰氧基硼氢化钠(95mg,0.45mmol)，室温搅拌2小时，加水(50mL)，用二氯甲烷(20mL x 3)萃取，有机相用饱和食盐水(60mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(甲醇:二氯甲烷＝1:9)纯化得到9-(((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝810.4.
步骤2：N-(1-((1R,4S)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
9-(((1S,4R)-4-(3-(二氟甲基)-4-(5-((3S)-3-((四氢-2H-吡喃-2-基)氧)哌啶-1-基)吡唑并[1,5-a]嘧啶-3-羧酰氨基)-1H-吡唑-1-基)环己基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(100mg,0.12mmol)用二氯甲烷(3mL)溶解，三氟乙酸(1mL)加入并室温搅拌1小时。反应液直接减压浓缩得到N-(1-((1R,4S)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝626.3.
步骤3：N-(3-(二氟甲基)-1-((1R,4S)-4-((9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
N-(1-((1R,4S)-4-((3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-3-(二氟甲基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(100mg,0.12mmol)用二甲亚砜(3mL)溶解，加入3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲基苯甲酸五氟苯酯(55mg,0.13mmol)和N,N-二异丙基乙胺(155mg,1.2mmol)，室温搅拌过夜。反应液中加水(30mL)，过滤，滤饼经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-((1R,4S)-4-((9-(3-(2,4-二氧代四氢嘧啶-1(2H)- 基)-4-甲基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)甲基)环己基)-1H-吡唑-4-基)-5-((S)-3-羟基哌啶-1-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝856.4.
1H NMR(400MHz,DMSO-d6)δ10.39(s,1H),9.38(s,1H),8.72(d,J＝8.0Hz,1H),8.36(s,1H),8.24(s,1H),7.36-7.29(m,2H),7.27-6.94(m,2H),6.86(d,J＝8.0Hz,1H),4.93-4.88(m,1H),4.20-4.10(m,1H),4.01-3.74(m,3H),3.70-3.44(m,6H),3.33-3.25(m,2H),2.84-2.64(m,2H),2.40-2.21(m,4H),2.21(s,3H),2.15-1.96(m,4H),1.87-1.70(m,6H),1.65-1.26(m,11H),1.10-0.95(m,2H).
实施例28：N-(3-(二氟甲基)-1-(1-(3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(200mg,0.79mmol)用二甲亚砜(4mL)溶解，搅拌下加入3-(2,4-二氧代基四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯基酯(340mg,0.79mmol)和N,N-二异丙基乙胺(413mg,3.24mmol)，反应液室温搅拌3小时。反应液加水(40mL)稀释，用乙酸乙酯(50mL x3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(乙酸乙酯:石油醚＝9:1)纯化得到9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝501.2.
步骤2：1-(2-甲氧基-5-(3,9-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-羧酸叔丁酯(230mg,0.46mmol)用二氯甲烷(3mL)溶解，加入三氟乙酸(0.3mL)并室温搅拌1小时。反应液直接减压浓缩得到1-(2-甲氧基-5-(3,9-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝401.1.
步骤3：1-(5-(9-(2-(1,3-二氧戊烷-2-基)乙基)-3,9-二氮杂螺[5.5]十一烷-3-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将1-(2-甲氧基-5-(3,9-氮杂螺[5.5]十一烷-3-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(180mg,0.45mmol)和2-(2-溴乙基)-1,3-二氧戊烷(121mg,0.67mmol)用DMF(5mL)溶解，搅拌下加入K2CO3(186mg,1.3mmol)，反应液加热至80℃并搅拌12小时。反应液加水(80mL)稀释，用乙酸乙酯(50mL x 3)萃取，有机相用饱和食盐水(80mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(甲醇:二氯甲烷＝1:7)纯化得到1-(5-(9-(2-(1,3-二氧戊烷-2-基)乙基)-3,9-二氮杂螺[5.5]十一烷-3-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝501.7.
步骤4：3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙醛的制备
1-(5-(9-(2-(1,3-二氧戊烷-2-基)乙基)-3,9-二氮杂螺[5.5]十一烷-3-羰基)-2-甲氧基苯基)二氢嘧啶-2,4(1H,3H)-二酮(200mg,0.5mmol)用丙酮(5mL)溶解，加入盐酸(2mol/L,10mL)，室温下搅拌6小时。反应液用碳酸氢钠调节反应液pH＝8，乙酸乙酯(50mL x 3)萃取，有机相经无水硫酸钠干燥，过滤，减压浓缩，得到3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙醛，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝457.7.
步骤5：N-(3-(二氟甲基)-1-(1-(3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙醛(70mg,0.15mmol)和N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰 胺(67mg,0.15mmol)用二氯乙烷(5mL)溶解，搅拌下加入三乙酰氧基硼氢化钠(126mg,0.6mmol)，反应液室温搅拌3小时。反应液加水(50mL)稀释，用二氯甲烷(60mL x 3)萃取，有机相经无水硫酸钠干燥，过滤，减压浓缩，粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(3-(二氟甲基)-1-(1-(3-(9-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)-3,9-二氮杂螺[5.5]十一烷-3-基)丙基)哌啶-4-基)-1H-吡唑-4-基)-5-吗啡啉吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝887.2.
1H NMR(400MHz,DMSO-d6)δ10.32(s,1H),9.40(s,1H),8.83(d,J＝7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.37(d,J＝8.4Hz,1H),7.32(d,J＝2.1Hz,1H),7.26-6.97(m,2H),6.91(d,J＝7.9Hz,1H),4.25-4.15(m,1H),3.84(s,3H),3.82-3.70(m,8H),3.62-3.33(m,6H),2.95-2.89(m,2H),2.72-2.62(m,2H),2.36-2.23(m,8H),2.05-1.80(m,6H),1.60-1.24(m,10H).
实施例29：N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(1,1-二氧代硫代吗啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺
将N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)-5-(1,1-二氧代硫代吗啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺(80mg,0.16mmol)和3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-甲醛(70mg,0.16mmol)溶于THF(3mL)中，加入三乙酰氧基硼氢化钠(102mg,0.48mmol)并室温搅拌16小时。反应液中加入水(50mL)，EA(30mL x 3)萃取，有机相用饱和食盐水(30mL)洗，无水硫酸钠干燥，过滤，减压浓缩，粗品经Prep-HPLC分离纯化得到N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)-5-(1,1-二氧代硫代吗啉基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝910.4.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),9.25(s,1H),8.92(d,J＝7.9Hz,1H),8.39(s,1H),8.34(s,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.8Hz,1H),7.39(d,J＝8.2Hz,1H),7.25-6.97(m,2H),4.31-4.20(m,5H),3.77-3.70(m,1H),3.67-3.57(m,3H),3.33-3.23(m,5H),2.90(d,J＝9.3Hz,2H),2.75-2.67(m,3H),2.18-2.11(m,2H),2.07-1.87(m,6H),1.73-1.65(m,2H),1.62-1.23(m,7H),1.15-0.92(m,4H).
实施例30：(S)-5-(3-氨基哌啶-1-基)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：(S)-(1-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁 酯的制备
(S)-(1-(3-((3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)氨基甲酸叔丁酯(70mg,0.12mmol)和3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-甲醛(70mg,0.15mmol)用THF(2mL)溶解，加入NaBH(OAc)3(56mg,0.37mmol)，反应液室温搅拌2h。反应液用水(20mL)稀释，乙酸乙酯(20mL x 3)，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗品使用柱层析(甲醇:二氯甲烷＝1:24)纯化得到(S)-(1-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝975.4.
步骤2：(S)-5-(3-氨基哌啶-1-基)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将(S)-(1-(3-((1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)氨甲酰基)吡唑并[1,5-a]嘧啶-5-基)哌啶-3-基)羧酸叔丁酯(30mg,0.03mmol)用DCM(5mL)溶解，加入TFA(1mL)，反应液室温搅拌1小时。反应液直接减压浓缩，粗品使用Prep-HPLC纯化得到(S)-5-(3-氨基哌啶-1-基)-N-(1-(1-((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)-3-氮杂螺[5.5]十一烷-9-基)甲基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝875.3.
1H NMR(400MHz,DMSO-d6)δ8.77(d,J＝7.9Hz,1H),8.37(s,1H),8.34-8.30(m,2H),7.65(d,J＝8.2Hz,1H),7.53(s,1H),7.40(d,J＝8.4Hz,1H),7.26-6.95(m,1H),6.85(d,J＝7.9Hz,1H),4.23-4.13(m,2H),3.82-3.74(m,1H),3.70-3.40(m,7H),3.30-3.10(m,3H),2.97-2.92(m,2H),2.77-2.73(m,2H),2.25-1.75(m,11H),1.75-1.15(m,12H),1.15-0.96(m,4H).
实施例31：5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲 酰胺
步骤1：4-(3-羟基丙氧基)哌啶-1-羧酸苄酯的制备
4-(3-甲氧基-3-氧丙基)哌啶-1-羧酸苄酯(500mg,1.49mmol)溶解于无水THF(10mL)中，0℃下加入BH3/THF溶液(15mL,14.9mmol)，室温搅拌16小时。向反应液加入甲醇淬灭，减压浓缩得到粗品。粗品使用柱层析(乙酸乙酯:石油醚＝9:1)纯化得到4-(3-羟基丙氧基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝294.2.
步骤2：3-(哌啶-4-基氧)丙烷-1-醇的制备
4-(3-羟基丙氧基)哌啶-1-羧酸苄酯(200mg,0.68mmol)溶解于乙酸乙酯(10mL)中，加入Pd(OH)2/C(80mg)，在氢气氛围下升温至70℃，搅拌过夜。反应液冷却至室温，过滤，滤液减压浓缩得到3-(哌啶-4-基氧)丙烷-1-醇，粗品无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝160.2.
步骤3：1-(2-氯-5-(4-(3-羟基丙氧基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
3-(哌啶-4-基氧)丙烷-1-醇(100mg,0.62mmol)溶解于DMSO(10mL)中，加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(300mg,0.69mmol)和DIEA(240mg,1.86mmol)，室温下搅拌2小时。向反应液加入水(100mL)，用二氯甲烷(30mL x 3)萃取，有机相经无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗品使用柱层析(甲醇:二氯甲烷＝1:9)纯化得到1-(2-氯-5-(4-(3-羟基丙氧基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝410.1.
