RET degradation agent with RET inhibitory activity as well as synthesis and application of RET degradation agent

Abstract

The invention discloses a method for targeted degradation of RET kinase (RET kinase). The method comprises the following steps: 1, preparing a recombinant plasmid; the invention relates to a transfection rearrangement kinase compound and application of a pharmaceutical composition thereof. Specifically, the invention discloses a compound, and the application of the compound to RET related cancers is described. A series of RET degradation agents are designed and synthesized, and can be used for treating related tumors with drug-resistant mutation.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to compounds that degrade RET kinase (rerangedduring transfection kinase) via the proteasome pathway, their uses, and their manufacture. Background Technology

[0002] The gene encoding the RET protein in humans is located on the long arm of chromosome 10. Abnormalities in RET (gene fusion, mutation, amplification) can cause various diseases, including papillary thyroid carcinoma (PTC), medullary thyroid carcinoma (MTC), lung adenocarcinoma, congenital megacolon, and irritable bowel syndrome. Chromosomal rearrangements of the RET gene can lead to RET gene breaks. After a break, the 3' end of the RET gene can fuse with different genes such as KIF5B, TRIM33, CCDC6, or NCOA4 to form fusion genes. The expressed fusion protein is persistently activated, driving tumorigenesis. RET is a single-transmembrane receptor belonging to the tyrosine kinase superfamily and is required for the normal development, maturation, and maintenance of various tissues and cells. RET signaling is mediated by the binding of glial cell-derived neurotrophic factor (GDNF) and its family ligand (GFL) to soluble proteins. RET kinase activation differs from the direct activation of other kinase receptors; it is indirectly activated. Glial cell-derived neurotrophic factor (GDNF) family ligands (GFLs) first form a co-receptor complex with GDNF family receptor α (GFRα), which then catalyzes RET homodimerization, leading to autophosphorylation of the intracellular region of RET. This process then recruits adaptor proteins and pathway proteins to activate multiple signaling pathways, including MAPK, PI3K, JAK-STAT, PKA, and PKC, thereby participating in cell proliferation, neural transmission, cell migration, and cell differentiation (Nature Reviews Clinical Oncology, 2018, 15:151–167). These pathways play a crucial role in regulating cell survival, differentiation, proliferation, migration, and chemotaxis. Several multi-target kinase inhibitors exhibit some inhibitory activity against RET, such as cabozantinib, vandetanib, lenvatini, and ponatinib, but these are all non-specific RET inhibitors. Furthermore, since RET and VEGFR2 share a high degree of homology in their kinase domains, these compounds, in addition to inhibiting RET, also have a certain inhibitory effect on multiple targets, including VEGFR2, leading to off-target toxicity risks and making it difficult to achieve satisfactory therapeutic effects.

[0003] Currently, among RET inhibitors, multi-target kinase inhibitors such as sunitinib, sorafenib, and cabozantinib have shown some RET inhibitory activity in clinical practice. However, their clinical toxicity and off-target effects prevent them from effectively treating RET-related diseases. Septinib (LOXO-292), approved in 2020, is a potent and highly selective small-molecule RET inhibitor with nanomolar inhibitory activity against wild-type or V804M / L mutations. Despite significant progress in selective RET kinase inhibitors in human cancer patients, acquired resistance caused by RET G810C / S / R mutations has emerged. Extensive efforts have been invested in developing next-generation inhibitors, but they are still in early stages, and no drugs have been approved to overcome RET G810 mutations. Therefore, the development of new RET inhibitors is still needed to treat tumors with related resistance mutations.

[0004] The ubiquitin-proteasome (UPS) pathway mediates cell cycle, signal transduction, DNA damage repair, and apoptosis, and is a key cellular mechanism for maintaining intracellular protein homeostasis. PROTAC technology, through UPS degradation of target proteins (POIs), can serve as an alternative for targeted anti-tumor therapy. PROTACs are heterobifunctional molecules composed of POI ligands, linker chains, and E3 ligase ligands. VHL and CRBN are widely used E3 ligase ligands for the design and synthesis of PROTACs. PROTACs induce ubiquitination of target proteins, thereby triggering subsequent proteasome degradation. To date, numerous VHL and CRBN-based PROTAC drugs have emerged, representing significant progress in cancer treatment. In particular, the emergence of PROTACs for overcoming drug-resistant mutants suggests that synthesizing PROTAC degraders targeting RET is an effective strategy for overcoming RET mutation resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a novel class of RET degraders. Activity tests show that these compounds not only have good anti-proliferation activity against RET wild-type and gatekeeper V804 mutant cells, but also have good anti-proliferation activity against solvent-front G810 mutant cells, while also exhibiting good degradation activity.

[0006] The purpose of this invention is to provide a pharmaceutical composition for preparing a drug for the prevention or treatment of cancer (non-small cell lung cancer, medullary thyroid carcinoma, thyroid cancer, ovarian epithelial cancer, salivary gland adenocarcinoma, pancreatic ductal carcinoma, breast cancer, prostate cancer, congenital megacolon cancer, neuroblastoma, irritable bowel syndrome).

[0007] The object of this invention is to provide a pharmaceutical composition for preparing a medicament for treating or preventing diseases related to RET activity or expression levels.

[0008] A first aspect of the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or an optical isomer thereof:

[0009]

[0010] Among them, RET inhibitors are the inhibitory portion of the RET protein, which has the following structure:

[0011]

[0012] Wherein, A is selected from the group consisting of: benzene ring; 5-7 membered nitrogen-containing heterocyclic group; chemical bond; wherein, when A is a benzene ring or a 5-7 membered nitrogen-containing heterocyclic group, A may be substituted by a substituent selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -O C1-C4 haloalkyl, -CN;

[0013] Y is selected from CH or N;

[0014] M is a divalent linker with -(L) x - The structure shown, wherein each L is independently selected from the group consisting of -O-, -S-, -NH-, -C(O)-, -CH2-; x is 0, 1, 2, 3 or 4 (when x is 0, the M is a chemical bond);

[0015] The B ring is a 5-12 member nitrogen-containing heterocyclic group; wherein the B ring can be composed of 0, 1, 2, 3, or 4 R groups. 1b Substituent substitution;