步骤4：3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙醛的制备
1-(2-氯-5-(4-(3-羟基丙氧基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(60mg,0.147mmol)溶解于DCM(5mL)中，加入PCC(63mg,0.294mmol)，室温下搅拌16小时。反应液减压浓缩，粗品使用柱层析(甲醇:二氯甲烷＝1:9)纯化得到3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙醛。
LC-MS:(ESI,m/z):[M+H]+＝408.1.
步骤5：5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙醛(55mg,0.135mmol)溶于THF(5mL)中，加5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(3-(二氟甲基)-1-(哌啶-4-基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(粗产品:70mg,0.135mmol)和STAB(86mg,0.405mmol)，室温下搅拌2小时。向反应液加入水(100mL)，用乙酸乙酯(30mL x 3)萃取，有机相用饱和氯化钠水溶液(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到5-((1R,4R)-2-氧-5-氮杂双环[2.2.1]庚烷-5-基)-N-(1-(1-(3-((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)氧)丙基)哌啶-4-基)-3-(二氟甲基)-1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝850.4.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),9.50(d,J＝5.7Hz,1H),8.79(d,J＝7.8Hz,1H),8.40(d,J＝3.2Hz,1H),8.26(d,J＝5.5Hz,1H),7.67-7.55(m,2H),7.40(d,J＝8.7Hz,1H),7.30-6.92(m,1H),6.88-6.45(m,1H),5.28-5.08(m,1H),4.77(d,J＝17.7Hz,1H),4.30-4.10(m,1H),4.02-3.68(m,4H),3.62-3.47(m,7H),3.30-3.10(m,2H),3.00-2.85(m,2H),2.78-2.69(m,2H),2.45-2.31(m,2H),2.10-1.55(m,12H),1.52-1.37(m,2H).
实施例32：N-(3-(二氟甲基)-1-((1R,4R)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺
步骤1：4-(2-(((苄氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯的制备
将4-(2-(甲胺基)乙基)哌啶-1-羧酸叔丁酯(200mg,0.82mmol)溶于二氯甲烷(4mL)中，加入三乙胺(249,2.46mmol)和CbzCl(139mg,0.82mmol)，反应液室温搅拌1个小时。反应液加水(50mL)稀释，二氯甲烷(80mL x 3)萃取，用无水硫酸钠干燥，减压浓缩，粗品经柱层析(PE:EA＝3:7)纯化得到化合物4-(2-(((苄氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H-100]+＝277.2.
步骤2：苄基甲基(2-(哌啶-4-基)乙基)氨基甲酸酯的制备
将化合物4-(2-(((苄氧基)羰基)(甲基)氨基)乙基)哌啶-1-羧酸叔丁酯(200mg,0.53mmol)溶于氯化氢/1-4,二氧六环(4mL)溶液中，反应液室温下搅拌1小时。反应液直接减压浓缩得到苄基甲基(2-(哌啶-4-基)乙基)氨基甲酸酯。
LC-MS:(ESI,m/z):[M+H]+＝277.2.
步骤3：苄基(2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基甲酸酯的制备
将化合物苄基甲基(2-(哌啶-4-基)乙基)氨基甲酸酯(200mg,0.72mmol)和3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酸五氟苯酯(309mg,0.72mmol)加入到DMSO(4mL)溶液中，然后加入DIEA(278mg,2.1mmol)，反应液室温搅拌3小时。反应液用水(50mL)稀释，乙酸乙酯(70mL x 4)萃取，饱和食盐水洗，无水硫酸钠干燥，减压浓缩，粗品使用柱层析(PE:EA＝1:3)纯化得到化合物苄基(2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基甲酸酯。
LC-MS:(ESI,m/z):[M+H]+＝523.2.
步骤4：1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮的制备
将化合物苄基(2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基甲酸酯(120mg 0.23mmol)溶于甲醇(3mL)溶液中，加入钯碳(10％，24mg)，反应液室温搅拌1小时。反应液经硅藻过抽滤，滤液减压浓缩得到化合物1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮。
LC-MS:(ESI,m/z):[M+H]+＝389.2.
步骤5：N-(3-(二氟甲基)-1-((1R,4R)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺的制备
将化合物N-(3-(二氟甲基)-1-((1R,4R)-4-醛基环己基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺(80mg,0.165mmol)和1-(2-甲氧基-5-(4-(2-(甲基氨基)乙基)哌啶-1-羰基)苯基)二氢嘧啶-2,4(1H,3H)-二酮(64mg,0.165mmol)溶于DCE(3mL)中，STAB(70mg,0.330mmol)加入，反应液室温搅拌2小时。反应液用水(20mL)稀释，乙酸乙酯(25mL x 4)萃取，用饱和食盐水洗，无水硫酸钠干燥，减压浓缩，粗品使用反相制备得到化合物N-(3-(二氟甲基)-1-((1R,4R)-4-(((2-(1-(3-(2,4-二氧代四氢嘧啶-1(2H)-基)-4-甲氧基苯甲酰)哌啶-4-基)乙基)(甲基)氨基)甲基)环己基)-1H-吡唑-4-基)-5-(2-氧-6-氮杂螺[3.3]庚烷-6-基)吡唑并[1,5-a]嘧啶-3-甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝858.4.
1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.69(s,1H),8.76(d,J＝7.6Hz,1H),8.37(s,1H),8.25(s,1H),7.35(s,1H),7.42-7.00(m,3H),6.38(d,J＝7.6Hz,1H),4.78-4.70(m,4H),4.48-4.35(m,4H),4.25-4.10(m,1H),3.84(s,3H),3.59(t,J＝5.0Hz,2H),3.30-3.10(m,3H),2.75-2.63(m,2H),2.36-2.25(m,2H),2.20-1.99(m,7H),1.93-1.83(m,2H),1.77-1.50(m,7H),1.44-1.30(m,2H),1.20-0.96(m,4H).
实施例33：N-(2-((1S,4S)-4-(((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)甲氧基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺
步骤1：(1-氧杂螺[2.5]辛烷-6-基)氨基甲酸叔丁酯的制备
0℃时，搅拌下向Me3OS+I-(6.2g,28.2mmol)的DMSO(50mL)溶液中加入NaH(1.17g,29.3mmol)，反应混合物在室温搅拌1h，然后滴加(4-氧代环己基)氨基甲酸叔丁酯(5.0g,23.5mmol)的DMSO(20mL)溶液，滴加完毕，反应液在室温继续搅拌1h。反应液中加水(700mL)，用EtOAc(100mL×3)萃取。有机相用饱和食盐水(300mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。粗产物经柱层析(EA:PE＝0～1:1)纯化得到(1-氧杂螺[2.5]辛烷-6-基)氨基甲酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ6.79(d,J＝7.2Hz,1H),3.37(m,1H),2.57(s,2H),1.83(td,J＝13.3,4.1Hz,2H),1.77-1.64(m,2H),1.48-1.40(m,2H),1.38(s,9H),1.20(d,J＝13.1Hz,2H).
步骤2：(4-羟基-4-(羟基甲基)环己基)氨基甲酸叔丁酯的制备
搅拌下向(1-氧杂螺[2.5]辛烷-6-基)氨基甲酸叔丁酯(4.7g,20.7mmol的NMP(50mL)溶液中加入 2N NaOH(47mL)，然后反应液在100℃搅拌2h。反应液中加水(500mL)，用EA(100mL×3)萃取。有机相用饱和食盐水(300mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。粗品经flash柱(MeOH:DCM＝0～1:9)纯化得到(4-羟基-4-(羟基甲基)环己基)氨基甲酸叔丁酯。
1H NMR(400MHz,DMSO-d6)δ6.67(d,J＝7.9Hz,1H),4.46(t,J＝5.8Hz,1H),3.81(s,1H),3.11(t,J＝6.8Hz,3H),1.56-1.45(m,4H),1.44-1.34(m,13H).
步骤3：(4-羟基-4-((吡啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯的制备
0℃时向(4-羟基-4-(羟基甲基)环己基)氨基甲酸叔丁酯(600mg,2.4mmol)的THF(20mL)溶液中加NaH(292mg,7.3mmol)，反应混合物保持在0℃搅拌20min，然后在室温再继续搅拌30min后加入4-(溴甲基)吡啶溴化氢盐(617mg,2.4mmol)。反应液在室温搅拌过夜。反应液中加水(30mL)，用EA(20mL×3)萃取，有机相用饱和食盐水(60mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗产物经flash柱(EA:PE＝0～4:1)纯化得到(4-羟基-4-((吡啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝337.1.
1H NMR(400MHz,DMSO-d6)δ8.53(d,J＝5.8Hz,2H),7.34(d,J＝5.6Hz,2H),6.69(d,J＝7.6Hz,1H),4.55(s,2H),4.19(s,1H),3.23(s,2H),3.18-3.07(m,1H),1.60-1.48(m,6H),1.42-1.35(m,J＝9.2Hz,11H).
步骤4：(4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯的制备
将(4-羟基-4-((吡啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯(700mg,2.07mmol)和Pd/C(400mg)的i-PrOH/H2O(18mL/21mL)混合液在H2氛围中75℃下搅拌过夜。反应液经硅藻土过滤，滤液减压浓缩得到(4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯。所得粗品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝343.2.
步骤5：4-(((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯的制备
将(4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)氨基甲酸叔丁酯(630mg,1.83mmol)，CbzCl(376 mg,2.20mmol)的ACN(10mL)溶液和饱和NaHCO3水溶液(10mL)室温搅拌过夜。反应液先减压浓缩，再用DCM(50mL×3)萃取。有机相用饱和食盐水(150mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。粗品经flash柱(EA:PE＝0～7:3)纯化得到4-(((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+Na]+＝499.3.
步骤6：4-(((4-氨基-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯的制备
-10～0℃下，边搅拌边向4-(((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯(300mg,0.63mmol)的DCM(12mL)溶液中加入TFA(3mL)，然后反应液在室温搅拌2h。反应混合液用DIEA调节pH至9.0，然后直接减压浓缩得到4-(((4-氨基-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯。所得粗产品无需纯化，直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝377.2.