[0016] R 1a Selected from the following group: H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -OC1-C4 haloalkyl, -CN, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or unsaturated 3-10 membered heterocyclic groups containing 1-3 heteroatoms selected from O, N and S, C6-C 10 Aryl, a 5-10 membered heteroaryl group containing 1-3 heteroatoms selected from O, N, and S; and the R 1a It can be 0, 1, 2, 3 or 4 Rs 2a Substituent substitution;

[0017] R 1b and R 2a Each is independently selected from the following group: H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -O C1-C4 haloalkyl and -CN;

[0018] The linkers mentioned are selected from the following group (unless otherwise specified, the following linkers can be connected to RETinhibitors and E3 ligase ligands in any order):

[0019]

[0020] Wherein, D is independently -CH2-, -CH2CH2-, -C(O)- or a chemical bond;

[0021] Each E is independently -C(O)-, vinyl, Or chemical bonds; each J is independently -C(O)- or a chemical bond;

[0022] Each of F, G, L, and M is independently CH, C(OH), C(CF3), CF, or N;

[0023] Each P bond is independently a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -C(O)NH-, -C(O)CH2- or -C(O)CH2CH2-, -(CH2CH2O). p -、-NHC(O)NH-、-S(O)-、-S(O)2-、Vinyl、 Each Q is independently -C(O)-, -C(O)NH-, vinyl, -NH- or chemical bonds;

[0024] Each Cy is independently a substituted or unsubstituted benzene ring or a substituted or unsubstituted 5-10 membered heteroaryl ring containing 1-3 members selected from O, N and S; wherein the substitution refers to the H on the group being substituted by one or more groups selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated C1-C4 alkyl;

[0025] Each n and r is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0026] Each of m, j, s, and t is independently 0, 1, or 2;

[0027] p is 1, 2, 3, 4, 5;

[0028] The E3 ligase ligand is the ligand portion of the E3 ubiquitin ligase;

[0029] Unless otherwise specified, the terms “heteroaryl” or “heterocyclic” include 1 to 3 heteroatoms selected from the group consisting of N, S, or O; the cycloalkyl or heterocyclic group may be saturated or partially unsaturated, and may be monocyclic, fused, bridged, or spirocyclic structures, but excluding aromatic structures.

[0030] In another preferred embodiment, the E3 ligase ligand is selected from the group consisting of:

[0031]

[0032] Or the E3 ligase ligand described herein has the structure shown in the following formula:

[0033]

[0034] W is selected from the following group: -CH2-, -C(O)-, -SO2-;

[0035] R1 is independently 0, 1 or 2, and each R1 is independently selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated-C1-C4 alkyl.

[0036] Z is selected from the following group: -O-, -NH-, Chemical bond, Linker-C(O)NH-, Or a 6-10 spirocyclic group containing 1-2 N atoms.

[0037] In another preferred embodiment, the E3 ligase ligand is selected from the following structures:

[0038]

[0039] Among them, W is selected from the following groups: -CH2-, -C(O)-;

[0040] R1 is 0, 1 or 2, and each R1 is independently selected from the following group: D, halogen, C1-C4 alkyl and halo-C1-C4 alkyl;

[0041] Z is selected from the following group: -O-, -NH-, Chemical bond, Linker-C(O)NH-,

[0042] Ring B is selected from the following group:

[0043]

[0044] In another preferred embodiment, in Formula I, the RET inhibitors are:

[0045]

[0046]

[0047] In another preferred embodiment, the E3 ligase ligand is

[0048]

[0049] Among them, W is selected from the following groups: -CH2-, -C(O)-.

[0050] In another preferred embodiment, R1 is 0 or 1, and each R1 is independently selected from the group consisting of: D, halogens, C1-C4 alkyl groups and halogenated C1-C4 alkyl groups.

[0051] In another preferred embodiment, Z is Or chemical bonds.

[0052] In another preferred embodiment, the Linker is selected from the group consisting of:

[0053]

[0054] In another preferred embodiment, the Linker is a -C6-C12 alkyl-CO-, a -C2-C13 alkyl-(ethyl, vinyl, or ethynyl)-, or a -C2-C6 alkyl-(ethyl, vinyl, or ethynyl)-substituted or unsubstituted phenyl-CO-, The substitution refers to the H on the group being replaced by one or more groups selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated C1-C4 alkyl;

[0055] In another preferred embodiment, n1 is 6, 7, 8, 9, 10, 11, or 12.

[0056] In another preferred embodiment, n2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0057] In another preferred embodiment, n3 is 1, 2, 3, 4, 5, or 6.

[0058] In another preferred embodiment, the Linker is selected from the group consisting of:

[0059]

[0060]

[0061]

[0062]

[0063] .

[0064] A second aspect of the present invention provides a pharmaceutical composition comprising one or more compounds as described in the first aspect of the present invention, or pharmaceutically acceptable salts thereof or optical isomers thereof, and a pharmaceutically acceptable carrier.

[0065] A third aspect of the invention provides the use of compounds as described in the first aspect of the invention, or pharmaceutically acceptable salts thereof or optical isomers thereof, or pharmaceutical compositions as described in the second aspect of the invention, in the preparation of medicaments for the prevention or treatment of cancer, or diseases related to RET activity or expression levels; preferably, the cancer is selected from the group consisting of: non-small cell lung cancer, medullary thyroid carcinoma, thyroid cancer, ovarian epithelial carcinoma, salivary gland adenocarcinoma, pancreatic ductal carcinoma, breast cancer, prostate cancer, congenital megacolon cancer, neuroblastoma, and irritable bowel syndrome.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 The degradation activity of some compounds of the present invention after 6 hours of treatment in BaF3-KIF5B-RET-WT cells is shown.

[0068] Figure 2 The degradation activity of some compounds of the present invention on RET protein after incubation in BaF3-KIF5B-RET-WT cells for different times is shown.

[0069] Figure 3 The degradation activity of some compounds of the present invention on RET protein was observed after 6 hours of treatment in mutant cell lines BaF3-KIF5B-RET-G810C, BaF3-KIF5B-RET-G810R, and BaF3-KIF5B-RET-V804M.

[0070] Figure 4 The present invention provides a compound-dependent induction of apoptosis in Ba / F3-RET-KIF5B-WT and Ba / F3-RET-KIF5B-G810C cells. Detailed Implementation

[0071] Through long-term and in-depth research, the inventors have provided a class of compounds that target the degradation of RET, pharmaceutical compositions thereof, and applications. Specifically, the present invention provides compounds of formula (I), which are capable of degrading tyrosine protein kinase receptors (RET) via the proteasome pathway (UPP) and can be used to treat tumors with drug-resistant mutations. Based on the above findings, the inventors completed the present invention.

[0072] the term

[0073] In this document, unless otherwise specified, the term "substitution" refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of: halogen, amino, hydroxyl, nitro, cyano, trifluoromethyl, C1-C 12 Alkyl or cycloalkyl, C1-C 12 Alkoxy group, oxygen atom (i.e., =O), unsubstituted or C-substituted 1-4 Alkylamine-substituted C1-C 12 Alkylamine, C2-C6 ester, C2-C6 acyl, C2-C6 amide, thioC1-C 12 Alkyl, carboxyl, C5-C 12 Aryl or heteroaryl, C5-C 12 Heterocyclic group (containing 1-5, preferably 1-3, heteroatoms selected from N, O or S).

[0074] The term "C1-C" 12 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0075] The term "C1-C" 12 "Cycloalkyl" refers to a compound having 1-12 alkyl groups, preferably 3-12 (i.e., C12-12 alkyl groups). 3-12 ) A cycloalkyl group with a carbon atom, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or similar groups.

[0076] The term "C1-C" 12 "Alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 12 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups.

[0077] The term "halogen" refers to F, Cl, Br, and I.

[0078] The term "C1-C" 12 "Alkylamine group (or alkylamine group)" refers to a C1-C group that has been substituted with an amino group. 12 Alkyl groups, for example, those having "C1-C 12 Alkyl-NH- or (alkyl)2-N- (total number of carbon atoms is 1-12) or -C1-C12 Groups with the structures "alkylene-NH2", "alkyl-N-alkylene-(total number of carbon atoms 1-12)", or "(alkyl)2-N-alkylene-(total number of carbon atoms 1-12)", such as CH3NH-, C2H5NH-, C3H7NH-, (CH3)2N-, -CH2NH2, -C2H5NH2, -C3H7NH2, -C2H4N(CH3)2, or similar groups. Where C... 1-12 The definition of alkyl groups is as described above.

[0079] The term "C2-C6 ester group" refers to a substituent with a structure of "straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-oxy-" having 1-5 carbon atoms, such as ethyl ester, propyl ester, butyl ester, or similar groups.

[0080] The term "C1-C6 amide group" refers to a substituent with a structure of "a straight-chain or branched alkyl / cycloalkyl / aryl / heteroaryl-carbonyl-amine-" having 0-5 carbon atoms, such as acetamido, propionamido, butyramido, or similar groups.

[0081] The term "C6-C" 10 "Aryl" refers to a group having 1-12 (preferably 6-10, i.e., C) groups. 6-10 The aryl group of the carbon atom, such as phenyl, naphthyl, etc., may be substituted or unsubstituted.

[0082] The term "C1-C" 12 "Heteroaryl" refers to a heteroaryl group having 1-12 carbon atoms and one or more (preferably 1-3) heteroatoms selected from O, S and / or N, preferably C5-C8 heteroaryl. The heteroaryl group may be substituted or unsubstituted.

[0083] The term "5-7 membered heterocycle" refers to a cyclic saturated, partially unsaturated or aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0084] The term "5-7 membered heteroaryl" refers to a cyclic aromatic group having 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and / or N.

[0085] Specifically, expressions in the form "C1-Cn" indicate that the group has 1 to n carbon atoms. For example, expressions in the form "C1-C12" indicate that the group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms; "C6~C10" indicates that the group has 6, 7, 8, 9 or 10 carbon atoms.

[0086] In this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0087] In this invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is useless to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0088] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0089] As used herein, the term "compound of the invention" refers to a compound of Formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of compounds of Formula I.

[0090] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0091] Compounds that target the degradation of RET

[0092] This invention provides a compound that targets and degrades RET. Specifically, this invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or an optical isomer thereof:

[0093]

[0094] Among them, RET inhibitors are the inhibitory portion of the RET protein, which has the following structure:

[0095]

[0096] The definitions of each group are as described above. Preferably, the above-described compounds have the definitions of compounds synthesized as in any embodiment of this application.

[0097] Preparation method

[0098] The starting materials and chemical reagents required for the synthesis can be routinely synthesized according to literature or are commercially available. All are prepared using the following three general methods.

[0099] General Method 1:

[0100]

[0101] Examples 1-8 were synthesized according to Method 1. General Method 2:

[0102]

[0103] Examples 9-13 were synthesized according to Method 2. General Method 3:

[0104]

[0105] In this invention, Examples 14-18 are synthesized according to Method 3. General Method 4:

[0106]

[0107] In this invention, Example 19 is synthesized according to Method 4. General Method 5:

[0108]

[0109] In this invention, Examples 22-24 are synthesized according to Method 5.

[0110] General Method Six:

[0111]

[0112] In this invention, Examples 25-26 are synthesized according to Method Six.

[0113] General Method Seven:

[0114]

[0115] In this invention, Example 27 is synthesized according to Method 7.

[0116] General Method Eight:

[0117]

[0118] In this invention, Examples 28-29 are synthesized according to Method Eight.

[0119] Pharmaceutical Compositions and Administration

[0120] Because the compounds of the present invention have excellent targeted degradation activity of RET (reranged during transfection), the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate related diseases such as vascular damage caused by abnormal RET activity or expression (such as excessive activity or expression).

[0121] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 5-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0122] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0123] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0124] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0125] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0126] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0127] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0128] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0129] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0130] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0131] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0132] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0133] Compared with the prior art, the main advantages of the present invention include:

[0134] (1) A new class of compounds that target and degrade RET (reranged during transfection) have been discovered, which have excellent reversal activity for type II diabetes and its vascular damage symptoms.

[0135] Example 1

[0136] Synthesis of (2S,4R)-1-((2S)-2-(5-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)pentanoylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S1).