步骤7：4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯的制备
搅拌下向4-(((4-氨基-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸苄酯(237mg,0.63mmol)的甲苯(10mL)溶液中加2-叠氮-4-甲氧基-5-硝基苯甲醛(170mg,0.76mmol)。反应液在100℃搅拌3h。反应液直接减压浓缩。粗产物先经flash柱(EA:PE＝0～4:1)纯化，再经Prep-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝553.2.
1H NMR(400MHz,DMSO-d6)δ8.58(s,1H),8.37(s,1H),7.40-7.29(m,5H),7.26(s,1H),5.07(s,2H),4.45(t,J＝12.0Hz,1H),4.36(s,1H),4.06-3.99(m,2H),3.90(s,3H),3.29(d,J＝6.2Hz,2H),3.20(s,2H),2.85-2.75(m,2H),2.35-2.20(m,2H),1.89(d,J＝11.8Hz,2H),1.76-1.61(m,7H),1.15-1.02(m,2H).
步骤8：4-((((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸叔丁酯的制备
向4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯(200mg,0.36mmol)的EtOH(20mL)溶液中加Raney-Ni(0.3mL)和N2H4H2O(0.3mL)。反应液在室温搅拌2h。反应液经硅藻土过滤，滤液直接减压浓缩得到4-((((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸叔丁酯。所得粗产物直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝523.2.
步骤9：4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯的制备
搅拌下向4-((((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)甲基)哌啶-1-羧酸叔丁酯(190mg,0.36mmol)的DMF(5mL)溶液中加6-(三氟甲基)吡啶甲酸(90mg,0.47mmol)，HATU(178mg,0.47mmol)和DIEA(140mg,1.08mmol)。然后反应液在室温搅拌2h。反应液中加水(30mL)，用EA(20mL×3)。有机相用饱和食盐水(60mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(EA:PE＝0～4:1)纯化得到4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝696.3.
1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),8.69(s,1H),8.47(d,J＝7.7Hz,1H),8.41(t,J＝7.8Hz,1H),8.32(s,1H),8.22(d,J＝7.7Hz,1H),7.41-7.29(m,5H),7.16(s,1H),5.08(s,2H),4.40-4.29(m,2H),4.15-3.95(m 5H),3.29(d,J＝6.2Hz,2H),3.21(s,2H),2.91-2.70(m,2H),2.35-2.15(m,2H),1.90(d,J＝9.5Hz,2H),1.85-1.60(m,7H),1.12-.1.02(m,2H).
步骤10：N-(2-((1S,4S)-4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
向4-((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)甲基)哌啶-1-羧酸苄酯(170mg,0.24mmol)的EA(20mL)溶液中加入Pd(OH)2/C(80mg)。然后反应液在H2氛围中70℃下搅拌过夜。反应液经硅藻土过滤，滤液直接减压浓缩得到N-(2-((1S,4S)-4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝562.3.
步骤11：N-(2-((1S,4S)-4-(((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)甲氧基) 甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
将N-(2-((1S,4S)-4-羟基-4-((哌啶-4-基甲氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺(130mg,0.23mmol)，4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(121mg,0.28mmol)和DIEA(89mg,0.69mmol)在DMSO(6mL)中的混合液在室温搅拌过夜。反应液加水(50mL)稀释，用DCM(20mL×3)。有机相用饱和食盐水(60mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经Prep-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(2-((1S,4S)-4-(((1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)甲氧基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝812.1.
1H NMR(400MHz,DMSO-d6)δ10.51(d,J＝7.5Hz,2H),8.69(s,1H),8.46(d,J＝7.7Hz,1H),8.40(t,J＝7.8Hz,1H),8.32(s,1H),8.21(d,J＝7.6Hz,1H),7.65(d,J＝8.2Hz,1H),7.57(d,J＝1.4Hz,1H),7.40(dd,J＝8.2,1.5Hz,1H),7.16(s,1H),4.58-4.29(m,3H),3.99(s,3H),3.84-3.55(m,3H),3.33-3.28(m,2H),3.22(s,2H),3.13-3.03(m,1H),2.88-2.70(m,3H),2.35-2.20(m,2H),1.94-1.59(m,9H),1.21-1.15(m,2H).
实施例34：N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙氧基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺
步骤1：4-(2-((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
0℃向(4-羟基-4-(羟基甲基)环己基)氨基甲酸叔丁酯(770mg,3.13mmol)的THF(20mL)溶液中加NaH(376mg,9.4mmol)，并在0℃搅拌2h，然后加入4-(2-(对甲苯磺酰基)乙基)哌啶-1-羧酸苄酯(2.62g,6.26mmol)和KI(1.04g,6.26mmol)。反应液在70℃搅拌过夜。反应液加水(50mL)稀释，用EA(30mL×3)萃取。有机相用饱和食盐水(100mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(EA:PE＝0～3:2)得到4-(2-((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+Na]+＝513.1.
1H NMR(400MHz,DMSO-d6)δ7.41-7.29(m,5H),6.67(d,J＝7.5Hz,1H),5.06(s,2H),4.05-3.90(m,3H),3.42(t,J＝6.4Hz,2H),3.10(s,3H),2.88-2.68(m,2H),1.65(d,J＝12.6Hz,2H),1.55-1.32(m, 20H),1.13-0.92(m,2H).
步骤2：4-(2-((4-氨基-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
-10～0℃时，搅拌下向4-(2-((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(270mg,0.55mmol)的DCM(3mL)溶液中加TFA(1.2mL)，然后反应液在室温搅拌2h。反应液中加DIEA调节pH至9.0，减压浓缩得到4-(2-((4-氨基-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝391.2.
步骤3：4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
搅拌下向4-(2-((4-氨基-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(400mg,0.55mmol)的甲苯(15mL)溶液中加2-叠氮-4-甲氧基-5-硝基苯甲醛(150mg,0.66mmol)。反应液在100℃搅拌3h。反应液减压浓缩，所得粗品经flash柱(EA:PE＝0～9:1)纯化得到4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝567.3.
1H NMR(400MHz,DMSO-d6)δ8.59(s,1H),8.38(s,1H),7.41-7.29(m,5H),7.27(s,1H),5.07(s,2H),4.50-4.40(m,1H),4.34(s,1H),4.05-3.95(m,2H),3.91(s,3H),3.47(t,J＝6.4Hz,2H),3.21(s,2H),2.90-2.70(m,2H),2.35-2.19(m,2H),1.90(d,J＝9.7Hz,2H),1.75-1.50(m,7H),1.50-1.40(m,2H),1.11-0.98(m,2H).
步骤4：4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
向4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(250mg,0.44mmol)的EtOH(20mL)溶液中加Raney-Ni(0.5mL)和N2H4.H2O(0.5mL)。反应液在室温搅拌2h。反应液经硅藻土过滤，滤液减压浓缩得到4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。所得产物直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝537.3
步骤5：4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
边搅拌边向4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(236mg,0.44mmol)的DMF(5mL)溶液中加6-(三氟甲基)吡啶甲酸(101mg,0.53mmol)，HATU(217mg,0.57mmol)和DIEA(170mg,1.32mmol)。然后反应液在室温搅拌2h。反应液中加水(50mL)稀释，用EA(20mL×3)萃取。有机相用饱和食盐水(60mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(EA:PE＝0～4:1)纯化得到4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝710.2.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),8.69(s,1H),8.46(d,J＝7.7Hz,1H),8.40(t,J＝7.8Hz,1H),8.32(s,1H),8.21(d,J＝7.7Hz,1H),7.41-7.28(m,5H),7.16(s,1H),5.06(s,2H),4.40-4.20(m,2H),4.10-3.90(m,5H),3.47(t,J＝6.4Hz,2H),3.21(s,2H),2.90-2.70(m,2H),2.32-2.18(m,2H),1.90(d,J＝10.4Hz,2H),1.72-1.53(m,7H),1.52-1.40(m,2H),1.15-1.00(m,2H).
步骤6：N-(2-((1S,4S)-4-羟基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
向4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(200mg,0.28mmol)的EA(20mL)溶液中加Pd(OH)2/C(100mg)。然后反应液在H2氛围中70℃下搅拌过夜。反应液经硅藻土过滤，滤液浓缩得到N-(2-((1S,4S)-4-羟基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺，所得产品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝576.2.
步骤7：N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙氧基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
将N-(2-((1S,4S)-4-羟基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺(130mg,0.22mmol)，4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(118mg,0.27mmol)和DIEA(87mg,0.68mmol的DMSO(3mL)溶液在室温搅拌过夜。反应液加水(30mL)稀释， 用DCM(10mL×3)萃取。有机相用饱和食盐水(30mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(MeOH:DCM＝0～1:9)纯化得到N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)哌啶-4-基)乙氧基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝826.1.
1H NMR(400MHz,DMSO-d6)δ10.51(d,J＝5.7Hz,2H),8.69(s,1H),8.46(d,J＝7.8Hz,1H),8.41(t,J＝7.8Hz,1H),8.33(s,1H),8.22(d,J＝7.7Hz,1H),7.64(d,J＝8.2Hz,1H),7.55(d,J＝1.8Hz,1H),7.39(dd,J＝8.2,1.8Hz,1H),7.16(s,1H),4.50-4.26(m,3H),3.98(s,3H),3.83-3.53(m,3H),3.48(t,J＝6.3Hz,2H),3.21(s,2H),3.13-2.97(m,1H),2.86-2.71(m,3H),2.32-2.18(m,2H),1.90(d,J＝9.8Hz,2H),1.85-1.45(m,9H),1.15-1.05(m,2H).
实施例35：N-(2-((1S,4S)-4-((((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺
步骤1：(4-羟基-4-(甲基氨基)甲基)环己基)氨基甲酸叔丁酯的制备
向(1-氧杂螺[2.5]辛烷-6-基)氨基甲酸叔丁酯(4.2g,18.42mmol)的EtOH/H2O(90mL/15mL)混合液中加甲胺(40mL,25％～30％水溶液)。然后反应液在25℃搅拌过夜。反应液直接减压浓缩，所得粗品经flash(MeOH:DCM＝0～1:1)纯化得到(4-羟基-4-(甲基氨基)甲基)环己基)氨基甲酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝259.2.