[0137]

[0138] Compound 6-bromo-4-hydroxypyrazolo[1,5-a]pyridine-3-carboxylonitrile 1a (1.0 g, 4.22 mmol, 1.0 equiv) was dissolved in dichloromethane (20 mL), followed by the addition of pyridine (0.4 mL, 5.06 mmol, 1.2 equiv). Tf2O (0.8 mL, 5.06 mmol, 1.2 equiv) was then added at 0 °C, and the reaction was stopped after 1 h. The crude product was concentrated and purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (20:1) to give a pale yellow solid compound 1b (1.5 g, 96% yield). 1 H NMR(400MHz,Chloroform-d)δ8.75(s,1H),8.30(s,1H),7.59(s,1H)

[0139] Compound 1b (1 g, 2.7 mmol, 1.0 equiv) and 2-fluoropyridine-5-boronic acid (457 mg, 3.24 mmol, 1.2 equiv) were added to a three-necked flask, and the mixture was purged once under argon protection. Pd(dppf)Cl2 (198 mg, 0.271 mmol, 0.1 equiv) was added, followed by 1,4-dioxane (20 mL). Then, potassium acetate solution (795 mg, 8.1 mmol, 3.0 equiv) was added at 0 °C until the mixture returned to room temperature. The mixture was stirred overnight at 40 °C. The crude product was concentrated and filtered, and then slurried with DCM to obtain a milky white solid compound 1c (1 g, 85% yield). 1 H NMR (400MHz, DMSO-d6) δ9.49(s,1H),8.73(s,1H),8.50(s,1H),8.27(t,J=9.06Hz,1H),7.86(s,1H),7.40(dd,J=8.20,2.28Hz,1H).

[0140] Compound 1c (500 mg, 1.57 mmol, 1.0 equiv) and 6-N-Boc-3,6-diazabicyclo[3.1.1]heptane (375 mg, 1.89 mmol, 1.2 equiv) were dissolved in DMSO (5 mL), followed by the addition of K2CO3 (653 mg, 4.73 mmol, 3.0 equiv), and the mixture was stirred overnight at 110 °C. After cooling the crude product, H2O was added directly to precipitate a pale yellow solid, which was then filtered to give compound 1d (700 mg, yield 89.6%). 1H NMR(400MHz,Chloroform-d)δ8.68(d,J=1.58Hz,1H),8.35(d,J=2.08Hz,1H),8.25(s,1H),7.71(dd,J=8.84,2.54Hz,1H),7.39(d,J=1.58Hz, 1H),6.65(d,J=8.84Hz,1H),4.31(d,J=4.50Hz,2H),4.18-4.13(m,2H) ,3.58-3.49(m,2H),2.69-2.65(m,1H),1.54-1.52(m,1H),1.38(s,9H).

[0141] Compound 1d (500 mg, 1.01 mmol, 1.0 equiv) was dissolved in DCM (4 mL), and trifluoroacetic acid (1 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was concentrated and purified by silica gel rapid column chromatography with dichloromethane:methanol (6:1) to give a yellow solid compound 1e (400 mg, yield: 97%). 1 H NMR (600MHz, DMSO-d6) δ9.39(s,1H),8.72(s,1H),8.45(s,1H),7.92(d,J=8.82Hz,1H),7.68(s,1H),6.88(d,J=8.80Hz,1 H),4.53(brs,2H),4.01(d,J=12.56Hz,2H),3.93(d,J=12.72Hz,2H),2.95-2.89(m,1H),1.92(dd,J=10.09,5.64Hz,1H).

[0142] Compound 1e (850 mg, 2.15 mmol, 1.0 equiv) was dissolved in a mixed solvent of DCE (20 mL) and MeOH (5 mL), and 6-methoxy-3-pyridinecarboxaldehyde (443 mg, 3.22 mmol, 1.5 equiv) was added. After stirring for half an hour, sodium borohydride acetate (1.8 g, 8.6 mmol, 4 equiv) was added, and the reaction was stirred overnight. The reaction solution was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (20:1) to give a yellow oily liquid compound 1f (190 mg, yield 15%). 1H NMR(600MHz,Chloroform-d)δ8.69(s,1H),8.39(d,J=2.16Hz,1H),8.26(s,1H), 8.09(s,1H),7.76(dd,J=8.70,2.20Hz,1H),7.64(d,J=8.78Hz,1H),7.40(s,1H) ,6.71(d,J=8.52Hz,1H),6.68(d,J=8.82Hz,1H),3.91(s,3H),3.91-3.79(m,4H) ,3.63-3.61(m,2H),3.47(s,2H),2.73(d,J=7.04Hz,1H),1.66(d,J=8.80Hz,1H).

[0143] Compound 1f (150 mg, 0.29 mmol, 1.0 equiv) and 1-Boc-pyrazole-4-boronic acid pinacol ester (86 mg, 0.29 mmol, 1.0 equiv) were dissolved in 1,4-dioxane (3.6 mL) and H2O (0.9 mL), followed by the addition of Na2CO3 (92 mg, 0.87 mmol, 3.0 equiv). The mixture was purged three times under argon protection, and then Pd(dppf)Cl2 (32 mg, 0.043 mmol, 0.15 equiv) was added. The mixture was stirred overnight at 90 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography with dichloromethane:methanol (25:1) to give 1 g (130 mg, 89% yield) of a yellow solid compound. 1 H NMR(400MHz,Chloroform-d)δ8.70(s,1H),8.45(d,J=2.24Hz,1H),8.29(s,1H),8.11(d,J=1.92Hz,1H),7.92(s,2H),7.83(dd,J=8.76,2.34Hz,1H), 7.67(d,J=10.12Hz,1H),7.46(s,1H),6.72(dd,J=8.44,2.88Hz,2H),3.92 (s,3H),3.90-3.80(m,4H),3.65-3.60(m,4H),2.74(brs,1H),1.69(s,1H).

[0144] 1 g (50 mg, 0.10 mmol, 1.0 equiv) of the compound and tert-butyl 5-bromopentanoate (47 mg, 1.98 mmol, 2 equiv) were dissolved in DMF (1 mL), followed by the addition of K2CO3 (42 mg, 0.3 mmol, 3.0 equiv) and NaI (7 mg, 0.3 mmol, 3.0 equiv), and the reaction was carried out overnight at 80 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (20:1) to give a yellow oily liquid compound 1 h (40 mg, yield 61%). 1 H NMR(400MHz,Chloroform-d)δ8.65(d,J=1.14Hz,1H),8.44(d,J=2.22Hz,1H),8.27(s,1H),8.11-8.09 (m,1H),7.83-7.80(m,2H),7.72(s,1H),7.64(d,J=8.44Hz,1H),7.43(d,J=1.20Hz,1H),6.71(t,J=7.9 8Hz,2H),4.20(t,J=7.00Hz,2H),3.92(s,3H),3.86-3.84(m,2H),3.81-3.76(m,2H),3.59(s,2H),3.48 (s,2H),2.71-2.69(m,1H),2.27(t,J=7.30Hz,2H),1.99-1.93(m,2H),1.68-1.62(m,3H),1.44(s,9H).

[0145] Compound 1h (40 mg, 0.06 mmol, 1.0 equiv) was dissolved in DCM (1 mL), and trifluoroacetic acid (0.3 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was concentrated to give a yellow oily compound 1i (35 mg, yield 96%).

[0146] Compound 1i (35 mg, 0.06 mmol, 1.0 equiv) was dissolved in DMF (1 mL), followed by the addition of DIPEA (63 μL, 0.36 mmol, 6.0 equiv), then HATU (23 mg, 0.06 mmol, 1 equiv), and finally VHL (27 mg, 0.06 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 1 h before being stopped. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S1 (24 mg, 21% yield). 1HNMR(600MHz,Methanol-d4)δ9.08(s,1H),9.02(s,1H),8.51-8.45(m,1H),8.38(d,J= 15.16Hz,1H),8.28(s,1H),8.22(s,1H),8.08-8.02(m,1H),8.00(s,1H),7.89-7.81(m, 1H),7.75(s,1H),7.44(d,J=8.34Hz,1H),7.41(d,J=8.28Hz,1H),7.21-7.13(m,1H),6. 90(d,J=8.52Hz,1H),4.98(q,J=7.0Hz,1H),4.69(brs,2H),4.62-4.58(m,2H),4.55(t, J=8.32Hz,1H),4.42(s,1H),4.30(brs,1H),4.25-4.22(m,4H),4.07(d,J=11.30Hz,1H) ,3.94(s,3H),3.87(d,J=11.10Hz,1H),3.73(dd,J=10.98,3.90Hz,1H),3.64-3.55(m,1 H),2.49(s,3H),2.34-2.30(m,2H),2.22-2.15(m,2H),1.96-1.90(m,3H),1.64-1.59(m ,2H),1.51(d,J=7.02Hz,1H),1.48(d,J=7.02Hz,3H),1.02(s,9H).MS(ESI,[M+H]+)m / z 515.9.

[0147] Example 2

[0148] Synthesis of (2S,4R)-1-((2S)-2-(7-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)heptanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S2).

[0149]

[0150] The preparation was carried out by replacing tert-butyl 5-bromopentanoate with tert-butyl 7-bromoheptanoate, referring to Example 1. 1H NMR(600MHz,Methanol-d4)δ9.15(s,1H),9.03(s,1H),8.52-8.46(m,1H),8.41-8.38 (m,2H),8.23(s,1H),8.11-8.10(m,1H),8.00(s,1H),7.88-7.83(m,1H),7.77(s,1H), 7.45(d,J=8.40Hz,2H),7.42(d,J=8.34Hz,2H),7.33(s,1H),6.90(d,J=8.58Hz,1H), 4.99(q,J=7.10Hz,1H),4.70(brs,2H),4.61(s,2H),4.58-4.55(m,1H),4.43-4.42(m, 1H),4.35-4.32(m,1H),4.27(d,J=13.02Hz,2H),4.21(t,J=6.84Hz,2H),4.11(d,J=1 1.22Hz,1H),3.95(s,3H),3.88-3.87(m,1H),3.73(dd,J=11.04,3.90Hz,1H),3.64-3. 58(m,1H),2.50(s,3H),2.29-2.25(m,2H),2.22-2.19(m,2H),1.95-1.89(m,4H),1.6 3-1.58(m,2H),1.49(d,J=7.02Hz,3H),1.38-1.34(m,4H),1.03(s,9H).MS(ESI,[M+H] + )m / z 529.9.

[0151] Example 3

[0152] Synthesis of (2S,4R)-1-((2S)-2-(9-(4-(3-cyano-4-(6-(6-(((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)nonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S3)

[0153]

[0154] The preparation was carried out by replacing tert-butyl 5-bromopentanoate with tert-butyl 9-bromononanoate, referring to Example 1. 1H NMR(600MHz,Methanol-d4)δ9.03(s,1H),9.00(s,1H),8.51-8.37(m,3H),8 .23(s,1H),8.01-8.00(m,2H),7.87-7.81(m,1H),7.74(brs,1H),7.44(d,J= 8.28Hz,2H),7.42(d,J=8.28Hz,2H),7.08-6.97(m,1H),6.91(d,J=8.46Hz, 1H),4.99(q,J=6.75Hz,1H),4.68(brs,2H),4.61-4.54(m,3H),4.42(brs,1H ),4.28(s,1H),4.26-4.20(m,4H),4.05-4.02(m,1H),3.95(s,3H),3.86(d, J=11.10Hz,1H),3.73(dd,J=11.10,3.90Hz,1H),3.60-3.56(m,1H),2.49(s, 3H),2.28-2.23(m,2H),2.22-2.18(m,2H),1.97-1.86(m,4H),1.61-1.55(m ,2H),1.49(d,J=7.02Hz,3H),1.36-1.33(m,8H),1.01(s,9H).MS(ESI,[M+H] + )m / z 543.9.