步骤2：((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯的制备
将(4-羟基-4-(甲基氨基)甲基)环己基)氨基甲酸叔丁酯(1.2g,4.61mmol)和氯甲酸苄酯(941mg,5.53mmol)的ACN/sat.NaHCO3(20mL/20mL)的混合液在室温搅拌过夜。反应液用水(50mL)稀释，然后用EA(50mL×3)萃取。有机相用饱和食盐水(150mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash(EA:PE＝0～7:3)纯化得到((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯。
LC-MS:(ESI,m/z):[M+Na]+＝415.2.
步骤3：((4-氨基-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯的制备
0℃时，边搅拌边向((4-((叔丁氧羰基)氨基)-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯(1.5g,3.81mmol)的DCM(15mL)溶液中加TFA(5mL)，然后反应液在0℃搅拌2h。反应液用DIEA调节pH至 9.0，然后直接减压浓缩得到((4-氨基-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯，所得粗品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝293.1.
步骤4：(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯的制备
搅拌下向((4-氨基-1-羟基环己基)甲基)(甲基)氨基甲酸苄酯(1.5g,3.8mmol)的甲苯(40mL)溶液中加2-叠氮-4-甲氧基-5-硝基苯甲醛(1.2g,5.7mmol)。反应液在100℃搅拌3h。反应液直接减压浓缩。所得粗品经flash柱(EA:PE＝0～1)纯化得到(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝469.2
1H NMR(400MHz,DMSO-d6)δ8.58(s,1H),8.38(s,1H),7.45-7.23(m,6H),5.09(s,2H),4.66-4.36(m,2H),3.91(s,3H),3.28(s,2H),3.10-2.95(m,3H),2.30-2.20(m,2H),1.95-1.60(m,2H),1.78-1.63(m,2H),1.59-1.41(m,2H).
步骤5：(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基甲基)(甲基)氨基甲酸苄酯的制备
向(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯(1.1g,2.34mmol)的EtOH(20mL)溶液中加N2H4H2O(1.2mL)和Raney-Ni(0.5mL)。反应液在室温搅拌2h。反应液经硅藻土过滤，滤液减压浓缩得到(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基甲基)(甲基)氨基甲酸苄酯，所得粗品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝439.2
步骤6：(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯的制备
搅拌下向(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲哚-2-基)-1-羟基环己基甲基)(甲基)氨基甲酸苄酯(940mg,2.14mmol)的DMF(10mL)溶液中加6-(三氟甲基)吡啶甲酸(538mg,2.78mmol)，HATU(1.06g,2.78mmol)和DIEA(828mg,6.42mmol)。然后反应液在室温搅拌过夜。反应液加水(100mL)稀释，用EA(30mL×3)萃取。有机相用饱和食盐水(100mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所 得粗品经flash柱(EA:PE＝0～9:1)纯化得到(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝612.2.
1H NMR(400MHz,DMSO-d6)δ10.50(s,1H),8.69(s,1H),8.46(d,J＝7.5Hz,1H),8.40(t,J＝7.8Hz,1H),8.31(s,1H),8.21(dd,J＝7.7,1.0Hz,1H),7.42-7.28(m,5H),7.16(s,1H),5.09(s,2H),4.60-4.31(m,2H),3.98(s,3H),3.28(s,2H),3.10-2.95(m,3H),2.30-2.15(m,2H),1.95-1.60(m,2H),1.67(t,J＝14.9Hz,2H),1.57-1.40(m,2H).
步骤7：N-(2-((1S,4S)-4-羟基-4-((甲基氨基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
向(((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基甲酸苄酯(500mg,0.81mmol)的EA(20mL)溶液中加Pd(OH)2/C(200mg)。然后反应液在H2氛围下70℃下搅拌过夜。反应液经硅藻土过滤，滤液减压浓缩得到N-(2-((1S,4S)-4-羟基-4-((甲基氨基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺，所得粗品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝478.1.
步骤8：9-(((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯的制备
将N-(2-((1S,4S)-4-羟基-4-((甲基氨基)甲基)环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺(200mg,0.41mmol)，9-醛基-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(141mg,0.50mmol)和STAB(434mg,2.05mmol)在THF(10mL)中的混合液在室温搅拌过夜。反应液加水(30mL)稀释，然后用DCM(10mL×3)萃取。有机相用饱和食盐水(30mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经flash柱(MeOH:DCM＝0～1:9)纯化得到9-(((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯。
LC-MS:(ESI,m/z):[M+H]+＝743.3.
步骤9：N-(2-((1S,4S)-4-((((3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
0℃向9-(((((1S,4S)-1-羟基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲哚-2-基)环己基)甲基)(甲基)氨基)甲基)-3-氮杂螺[5.5]十一烷-3-羧酸叔丁酯(200mg,0.27mmol)的DCM(5mL)溶液中加TFA(2mL)。然后反应液在室温搅拌2h。反应液用DIE调节pH至9.0。有机相用水(100mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩得到N-(2-((1S,4S)-4-((((3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺，所得粗品直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝643.3.
步骤10：N-(2-((1S,4S)-4-((((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
将N-(2-((1S,4S)-4-((((3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺(130mg,0.20mmol)，4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(105mg,0.24mmol)和DIEA(77mg,0.60mmol)在DMSO(5mL)中的混合液在室温搅拌过夜。反应液加水(50mL)稀释，然后用DCM(20mL×3)萃取，有机相用饱和食盐水(60mL)洗涤，无水Na2SO4干燥，过滤，减压浓缩。所得粗品经Prep-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(2-((1S,4S)-4-((((3-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰基)-3-氮杂螺[5.5]十一烷-9-基)甲基)(甲基)氨基)甲基)-4-羟基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝893.1.
1H NMR(400MHz,DMSO-d6)δ10.51(s,2H),8.69(s,1H),8.47(d,J＝7.8Hz,1H),8.41(t,J＝7.8Hz,1H),8.32(s,1H),8.22(d,J＝7.8Hz,1H),7.63(d,J＝8.2Hz,1H),7.55(d,J＝1.7Hz,1H),7.39(d,J＝8.3Hz,1H),7.15(s,1H),4.37-4.27(m,1H),4.10-3.90(m,4H),3.82-3.49(m,4H),3.35-3.25(m,1H),2.80-2.70(m,2H),2.36-2.15(m,9H),1.95-1.85(m,2H),1.78-1.22(m,14H),1.18-0.98(m,4H).
实施例36：N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙氧基)甲基)-4-甲氧基环己基)-6-甲氧基-2H-吲哚-5-基)-6-(三氟甲基)吡啶酰胺
步骤1：4-(2-(((1S,4S)-4-(6-甲氧基-5-硝基-2H-吲唑-2-基)-1-((甲硫基)甲氧基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
将化合物4-(2-(((1S,4S)-1-羟基-4-(6-甲氧基-5-硝基-2H-吲唑-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(700mg,1.24mmol)和Ac2O(151mg,1.48mmol)溶于无水DMSO(20mL)中，氮气保护下室温搅 拌16h。反应液中加入水(50mL)，用乙酸乙酯(50mL x 3)萃取，有机相用饱和食盐水(150mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。粗品使用柱层析(乙酸乙酯:石油醚＝1:9)纯化，得到4-(2-(((1S,4S)-4-(6-甲氧基-5-硝基-2H-吲唑-2-基)-1-((甲硫基)甲氧基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝627.2.
步骤2：4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲唑-2-基)-1-甲氧基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
将化合物4-(2-(((1S,4S)-4-(6-甲氧基-5-硝基-2H-吲唑-2-基)-1-((甲硫基)甲氧基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(600mg,0.96mmol)和预处理后的Raney-Ni(200mg)(预处理方法：先用水洗3次，再用丙酮洗3次，最后用乙醇洗3次)溶于无水乙醇(50mL)中，氢气气氛下，室温搅拌3h，过滤，滤液直接减压浓缩，粗品使用反相硅胶柱(ACN:H2O＝1:1，流速40mL/min)纯化，得到4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲唑-2-基)-1-甲氧基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝551.3.
步骤3：4-(2-(((1S,4S)-1-甲氧基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲唑-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯的制备
将化合物6-(三氟甲基)吡啶甲酸(60mg,0.3mmol),HATU(114mg,0.3mmol)和DIEA(100mg,0.78mmol)溶于DMF(10mL)中，室温搅拌20min后，加入4-(2-(((1S,4S)-4-(5-氨基-6-甲氧基-2H-吲唑-2-基)-1-甲氧基环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(150mg,0.26mmol)，室温反应1h。反应液中加入水(100mL)，用乙酸乙酯(30mL x 3)萃取，有机相用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。粗品使用柱层析(乙酸乙酯:石油醚＝1:1)纯化，得到4-(2-(((1S,4S)-1-甲氧基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲唑-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯。
LC-MS:(ESI,m/z):[M+H]+＝724.4.
步骤4：N-(6-甲氧基-2-((1S,4S)-4-甲氧基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-2H-吲唑-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
4-(2-(((1S,4S)-1-甲氧基-4-(6-甲氧基-5-(6-(三氟甲基)吡啶甲酰胺)-2H-吲唑-2-基)环己基)甲氧基)乙基)哌啶-1-羧酸苄酯(70mg,0.097mmol)溶解于三氟乙酸(3mL)中，升温至90℃搅拌1小时，反应液直接减压浓缩得到N-(6-甲氧基-2-((1S,4S)-4-甲氧基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-2H-吲唑-5-基)-6-(三氟甲基)吡啶甲酰胺，无需纯化直接用于下一步反应。
LC-MS:(ESI,m/z):[M+H]+＝590.4.
步骤5：N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙氧基)甲基)-4-甲氧基环己基)-6-甲氧基-2H-吲唑-5-基)-6-(三氟甲基)吡啶甲酰胺的制备
N-(6-甲氧基-2-((1S,4S)-4-甲氧基-4-((2-(哌啶-4-基)乙氧基)甲基)环己基)-2H-吲唑-5-基)-6-(三氟甲基)吡啶甲酰胺(70mg,0.097mmol)用二甲亚砜(3mL)溶解，搅拌下加入4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酸五氟苯酯(48mg,0.11mmol)和N,N-二异丙基乙胺(37mg,0.29mmol)。室温下搅拌2小时后，加水(50mL)，用二氯甲烷(20mL x 3)萃取，饱和食盐水(3x 60mL)洗涤有机相，经无水硫酸钠干燥，过滤，减压浓缩得到粗品，粗品经pre-HPLC(CH3CN/0.08％NH4HCO3水溶液,5％～95％)纯化得到N-(2-((1S,4S)-4-((2-(1-(4-氯-3-(2,4-二氧代四氢嘧啶-1(2H)-基)苯甲酰)哌啶-4-基)乙氧基)甲基)-4-甲氧基环己基)-6-甲氧基-2H-吲唑-5-基)-6-(三氟甲基)吡啶甲酰胺。
LC-MS:(ESI,m/z):[M+H]+＝840.3.
1H NMR(400MHz,DMSO-d6)δ10.51(d,J＝4.5Hz,2H),8.68(s,1H),8.49-8.37(m,2H),8.32(s,1H),8.22(d,J＝7.6Hz,1H),7.64(d,J＝8.2Hz,1H),7.55(d,J＝1.9Hz,1H),7.39(dd,J＝8.2,2.0Hz,1H),7.18(s,1H),4.52-4.30(m,2H),3.98(s,3H),3.80-3.52(m,3H),3.47(t,J＝6.2Hz,2H),3.34(s,2H),3.19(s,3H),3.15-2.95(m,1H),2.85-2.70(m,3H),2.19-1.97(m,2H),1.96-1.85(m,4H),1.85-1.55(m,3H),1.55-1.45(m,4H),1.25-1.05(m,2H).
Ⅱ生物活性测试实施例
测试实施例1：IRAK4激酶活性测试
用KinEASE-STK S1丝/苏氨酸激酶试剂盒(Cisbio)检测化合物对IRAK4激酶活性的抑制作用，具体方法为：化合物溶解于二甲亚砜中，再用试剂盒的缓冲液进行等梯度稀释，使受试化合物在反应体系中的终浓度范围为10000nM～0.038nM，再依次加入2.5nM IRAK4激酶(Carna)、1μM生物素化的多肽底物(Cisbio)和7μM三磷酸腺苷(Sigma-Aldrich)在37℃下孵育120min。随后向反应体系中加入偶联有铕系元素化合物的抗磷酸化丝/苏氨酸抗体(Cisbio)和偶联有修饰化的XL665链霉亲和素(Cisbio)以终止反应，在室温下孵育1h后，在酶标仪EnVision(PerkinElmer)上以HTRF模式测定各孔在激发波长 为337nm，读取各孔在发射波长为620nm和665nm的荧光强度，使用公式Ratio＝(665nm/620nm)×104算出Ratio值。通过与对照组荧光强度比值进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 7以对数浓度-抑制率进行非线性曲线拟合，得出化合物的IC50值。
表1：

实验结果：本发明所述化合物能够有效的与靶蛋白相结合，且抑制IRAK4激酶活性。
测试实施例2：化合物对THP-1细胞中IRAK4的降解
24孔细胞培养板每孔接种0.95mL THP-1细胞(中国科学院干细胞库)，细胞密度为5×105个/孔；细胞板置于5％二氧化碳培养箱中37℃培养过夜，然后再加入50μL化合物二甲亚砜溶液，化合物终浓度在0.03～3000nM范围内，继续培养24小时后，收集细胞至1.5mL的离心管中，1000rpm、4℃离心5分钟。细胞沉淀用1×DPBS清洗2次，重悬的细胞用200μL裂解液(细胞裂解液为Western及IP细胞裂解液(碧云天)，补充1mM苯甲基磺酰氟和蛋白酶抑制剂混合物(碧云天))裂解，冰上静置30分钟后14000g、4℃离心10分钟，取上清液用蛋白免疫印迹法(Western Blot)检测IRAK4蛋白水平。
人IRAK4 AlphaLISA检测
上述细胞裂解液同时使用人IRAK4 AlphaLISA检测试剂盒(PerkinElmer，AL3117C)测定IRAK4降解作用，具体方法为取2μL待测的细胞裂解上清液样品加入白色384孔板(PerkinElmer，6007299)，然后再加入4μL的5×抗IRAK4受体微珠(PerkinElmer,终浓度为10μg/mL)，23℃孵育30分钟。接着再加入4μL的5×生物素标记的抗IRAK4抗体(PerkinElmer,终浓度为1nM)，23℃孵育60分钟。最后在各孔中加入10μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，23℃避光孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer，2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值，据此计算IRAK4浓度。再用GraphPad Prism 7以对数浓度-抑制率进行非线性曲线拟合，得出化合物的DC50和Dmax值。
表2：


实验结果：本发明所述化合物能够有效的降解细胞中的IRAK4激酶蛋白，且具有优良的降解活性。
测试实施例3：LPS诱导正常人全血﹑PBMC分泌的细胞因子浓度测定
(1)LPS诱导正常人全血分泌的细胞因子浓度测定
在96孔透明细胞板(Labserv，310109008)每孔中铺190μL肝素钠抗凝的新鲜的正常人全血(年龄为22-50的健康人，现采集的血液)，然后在相应的孔中加入10μL稀释后的化合物溶液，使化合物的终浓度在10～20000nM范围内。二甲亚砜终浓度为0.1％。加化合物处理的细胞板贴上封板膜，置于5％二氧化碳培养箱中37℃培养20小时后，加入10μL LPS(0111:B4)(Sigma，L4391)，终浓度为100ng/mL。细胞板贴上封板膜，再次置于5％二氧化碳培养箱中37℃培养5小时后，2000rpm离心10分钟，每孔取30μL上清血浆转移到一块新的96孔透明细胞板，用于细胞因子浓度测试，冻存于-80℃待测。
白介素-6(IL-6)AlphaLISA检测。
使用白介素-6(IL-6)AlphaLISA检测试剂盒(PerkinElmer,AL223C)测定LPS诱导的人全血(年龄为22-50的健康人,现采集的血液)的上清血浆样品中的IL-6浓度。
按照产品说明书配制不同浓度的IL-6标准液，浓度范围为0～100000pg/mL。取2μL各浓度的IL-6标准液和待测的细胞上清液样品分别加入白色384孔板(PerkinElmer，6007299)，然后在各孔中加入8μL的5×抗IL-6受体微珠(PerminElmer,终浓度为10μg/mL)和生物素标记的抗IL-6抗体(PerminElmer,终浓度为1nM)混合溶液，23℃孵育60分钟。最后在各孔中加入10μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再23℃避光孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer，2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IL-6标准液的AlphaLISA信号值绘制标准曲线，再根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，从而确定各细胞裂解上清液的IL-6浓度。再与对照组IL-6浓度进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 7以对数浓度-抑制率进行非线性曲线拟合，计算化合物的IC50值。
(2)LPS诱导人PBMC分泌的细胞因子浓度测定
冻存的人PBMC(妙顺,PB010C)复苏后重悬至RPMI 1640培养基(Gibco,A1049101)，补充10％胎牛血清(Gibco,10099141)，100U/mL青霉素和100μg/mL链霉素(Gibco,15140122)。同一天在96孔透明细胞板(Labserv,310109008)每孔中铺150μL的PBMC，使细胞密度为2×105个/孔。然后在相应的铺有细胞的孔中加入50μL稀释后的化合物溶液，使化合物的终浓度在0.026～10000nM范围内。DMSO终浓度为0.25％。加药处理的细胞板置于5％CO2培养箱中37℃培养1小时或者20小时后，加入10μL LPS(0111:B4)(Sigma,L4391)，终浓度为100ng/mL。细胞板再次置于5％CO2培养箱中37℃培养5小时后，2000rpm离心10分钟，每孔取100μL细胞上清液转移到一块新的96孔透明细胞板，用于细胞因子浓度测试，冻存于-80℃待测。
白介素-6(IL-6)AlphaLISA检测
使用白介素-6(IL-6)AlphaLISA检测试剂盒(PerkinElmer,AL223C)测定LPS诱导人PBMC细胞上清液中的IL-6浓度。
按照产品说明书配制不同浓度的IL-6标准液，浓度范围为0～100000pg/mL。取2μL各浓度的IL-6标准液和待测的细胞上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入8μL的5×抗IL-6受体微珠(终浓度为10μg/mL)和生物素标记的抗IL-6抗体(终浓度为1nM)混合溶液，23℃孵育60分钟。最后在各孔中加入10μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再23℃避光孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IL-6标准液的AlphaLISA信号值绘制标准曲线，再根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，从而确定各细胞裂解上清液的IL-6浓度。再与对照组IL-6浓度进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 7以对数浓度-抑制率进行非线性曲线拟合，计算化合物的IC50值。