[0155] Example 4

[0156] Synthesis of (2S,4R)-1-((2S)-2-(11-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)undecanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S4)

[0157]

[0158] The preparation was carried out by replacing tert-butyl 5-bromopentanoate with tert-butyl 11-bromoundecanoate, referring to Example 1. 1HNMR(400MHz,Methanol-d4)δ9.02(s,1H),8.96(s,1H),8.55-8.50(m,1H),8 .48-8.37(m,2H),8.23(s,1H),8.02(s,1H),7.96(brs,1H),7.85-7.79(m,1H ),7.73(brs,1H),7.46-7.40(m,4H),7.04-6.94(m,1H),6.91(d,J=8.60Hz,1 H),5.01-4.98(m,1H),4.68(s,2H),4.62-4.60(m,1H),4.58-4.54(m,2H),4. 42(s,1H),4.29-4.25(m,2H),4.22-4.19(m,3H),4.02(d,J=13.20Hz,1H),3. 95(s,3H),3.87(d,J=11.80Hz,1H),7.43(dd,J=11.04,3.92Hz,1H),3.60-3. 55(m,1H),2.48(s,3H),2.23-2.18(m,3H),1.97-1.89(m,3H),1.60-1.55(m, 2H),1.50(d,J=7.04Hz,3H),1.37-1.30(m,14H),1.02(s,9H).MS(ESI,[M+H] + )m / z 557.9.

[0159] Example 5

[0160] Synthesis of (2S,4R)-1-((2S)-2-(15-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)pentadecaamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S5)

[0161]

[0162] The preparation was carried out by replacing tert-butyl 5-bromopentanoate with tert-butyl 15-bromopentadecanate, as described in Example 1. 1HNMR(400MHz,Methanol-d4)δ9.02(s,1H),8.95(s,1H),8.50-8.44(m,1H),8.42-8.37(m,2H),8.23(s,1H),8.02(s,1H),7.95(brs,1H),7.85(brs,1H ),7.74-7.72(m,1H),7.45-7.40(m,4H),7.04-6.94(m,1H),6.91(d,J=8.7 2Hz,1H),5.02-4.99m,1H),4.68(s,2H),4.62(s,1H),4.58-4.54(m,2H),4. 43(s,1H),4.29(brs,1H),4.25(s,1H),4.22-4.19(m,2H),4.03-4.00(m,1 H),3.95(s,3H),3.88-3.86(m,2H),3.75(dd,J=10.88,4.12Hz,1H),3.57(b rs,1H),2.48(s,3H),2.27-2.18(m,4H),1.95-1.88(m,2H),1.60-1.56(m,2 H),1.50(d,J=7.04Hz,3H),1.33-1.26(m,22H),1.04(s,9H).MS(ESI,[M+H] + )m / z 586.0.

[0163] Example 6

[0164] Synthesis of (2S,4R)-1-((2S)-2-(3-(2-(2-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl))))methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)ethoxy)ethoxy)propamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S6)

[0165]

[0166] Referring to Example 1, tert-butyl 5-bromopentanoate was replaced with tert-butyl 3-(2-(2-bromoethoxy)ethoxy)propionate for preparation. MS (ESI, [M+H] + )m / z 545.9.

[0167] Example 7

[0168] Synthesis of (2S,4R)-1-((14S)-14-(tert-butyl)-1-(4-(3-cyano-4-(6-(6-((6-methoxypyridin-3-yl)methyl))-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-acyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (S7)

[0169]

[0170] Referring to Example 1, tert-butyl 5-bromopentanoate was prepared by replacing it with tert-butyl 3-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)propionate. MS (ESI, [M+H] + )m / z 567.9

[0171] Example 8

[0172] Synthesis of 1-(2-(4-(4-(3-cyano-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3)-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-oxoethyl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)piperidin-4-carboxamide (S8)

[0173]

[0174] Synthesized according to Example 1. MS(ESI,[M+H) + )m / z 591.9.

[0175] Example 9

[0176] Synthesis of 6-(1-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)but-3-yn-1-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S9)

[0177]

[0178] The preparation was carried out by replacing 9-decyn-1-ol with 3-butyn-1-ol, as described in Example 12. 1 H NMR(600MHz, Methanol-d4)δ9.02(s,1H),8.46-8.37(m,3H),8.33(s,1H),8.07(s,1H),7.92(brs,1H),7.86(brs,1H),7.66(d,J=7.50Hz,1H), 7.59(s,1H),7.55(d,J=7.56Hz,1H),7.42(t,J=7.56Hz,1H),7.04-6.93(m,1H),6.91(d,J=8.46Hz,1H),4.98(dd,J=13.26,4.98Hz,1H),4.69( s,2H),4.59(brs,1H),4.47(t,J=6.06Hz,2H),4.33(s,2H),4.24(d,J=13.38Hz,2H),4.04-4.02(m,1H),3.95(s,3H),3.60(brs,1H),3.10(t,J =6.24Hz,2H),2.82-2.76(m,1H),2.66-2.63(m,1H),2.43-2.35(m,1H), 2.23-2.19(m,1H),2.05-2.03(m,1H),1.33-1.30(m,2H).MS(ESI,[M+H] + )m / z 798.3

[0179] Example 10

[0180] Synthesis of 6-(1-(6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)hex-5-yn-1-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S10)

[0181]

[0182] In Example 12, 9-decyn-1-ol was replaced with 5-hexyn-1-ol for preparation. 1H NMR(400MHz, Methanol-d4)δ8.96(s,1H),8.48-8.37(m,3H),8.24(s,1H),8.00(s,1H),7.94-7.86(m,2H),7.65(d,J=7.64Hz,2H),7.58(d,J =7.64Hz,1H),7.44(t,J=7.64Hz,1H),7.02-6.94(m,1H),6.91(d,J=8.52Hz,1H),4.98(dd,J=13.28,5.16Hz,1H),4.69(s,2H),4.59(brs,1H ),4.46(d,J=5.92Hz,2H),4.30(t,J=6.6Hz,3H),4.23(d,J=13.36Hz,2H),4.04-4.00(m,1H),3.95(s,3H),3.60(brs,1H),2.88-2.83(m,1H) ,2.77-2.72(m,1H),2.53(t,J=6.76Hz,2H),2.21(d,J=12.48Hz,1H),2.15-2.10(m,3H),1.70-1.62(m,2H),1.35-1.30(m,2H).MS(ESI,[M+H] + )m / z 826.4

[0183] Example 11

[0184] Synthesis of 6-(1-(8-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)oct-7-yn-1-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S11)

[0185]