测试实施例4：化合物对IRAK4的降解作用
(1)化合物对正常人PBMC中IRAK4蛋白降解作用
冻存的正常人PBMC(妙顺,PB010C)复苏后重悬至RPMI 1640培养基(Gibco,A1049101)，补充10％胎牛血清(Gibco,10099141)，100U/mL青霉素和100μg/mL链霉素(Gibco,15140122)。在24孔细胞培养板每孔中铺0.95mL细胞，然后在相应铺有细胞的孔中加入50μL稀释后的化合物溶液，使化合物的终浓度在0.01～3000nM范围内。DMSO终浓度为0.25％。给药后的细胞板置于5％CO2培养箱中37℃培养24小时后，收集细胞至1.5mL的离心管中，1000rpm、4℃离心5分钟。细胞沉淀用1×DPBS清洗2次，重悬的细胞用100μL裂解液裂解，冰上静置30分钟后14000g、4℃离心10分钟。离心后的细胞裂解上清样品冻存于-80℃待测。细胞裂解液为Western及IP细胞裂解液(碧云天,P0013)，补充1mM苯甲基磺酰氟和蛋白酶抑制剂混合物(碧云天,P1008)。
细胞裂解样品中总蛋白浓度使用BCA蛋白质定量试剂盒(天根,PA115-02)测定。
制备的PBMC裂解样品使用人IRAK4 AlphaLISA检测试剂盒(PerkinElmer,AL3117C)测定IRAK4浓度，从而确定化合物对IRAK4的降解作用。方法如下：
取5μL各浓度的IRAK4标准液和待测的细胞裂解上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入10μL的5×抗IRAK4受体微珠(终浓度为10μg/mL)，室温孵育30分钟。接着在各孔中加入10μL的5×生物素标记的抗IRAK4抗体(终浓度为1nM)，再室温孵育60分钟。最后在各孔中加入25μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再室温孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IRAK4标准液的AlphaLISA信号值绘制标准曲线，根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，再用BCA定量得到的各样品的总蛋白浓度进行相应的校正，从而确定各细胞裂解上清液的IRAK4浓度。再与对照组IRAK4浓度进行对比，计算化合物在各浓度下的降解率，进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，得出化合物的DC50和Dmax 值。
表4：

(2)化合物对病人全血中IRAK4蛋白降解作用
采集正常人或者病人(类风湿性关节炎、皮肌炎)志愿者的新鲜全血用肝素钠抗凝。在24孔细胞培养板每孔中铺0.95mL全血，在相应铺有全血的孔中加入50μL稀释后的化合物溶液，使化合物的终浓度在10～10,000nM范围内。DMSO终浓度为0.1％。给药后的细胞板置于5％CO2培养箱中37℃培养24小时后，收集每孔中的全血样品，采用FICOLL分离液(GE,17-1440-02)，按照产品说明书步骤从全血中分离出PBMC，1×DPBS清洗一次分离的细胞，然后采用红细胞裂解液(南京森贝伽,BL-O51-100ml)裂解红细胞5分钟，用1×DPBS清洗一次细胞。最后3000转/分钟离心5分钟后收集的白色沉淀细胞即为PBMC。
分离得到的PBMC移去上清液后用细胞裂解液裂解，冰上静置30分钟后14000g、4℃离心10分钟。细胞裂解液为Western及IP细胞裂解液(碧云天,P0013)，补充1mM苯甲基磺酰氟和蛋白酶抑制剂混合物(碧云天,P1008)。离心后的细胞裂解上清蛋白样品冻存于-80℃待测。
制备的正常人或病人(类风湿性关节炎、皮肌炎)全血细胞裂解样品中总蛋白浓度使用BCA蛋白质定量试剂盒(天根,PA115-02)测定。
制备的正常人或病人(类风湿性关节炎、皮肌炎)全血细胞裂解样品使用人IRAK4 AlphaLISA检测试剂盒(PerkinElmer,AL3117C)测定IRAK4浓度，从而确定化合物对IRAK4的降解作用。方法如下：
取5μL各浓度的IRAK4标准液和待测的细胞裂解上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入10μL的5×抗IRAK4受体微珠(终浓度为10μg/mL)，室温孵育30分钟。接着在各孔中加入10μL的5×生物素标记的抗IRAK4抗体(终浓度为1nM)，再室温孵育60分钟。最后在各孔中加入25μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再室温孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IRAK4标准液的AlphaLISA信号值绘制标准曲线，根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，再用BCA定量得到的各样品的总蛋白浓度进行相应的校正，从而确定各细胞裂解上清液的IRAK4浓度。再与对照组IRAK4浓度进行对比，计算化合物在各浓度下的降解率，进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，得出化合物的DC50和Dmax 值。
图1A和图1B表明本发明化合物A对正常人外周血单个核细胞﹑皮肌炎病人﹑类风湿关节炎病人的IRAK4均有较好的降解作用。
测试实施例5：各类病人PBMC胞内IRAK4蛋白表达水平的测定
采集志愿者的新鲜全血用肝素钠抗凝，然后采用FICOLL分离液(GE，17-1440-02)，按照产品说明书步骤从全血中分离出PBMC，1×DPBS清洗一次分离的细胞，然后采用红细胞裂解液(南京森贝伽,BL-O51-100ml)裂解红细胞，用1×DPBS清洗一次细胞。最后3000转/分钟离心5分钟后收集的白色沉淀细胞即为PBMC。此方法适用于从骨关节炎、类风湿性关节炎、系统性红斑狼疮、皮肌炎、系统性硬化症等病人全血，以及正常人全血中分离得到PBMC。
分离得到的PBMC移去上清液后用细胞裂解液裂解，冰上静置30分钟后14000g、4℃离心10分钟。细胞裂解液为Western及IP细胞裂解液(碧云天,P0013)，补充1mM苯甲基磺酰氟和蛋白酶抑制剂混合物(碧云天,P1008)。离心后的细胞裂解上清蛋白样品冻存于-80℃待测。
细胞蛋白样品中总蛋白浓度使用BCA蛋白质定量试剂盒(天根,PA115-02)测定。
制备的细胞蛋白样品使用人IRAK4 AlphaLISA检测试剂盒(PerkinElmer,AL3117C)测定各类病人或正常人PBMC中IRAK4蛋白的表达水平。方法如下：
取5μL各浓度的IRAK4标准液和待测的细胞裂解上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入10μL的5×抗IRAK4受体微珠(终浓度为10μg/mL)，室温孵育30分钟。接着在各孔中加入10μL的5×生物素标记的抗IRAK4抗体(终浓度为1nM)，再室温孵育60分钟。最后在各孔中加入25μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再室温孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IRAK4标准液的AlphaLISA信号值绘制标准曲线，根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，再用BCA定量得到的各样品的总蛋白浓度进行相应的校正，从而确定各细胞裂解样品中的IRAK4浓度。通过GraphPad Prism 8软件绘制柱状图谱，结果如图2所示。
图2表明类风湿关节炎病人﹑皮肌炎病人﹑系统红斑狼疮病人和系统性硬化症病人的PBMC胞内IRAK4蛋白具有高水平的表达。
测试实施例6：化合物对LPS诱导的正常人PMBC的IL-6抑制作用
冻存的正常人PBMC(妙顺,PB010C)复苏后重悬至RPMI 1640培养基(Gibco,A1049101)，补充10％胎牛血清(Gibco,10099141)，100U/mL青霉素和100μg/mL链霉素(Gibco,15140122)。在96孔透明细胞板(Labserv,310109008)每孔中铺150μL的PBMC，使细胞密度为2×105个/孔。然后在相应的铺有细胞的孔中加入50μL稀释后的化合物溶液，使化合物的终浓度在0.026～10000nM范围内。DMSO终浓度为0.25％。加药处理的细胞板置于5％CO2培养箱中37℃培养20小时后，加入10μL LPS(0111:B4)(Sigma,L4391)。细胞板再次置于5％CO2培养箱中37℃培养5小时后，2000rpm离心10分钟，每孔取100μL细胞上清液转移到一块新的96孔透明细胞板，用于细胞因子浓度测试，冻存于-80℃待测。
使用人IL-6AlphaLISA检测试剂盒(PerkinElmer,AL223C)测定正常人PBMC细胞上清液中的IL-6浓度。方法如下：
按照产品说明书配制不同浓度的IL-6标准液，浓度范围为0～100000pg/mL。取2μL各浓度的IL-6标准液和待测的细胞上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入8μL的5×抗IL-6受体微珠(终浓度为10μg/mL)和生物素标记的抗IL-6抗体(终浓度为1nM)混合溶液，23℃孵育60分钟。最后在各孔中加入10μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再23℃避光孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IL-6标准液的AlphaLISA信号值绘制标准曲线，再根据待测样品的AlphaLISA信号值在标准曲线上相应的浓度，从而确定各细胞裂解上清液的IL-6浓度。再与对照组IL-6浓度进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 8以对数浓度-抑制率进行非线性曲线拟合，计算化合物的IC50值。
图3说明本发明化合物A对由脂多糖刺激的人外周血单个核细胞中的IL-6具有很好的抑制作用。
测试实施例7：人PBMC细胞因子测试
测试仪器及试剂：
正常人外周血单个核细胞(PBMC)(TPCS，PB010C)
RPMI 1640培养基(Gibco，A1049101)
胎牛血清(Gibco，10099141)
双抗(Gibco，15140122)
脂多糖(LPS)(0111:B4)(Sigma-Aldrich，L4391)
雷希莫特(R848)(MCE，HY-13740)
DMSO(Sigma-Aldrich，D8418-500ML)
Ficoll分离液(Cytiva，17544602)
ACK红细胞裂解液(无菌)(Sbjbio，BL-O51)
96孔细胞培养板(Corning，3596)
真空采血管(肝素锂抗凝)(BD，367880)
离心机(Thermo Scientific，ST16R)
移液器(Eppendorf)
(1)脂多糖或雷希莫特诱导的正常人PMBC细胞因子测试
冻存的正常人PBMC复苏后重悬至RPMI 1640培养基(补充10％胎牛血、100U/mL青霉素和100μg/mL链霉素)。在96孔透明细胞板铺PBMC，使细胞密度为2×105个/孔。然后在化合物孔中加入培养基稀释的待测化合物，使化合物终浓度为500nM(含0.25％DMSO)，同时在阳性和阴性照孔中分别加入含0.25％DMSO的培养基。加样后的细胞板放置于5％CO2培养箱中37℃孵育20小时后，在化合物孔和阳性对照孔中加LPS或R848。加样后细胞板再次放置于5％CO2培养箱中37℃孵育5小时 后，2000rpm离心10分钟。最后每孔取细胞上清液，冻存于-80℃备用。
人48种细胞因子的检测(上海优宁维生物科技股份有限公司)按照Bio-Plex Pro Human Cytokine Screening Panel试剂盒(Bio-Rad,12007283)产品说明书操作，采用Luminex(X-200)上机检测。样本中各因子表达检测结果见附图4和5。
图4﹑5表明在500nM浓度时，本发明降解剂化合物A对脂多糖或雷希莫特刺激人外周血单个核细胞分泌的细胞因子普遍具有抑制作用，且优于同浓度的抑制剂化合物(对照I和对照Ⅱ)。
测试实施例8：化合物对正常人或病人全血IL-6抑制作用
采集正常人或者病人(类风湿性关节炎、系统性红斑狼疮、皮肌炎)志愿者的新鲜全血用肝素钠抗凝。在96孔透明细胞板(Labserv,310109008)中铺190μL全血，在相应铺有全血的孔中加入10μL稀释后的化合物溶液，使化合物的终浓度在10～10000nM范围内。DMSO终浓度为0.25％。加药处理的细胞板置于5％CO2培养箱中37℃培养20小时后，加入10μL LPS。细胞板再次置于5％CO2培养箱中37℃培养5小时后，2000rpm离心10分钟，每孔取50μL上清液转移到一块新的96孔透明细胞板，用于细胞因子浓度测试，冻存于-80℃待测。
使用人IL-6AlphaLISA检测试剂盒(PerkinElmer，AL223C)测定正常人或者病人(皮肌炎、类风湿性关节炎、系统性红斑狼疮)全血上清液中的IL-6浓度。方法如下：
按照产品说明书配制不同浓度的IL-6标准液，浓度范围为0～100000pg/mL。取2μL各浓度的IL-6标准液和待测的细胞上清液样品分别加入白色384孔板(PerkinElmer,6007299)，然后在各孔中加入8μL的5×抗IL-6受体微珠(终浓度为10μg/mL)和生物素标记的抗IL-6抗体(终浓度为1nM)混合溶液，23℃孵育60分钟。最后在各孔中加入10μL的2×链霉亲和素标记的供体微珠(终浓度为40μg/mL)，再23℃避光孵育30分钟。孵育结束后，在酶标仪EnVision(PerkinElmer,2105)上以AlphaScreen标准设置模式测定AlphaLISA信号值。通过各浓度IL-6标准液的AlphaLISA信号值绘制标准曲线，再根据待测样品的lphaLISA信号值在标准曲线上相应的浓度，从而确定各细胞裂解上清液的IL-6浓度。再与对照组IL-6浓度进行对比，计算化合物在各浓度下的抑制率，进而通过GraphPad Prism 7以对数浓度-抑制率进行非线性曲线拟合，计算化合物的IC50值。
图6A、图6B、图6C、图6D表明本发明化合物A对由脂多糖诱导的正常人全血中的IL-6﹑皮肌炎病人全血中的IL-6，类风湿关节炎病人全血中的IL-6和系统红斑狼疮病人全血中的IL-6均具有很好的抑制作用。
测试实施例9：化合物A在雄性BALB/C小鼠体内多次口服给药后对组织IRAK4蛋白表达影响的试验
受试者信息
雄性BALB/C小鼠，6-8周龄。
试验试剂
DMSO，Solutol，Glucose(批号20181102,AR,国药集团化学试剂有限公司)。
给药制剂配制
称取一定量的化合物A，先加入5％DMSO溶解化合物成澄清溶液，再加入15％Solutol震荡混匀，最后加入80％生理盐水混匀。
剂量设置及分组
化合物A剂量设置为30mpk，60mpk，90mpk；口服给药，每天2次；Vehicle为5％DMSO+15％Solutol+80％生理盐水，分组信息如表5所示：
表5化合物剂量设置及分组信息
实验流程
动物按照体重进行分组，分组信息见表5，然后给药。多次灌胃给药，3天后动物CO2安乐死。CO2安乐死后心脏采血，肝素抗凝，分离制备PBMC，检测IRAK4的降解；并取脾脏和皮肤组织，用于检测IRAK4蛋白水平。
检测指标
PBMC﹑脾脏﹑皮肤中IRAK4的蛋白水平。
试验结果
图7A﹑图7B和图7C显示了口服化合物A后，动物PBMC﹑脾脏﹑皮肤中IARK4均有很好的降解。随着给药时间的延长，PBMC﹑脾脏组织中的IRAK4降解率也增加，降解率达到80％以上。
测试实施例10：化合物A在咪喹莫特诱导的C57小鼠牛皮癣(银屑病)模型上的的药效作用
试剂
5％咪喹莫特乳膏，Aldara,3M Pharmaceuticals，LOT：GVJ005C
VecticalTM(calcitriol)Ointment，Galderma，LOT：321449
醋酸地塞米松片，辰欣药业，国药准字：H37021898
受试者信息
雌性C57BL/6小鼠(体重19～21g)，9周，每组10只。
实验方法
A.试剂制备
i.咪喹莫特乳膏：每只小鼠对应称量62.5mg咪喹莫特乳膏直接涂抹背部，称量20mg咪喹莫特乳膏直接涂抹右耳；
ii.骨化三醇软膏(calcitriol)：每只小鼠对应称量75mg骨化三醇软膏直接涂抹背部，称量24mg骨化三醇软膏直接涂抹右耳；
iii.受试化合物Dex配制成相应浓度进行口服给药；
5mg/kg：取5片Dex药片，加入7.5ml生理盐水，涡旋超声至均匀混悬，4℃保存备用。给药前注意提前涡旋超声至均匀，三天一配；
iv.受试化合物A配制成相应浓度进行口服给药。
Vehicle：1％DMSO+10％Solutol+89％(5％Dextrose)，每次给药现配现用。
受试化合物A的10、100mg/kg组：在EP管里配制。称取化合物A，加入总体积的1％的DMSO，涡旋超声至完全溶解，再加入总体积10％的Solutol，涡旋超声至澄清，分步加入总体积89％的5％葡萄糖溶液，边加边斡旋，给药前，一直处于磁力搅拌子搅拌状态。化合物每次都现配现用。
B.咪喹莫特在C57BL/6J小鼠身上致敏和药物干预，具体给药时间见表6。
i.Day-1小鼠背部剃毛，通过使用模具保证面积一致，约2cm*3cm，挑选60只；
ii.根据体重和右耳厚度将小鼠随机分成6组，每组10只；
iii.为诱导模型小鼠出现银屑病症状，Day 0开始，中午在G2～G6组小鼠背部皮肤和右耳上涂抹咪喹莫特致敏，持续7天。
iv.Day 0天开始，每天上午给药前对小鼠背部皮肤进行临床评分，隔天测量耳朵厚度；并在上午9:00和下午17:00给予化合物A；下午13:30涂抹咪喹莫特致敏，详细的分组给药方案见表6。
v.Day5或者Day7对小鼠进行终点收样
表6分组信息
检测指标
临床评分和右耳厚。
试验结果
临床评分
本实验评价了化合物在咪喹莫特诱导的小鼠银屑病模型中对临床评分的改善作用。溶剂对照组的平均临床评分逐渐升高，至第5天达到8.00分，提示咪喹莫特诱导的小鼠银屑病模型的成功建立。
化合物A在10mg/kg和100mg/kg两种剂量对小鼠银屑病临床评分均有抑制作用，呈剂量依赖性。 化合物A在10mg/kg和100mg/kg两种剂量下从Day 3开始与溶剂对照组相比有显著性差异，并持续到实验结束(Day 5)。
通过分析每组每只动物的临床评分曲线，计算曲线下面积AUC，通过组间AUC平均值，计算各给药组相对于溶剂对照组的抑制率，结果如图8A﹑图8B所示。化合物A在10mg/kg﹑100mg/kg剂量下抑制率分别为19.2％﹑26.9％(p<0.0001)。阳性药骨化三醇﹑地塞米松组抑制率分别为44.5％﹑20.7％(p<0.0001)。
右耳厚度
图8C表明，第5天化合物A在10mg/kg和100mg/kg剂量下对耳厚差值Δ耳厚(给药当天耳厚数值-分组给药前耳厚数值)均有抑制作用，且化合物A在100mg/kg时，对耳厚差值Δ耳厚的抑制作用有显著性的统计学差异(p<0.0001)。
试验结论
综合以上所述生物活性测试实验数据可以发现：本发明化合物作为IRAK4降解剂可治疗风湿性关节炎、皮肌炎、系统性红斑狼疮、系统性硬化症和/或银屑病。

Claims (16)

  1. IRAK4降解剂，和/或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药和/或其药学上可接受的盐，其用于治疗和/或预防免疫性和/或炎症性疾病的用途。
  2. IRAK4降解剂，和/或其立体异构体、对映异构体、非对映异构体、氘代化物、水合物、溶剂化物、前药和/或其药学上可接受的盐，其用于治疗和/或预防由IL-1R/TLR通路中IRAK4及IRAK4相关蛋白的异常表达和/或IRAK4所介导的化学因子、细胞因子或免疫细胞异常分泌或增生引起的疾病的用途。
  3. 如权利要求1或2所述的用途，其中所述IRAK4降解剂为具有式Ⅰ或式Ⅱ所示的化合物或其药学上可以接受的盐：
    其中：
    环A为苯基或吡啶基；
    环B为C6-C10环烷基或含有1-2个选自N、O或S杂原子的6-10元杂环烷基，所述C6-C10环烷基和6-10元杂环烷基任选被选自卤素、氧代、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