[0186] The preparation was carried out by replacing 9-decyn-1-ol with 7-octyyn-1-ol, as described in Example 12. 1H NMR (400MHz, Methanol-d4) δ8.93(s,1H),8.48-8.37(m,3H),8.20(s,1H),7.98(s,1H),7.85(brs,1H),7.67(s,1H),7.61(d,J=7.48Hz,1H),7.52(d,J =7.20Hz,1H),7.39(t,J=7.64Hz,1H),7.07-6.96(m,1H),6.90(d,J=8.52Hz ,1H),5.13(dd,J=13.32,5.16Hz,1H),4.69(s,2H),4.59(brs,1H),4.43(d, J=4.00Hz,2H),4.29(s,1H),4.25(s,1H),4.21(t,J=6.44Hz,3H),4.05-4. 02(m,1H),3.95(s,3H),3.60(brs,1H),2.92-2.83(m,1H),2.77-2.73(m,1H ),2.48-2.44(m,2H),2.22-2.13(m,2H),1.97-1.90(m,2H),1.65-1.58(m,2 H),1.55-1.48(m,2H),1.43-1.38(m,2H),1.31-1.29(m,2H).MS(ESI,[M+H] + )m / z 854.4.

[0187] Example 12

[0188] Synthesis of 6-(1-(10-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)dec-9-yn-1-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S12)

[0189]

[0190] Step 1: Triphenylphosphine (443 mg, 1.7 mmol, 1.3 equiv) was dissolved in DCM (2 mL). Imidazole (115 mg, 1.7 mmol, 1.3 equiv) and I2 (428 mg, 1.7 mmol, 1.3 equiv) were added at 0 °C. Finally, 9-decyn-1-ol 12a (200 mg, 1.3 mmol, 1.0 equiv) was added, and the reaction was stopped after returning to room temperature for 2 hours. The reaction solution was concentrated and purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (40:1) to give a colorless oily compound 12b (360 mg, yield: 95%).

[0191] 1 H NMR(400MHz,Chloroform-d)δ3.17(t,J=7.04Hz,2H),2.17(td,J=7.04,2.64Hz,2H),1.93(t ,J=2.64Hz,1H),1.81(p,J=7.04Hz,2H),1.51(dt,J=14.63,6.86Hz,2H),1.43-1.24(m,8H).

[0192] Step 2: 1 g (40 mg, 0.079 mmol, 1.0 equiv) of compound and K2CO3 (33 mg, 0.24 mmol, 3 equiv) were added to ultra-dry DMF (1 mL), followed by the addition of 12b (42 mg, 0.16 mmol, 2.0 equiv). The mixture was stirred overnight at 60 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (40:1) to obtain a pale yellow oily liquid compound 12c (38 mg, yield: 54%). 1 H NMR(400MHz,Chloroform-d)δ8.64(s,1H),8.43(s,1H),8.26(s,1H),8.09(s,1H),7.80(d,J=8 .76Hz,2H),7.70(s,1H),7.65(d,J=8.16Hz,2H),7.42(s,1H),6.71-6.69(m,2H),4.16(t,J=6. 88Hz,2H),3.90(s,3H),3.87-3.80(m,3H),3.60(s,3H),3.50(brs,1H),3.35(s,1H),2.72(brs ,1H),2.17-2.14(m,2H),1.92(s,2H),1.71-1.65(m,2H),1.51-1.48(m,2H),1.38-1.33(m,8H).

[0193] Step 3: Compound 12c (15 mg, 0.02 mmol, 1.0 equiv) and 3-(4-iodo-1-oxoisoindolin-2-yl)piperidin-2,6-dione (13 mg, 0.04 mmol, 2.0 equiv) were dissolved in DMF (1 mL), and TEA (17 μL, 0.12 mmol, 6.0 equiv) was added. The mixture was purged once under argon protection. CuI (1 mg, 0.005 mmol, 0.2 equiv) and Pd(dppf)Cl2 (3 mg, 0.03 mmol, 0.15 equiv) were added, and the mixture was purged three times under argon protection. The reaction was carried out overnight at 80 °C. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S12. 1 H NMR(400MHz,Methanol-d4)δ8.96(s,1H),8.48-8.37(m,3H),8.20(s,1H),7 .99(s,1H),7.96-7.91(m,1H),7.89-7.84(m,1H),7.69(s,1H),7.64(d,J=7. 52Hz,1H),7.51(d,J=7.00Hz,1H),7.41(t,J=7.64Hz,1H),7.04-6.93(m,1H) ,6.90(d,J=8.64Hz,1H),5.15(dd,J=13.32,5.12Hz,1H),4.68(s,2H),4.58( brs,1H),4.42(dd,J=17.48,7.28Hz,2H),4.29-4.18(m,3H),4.04-4.01(m,1 H),3.95(s,3H),3.59(brs,1H),3.35(s,2H),2.87(dd,J=13.16,5.00Hz,1H) ,2.80-2.75(m,1H)2.44(t,J=6.48Hz,2H),2.21-2.16(m,2H),1.94-1.87(m, 2H),1.62-1.55(m,2H),1.49-1.42(m,2H),1.37-1.29(m,8H).MS(ESI,[M+H] + )m / z 882.5.

[0194] Example 13

[0195] Synthesis of 6-(1-(12-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)dodecane-11-yn-1-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S13)

[0196]

[0197] The preparation was carried out by replacing 9-decyn-1-ol with dodecyl-11-yn-1-ol according to Example 12. 1 H NMR(400MHz,Methanol-d4)δ8.98(s,1H),8.48-8.39f(m,3H),8.20(s,1H),8. 00(s,1H),7.93-7.86(m,2H),7.70(s,1H),7.67(d,J=7.52Hz,1H),7.54(d,J=7 .24Hz,1H),7.44(t,J=7.60Hz,1H),7.00-6.94(m,1H),6.90(d,J=8.08Hz,1H), 5.14(dd,J=13.36,5.16Hz,1H),4.68(s,2H),4.58(brs,1H),4.43(d,J=8.88Hz ,2H),4.27(brs,1H),4.22(s,1H),4.17(t,J=6.80Hz,2H),4.03-4.00(m,1H), 3.95(s,3H),3.59(brs,1H),2.94-2.95(m,1H),2.79-2.74(m,1H),2.47(dd,J= 13.12,4.72Hz,1H),2.42(t,J=6.76Hz,1H),2.21-2.15(m,2H),1.90-1.85(m,2 H),1.60-1.53(m,2H),1.46-1.43(m,2H),1.35-1.28(brs,11H).MS(ESI,[M+H] + )m / z 910.5.