    环C为C6-C12环烷基或含有1-2个选自N、O或S杂原子的6-12元杂环烷基，所述C6-C12环烷基和6-12元杂环烷基任选被选自卤素、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
    环D为C6-C10芳基，所述C6-C10芳基任选被卤素、氰基、氨基、羟基、C1-C6烷基、C1-C6卤代烷基、C1-C6羟基烷基或-O-(C1-C6烷基)的取代基取代；
    环Y为5-6元杂芳基；
    X为键、-O-、-NH-、-C(O)-、-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-；
    L为-(CH2)j-，所述-(CH2)j-中的1个或多个亚甲基任选的被选自-NR3’-、-O-、-S-、-S(O)-、-S(O)2-、 -S(O)2NR3’-、-CR1’R2’-、-C(O)-、-C(O)O-、-OC(O)-、-NR3’C(O)O-、-OC(O)NR3’-、-C(O)NR3’-、-NR3’C(O)-、-NR4’C(O)NR3’-、亚乙烯基或亚乙炔基的基团替代；
    R1’、R2’各自独立为卤素、-OH、-NH2、C1-C4烷基、C1-C4卤代烷基、C1-C4羟基烷基、-O(C1-C4烷基)或-NH(C1-C4烷基)；
    R3’、R4’各自独立的为氢或C1-C6烷基；
    Rd各自独立的为氢、氘、卤素、氰基、C1-C6烷基，所述烷基任选的被1个或多个选自卤素、羟基、氨基的基团取代；
    Rc为-O(C1-C3烷基)、-N(C1-C3烷基)1-2或C1-C6烷基，所述烷基任选被1个或多个独立的选自羟基、氨基、卤素、氰基或-O-(C1-C3烷基)的基团取代；
    Rb为氢或C1-C6烷基，所述烷基任选被1个或多个独立的选自羟基、氨基、卤素、氰基的基团取代；
    Ra为氢、卤素、C1-C6烷基或-O-(C1-C6烷基)，所述烷基任选被卤素或羟基取代；
    各个R1独立地选自：C1-C4烷基、-O(C1-C4烷基)、-N(C1-C4烷基)1-2、CN、卤素、-OH、-NH2，所述C1-C4烷基任选被选自卤素、氰基、-OH、C1-C4烷基、-O(C1-C4烷基)的基团取代；
    各个R2独立地为氢、C1-C4烷基、-O(C1-C4烷基)、C3-C8环烷基、3-8元杂环烷基、C6-C10芳基、5-6元杂芳基、CN、卤素、-OH，所述C3-C8环烷基、3-8元杂环烷基、C6-C10芳基、5-6元杂芳基任选被选自卤素、氰基、-OH、-NH2、C1-C4烷基和-O(C1-C4烷基)；
    X’为CH或N；
    m是0、1、2、3或者4；
    n是0、1、2、3或者4；
    p为1或者2；
    j为0、1、2、3、4或者5。
  4. 如权利要求3所述的用途，所述IRAK4降解剂为式Ⅰ-1、Ⅰ-2或Ⅱ-1所示的化合物或其药学上可以接受的盐：

    其中：
    R3为H、卤素、C1-C6烷基或-O-(C1-C6烷基)；
    Rc、Rd、环B、L、环C、X、X’、p、R2和m如权利要求3所定义和描述。
  5. 如权利要求3或4所述的用途，其特征在于，所述IRAK4降解剂为选自A1-A52，B1-B5和C1-C36的化合物：























    或其药物学上可接受的盐、氘代化物、溶剂化物或前药。
  6. 如权利要求1所述的用途，其特征在于，所述IRAK4降解剂用于治疗和/或预防免疫性疾病的用途。
  7. 如权利要求6所述的用途，其特征在于，所述免疫性疾病选自：成人斯蒂尔病、斑秃、强直性脊柱炎、自身免疫性肝炎、自身免疫性心肌炎、自身免疫性胰腺炎、自身免疫性视网膜病变、自身免疫性荨麻疹、白塞病、良性黏膜类天疱疮(黏膜类天疱疮)、大疱性类天疱疮、卡斯尔曼病(CD)、乳糜泻、柯萨奇心肌炎、克罗恩病、疱疹样皮炎、特应性皮炎、皮肌炎、盘状狼疮、子宫内膜异位症、嗜酸性筋膜炎、结节性红斑、纤维化肺泡炎、原发肾小球肾炎、Goodpasture综合征、自身免疫性溶血性贫血、过敏性紫癜(HSP)、化脓性汗腺炎(HS)、IgG4相关的硬化性疾病、包涵体肌炎(IBM)、间质性膀胱炎(IC)、兰伯特-伊顿综合征、线性IgA疾病(LAD)、系统性红斑狼疮、慢性莱姆病、多发性硬化症、系统性硬化症、特发炎症性肌病(IIM)、眼瘢痕性类天疱疮视神经炎、回纹风湿病(PR)、天疱疮、自身免疫性脑脊髓炎、POEMS综合征、结节性多动脉炎、原发性胆汁性胆管炎、原发性硬化性胆管炎、银屑病、银屑病关节炎、反应性关节炎、复发性多软骨炎、腹膜后纤维化、风湿热、类风湿关节炎、结节病、自身免疫性巩膜炎、硬斑病、干燥综合征、大动脉炎、颞动脉炎、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、甲状腺眼病(Ted)、Tolosa-Hunt综合征(THS)、横贯性脊髓炎、溃疡性结肠炎(UC)、未分化结缔组织病(UCTD)、葡萄膜炎、系统性血管炎、白癜风、Vogt-小柳-原田病、重症肌无力、痛风、脊柱关节病、晶型关节病、骨关节炎、类风湿性关节炎、多发性肌炎、间质性肺疾病、巨细胞动脉炎、风湿性多肌痛、肉芽肿性血管炎、嗜酸性肉芽肿性血管炎、嗜酸性粒细胞血管炎、显微镜下多血管炎、冷球蛋白血症、皮肤白细胞破碎性血管炎、混合性结缔组织病、史蒂芬-约翰逊综合症、肺动脉高压、心内膜炎、动脉粥样硬化、多形性红斑、急性冠脉综合征、特发性肺纤维化、非酒精性脂肪肝、肾纤维化、1型糖尿病、初级干燥、川崎病、脓疱型银屑病、慢性肉芽肿病、视神经脊髓炎荨麻疹、掌跖脓疱病、败血症、大疱性皮肤病、阿茨海默病、慢性自发性荨麻疹、慢性牛皮癣、幼年特受性关节炎、中轴性脊柱关节炎或格雷夫斯病。
  8. 如权利要求7所述的用途，其特征在于，所述免疫性疾病选自：类风湿性关节炎、特应性皮炎、化脓性汗腺炎(HS)、白癜风、皮肌炎、斑秃、荨麻疹、多发性肌炎、间质性肺疾病、系统性红斑狼疮、系统性硬化症和/或银屑病。
  9. 如权利要求1所述的用途，其特征在于，所述IRAK4降解剂用于治疗和/或预防炎症性疾病的用途。
  10. 如权利要求9所述的用途，其特征在于，所述炎症性疾病选自：家族性地中海热、肿瘤坏死因子相关周期性综合征、甲羟戊酸激酶缺乏症、吡啶相关自身炎症伴中性粒细胞性皮肤病(PAAND)、化脓性无菌关节炎、坏疽性脓皮病和痤疮(PAPA)、家族性感冒自身炎症综合征(FCAS)、家族性慢性地衣样角化病(FKLC)、NLRP1相关自身炎症伴关节炎和角化不良(NAIAD)、IL-1受体拮抗剂(DIRA)缺乏症、IL-36受体拮抗剂(DITRA)缺乏症、过敏性接触性皮炎、CAR-T细胞诱导的细胞因子释放综合征、克罗恩氏病、慢性支气管炎、COPD、活动性强直性脊柱炎、中轴型脊柱关节、毛发红糠疹、炎性肠病、脊柱关节炎、急性肺损伤、泛发性脓疱型银屑病、寻常痤疮或结肠炎。
  11. 如权利要求2所述的用途，其特征在于，所述IRAK4降解剂用于治疗和/或预防由IL-2R alpha﹑IL-6﹑IFA-alpha2﹑IFN-gamma﹑IL-1ra﹑MCP-3﹑IL-16﹑IL-12(p40)﹑LIF﹑IL-5﹑GM-CSF﹑TNF-alpha﹑IL-2﹑IL-1alpha、IL-1beta﹑IL-18﹑Eotaxin﹑Basic FGF﹑beta-NGF﹑PDGF-BB﹑IL-4﹑MCP-1﹑IL-8﹑IL-10﹑GRO-alpha﹑HGF﹑IL-1alpha、IL-1beta﹑IL-3﹑SCF﹑TRAIL﹑M-CSF﹑CTACK﹑IL-15﹑IL-12(P70)、IL-17、IL-23、IL-33和/或IL-36细胞因子所介导的疾病的用途。
  12. 如权利要求11所述的用途，其特征在于，所述IRAK4降解剂用于治疗和/或预防由IL-4、IL-6﹑IL-12(p40)﹑GM-CSF﹑TNF-alpha﹑IL-2﹑IL-1alpha、IL-1beta、IL-18﹑IL-8﹑IL-10﹑IL-17、IL-23、IL-33和/或IL-36细胞因子所介导的疾病中的用途。
  13. 如权利要求1-12任意一项所述的用途，其特征在于，所述疾病的样品是脾脏、皮肤和/或血液样品。
  14. 如权利要求13所述的用途，其特征在于，所述血液样品是正常人全血和/或病人全血。
  15. 治疗1-12任一项所述疾病的方法，包括将有效量的IRAK4降解剂施用于所述对象。
  16. 如权利要求15所述的治疗方法，其中所述IRAK4降解剂为权利要求5所述的单一化合物或与其它药物的联用。
PCT/CN2023/129712 2022-11-04 2023-11-03 Irak4降解剂及其用途 WO2024094190A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021213287A1 (zh) * 2020-04-20 2021-10-28 上海领泰生物医药科技有限公司 Irak4抑制剂治疗ali/ards中的应用
WO2022028547A1 (zh) * 2020-08-05 2022-02-10 上海领泰生物医药科技有限公司 靶向蛋白降解化合物及其制备方法和应用
WO2022088551A1 (zh) * 2020-10-26 2022-05-05 上海领泰生物医药科技有限公司 吲唑类衍生物及其制备方法和应用
CN115710274A (zh) * 2021-08-23 2023-02-24 上海领泰生物医药科技有限公司 Irak4降解剂及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021213287A1 (zh) * 2020-04-20 2021-10-28 上海领泰生物医药科技有限公司 Irak4抑制剂治疗ali/ards中的应用
WO2022028547A1 (zh) * 2020-08-05 2022-02-10 上海领泰生物医药科技有限公司 靶向蛋白降解化合物及其制备方法和应用
WO2022088551A1 (zh) * 2020-10-26 2022-05-05 上海领泰生物医药科技有限公司 吲唑类衍生物及其制备方法和应用
CN115710274A (zh) * 2021-08-23 2023-02-24 上海领泰生物医药科技有限公司 Irak4降解剂及其制备方法和应用

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