[0198] Example 14

[0199] Synthesis of 6-(1-(1-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)prop-2-yn-1-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S14)

[0200]

[0201] The preparation was carried out by replacing 9-decyn-1-ol with 3-bromo-1-propyne, as described in Example 12. 1 H NMR(600MHz, Methanol-d4)δ9.00(s,1H),8.50-8.42(m,2H),8.37-8.28(m,2H),8.07(s,1H),7.99-7.94(m,1H),7.87-7.86(m,2H),7.80( d,J=7.50Hz,1H),7.72(brs,1H),7.59(t,J=6.90Hz,1H),7.04-6.94(m,1H),6.91(d,J=8.58Hz,1H),5.19(dd,J=13.44,4.80Hz,1H),4.68( s,3H),4.64-4.55(m,3H),4.47(m,2H),4.28(brs,1H),4.23(d,J=13.32Hz,2H),4.02(d,J=12.24Hz,1H),3.95(s,3H),3.91-3.88(m,2H), 3.59(brs,1H),3.47(s,2H),2.95-2.89(m,1H),2.80-2.77(m,1H),2.53-2.49(m,4H),2.21-2.19(m,2H),1.33-1.30(m,2H).MS(ESI,[M+H] + )m / z 867.4.

[0202] Example 15

[0203] Synthesis of 6-(1-(1-(5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)pent-4-yn-1-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S15)

[0204]

[0205] The preparation was carried out by replacing 9-decyn-1-ol with 4-pentyn-1-ol, as described in Example 12. 1 H NMR(400MHz,Methanol-d4)δ9.02(s,1H),8.50-8.42(m,2H),8.37-8.31(m,2H),8.08(s,1H),7.96(brs,1H),7.86(brs,1H),7.78-7 .74(m,2H),7.66(d,J=7.52Hz,1H),7.53(t,J=7.16Hz,1H),7.04-6.94(m,1H),6.91(d,J=8.52Hz,1H),5.22-5.18(m,1H),4.68(s,2H ),4.59(s,2H),4.53(d,J=9.48Hz,2H),4.28-4.22(m,3H),4.04(brs,1H),3.95(s,3H),3.84(d,J=12.24Hz,2H),3.59(brs,1H),2.9 3-2.89(m,1H),2.81-2.77(m,1H),2.69(t,J=6.36Hz,2H),2.54-2.43(brs,4H),2.21-2.12(m,5H),1.39-1.30(m,5H).MS(ESI,[M+H] + )m / z 895.4

[0206] Example 16

[0207] Synthesis of 6-(1-(1-(6-(2-(6-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)hexyl-5-yn-1-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1]heptane-3-yl)pyridin-3-yl]pyrazol[1,5-a]pyridine-3-onitrile (S16)

[0208]

[0209] In Example 12, 9-decyn-1-ol was replaced with 5-hexyn-1-ol for preparation. 1H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.50-8.41(m,2H),8.37-8.30(m,2H),8.07(s ,1H),8.00-7.95(m,1H),7.87-7.80(m,1H),7.75(t,J=7.74Hz,2H),7.64(d,J=7.50Hz,1 H),7.51(t,J=7.80Hz,1H),7.04-6.94(m,1H),6.91(d,J=7.86Hz,1H),5.19(dd,J=12.6 0,4.50Hz,1H),4.68(s,2H),4.62-4.58(m,2H),4.52(q,J=16.92Hz,2H),4.28(brs,1H), 4.23(d,J=13.20Hz,2H),4.03(d,J=12.18Hz,1H),3.95(s,3H),3.80(d,J=11.82Hz,2H) ,3.59-3.55(m,1H),3.26-3.19(m,3H),2.94-2.90(m,1H),2.81-2.78(m,1H),2.62(t,J= 6.66Hz,2H),2.56-2.49(m,1H),2.42-2.36(m,4H),2.20-2.18(m,2H),2.03-1.97(m,2H ),1.75(p,J=7.14Hz,2H),1.37(t,J=6.24Hz,1H),1.31(t,J=7.38Hz,1H).MS(ESI,[M+H] + )m / z 909.5.

[0210] Example 17

[0211] Synthesis of 6-(1-(1-(7-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)hepta-6-yn-1-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hepta-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S17)

[0212]

[0213] Compound 1f (236 mg, 0.46 mmol, 1.0 equiv) and tert-butyl 4-(4-boronic acid pinacol-1H-pyrazole-1-yl)piperidine-1-carboxylic acid (260 mg, 0.69 mmol, 1.5 equiv) were dissolved in dioxane (8 mL) and H2O (2 mL). Then, Na2CO3 (146 mg, 1.37 mmol, 3.0 equiv) was added, and the mixture was purged three times under argon protection. Pd(dppf)Cl2 (35 mg, 0.05 mmol, 0.1 equiv) was added, and the mixture was stirred overnight at 90 °C. The reaction mixture was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (40:1) to give a greenish-brown oily compound 17a (256 mg, yield: 82%). 1 H NMR(600MHz,Chloroform-d)δ8.64(s,1H),8.43(s,1H),8.27(s,1H),8.10(s,1H),7.82-7.80(m, 2H),7.74(s,1H),7.62(d,J=8.34Hz,1H),7.42(s,1H),6.70(t,J=6.72Hz,2H),4.11(dd,J=13.92, 7.02Hz,1H),3.91(s,3H),3.83(brs,2H),3.76(d,J=4.2Hz,2H),3.60-3.57(m,4H),2.92(brs,2H) ,2.68(brs,1H),2.18(d,J=12.36Hz,1H),2.03-1.93(m,4H),1.65(d,J=8.58Hz,1H),1.48(s,9H).

[0214] Compound 17a (256 mg, 0.37 mmol, 1.0 equiv) was dissolved in DCM (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was evaporated to dryness and concentrated to give a yellow oily compound 17b (212 mg, 97% yield).

[0215] In Example 12, 9-decyn-1-ol was replaced with 6-heptynol for preparation. 1H NMR(600MHz,Methanol-d4)δ9.01(s,1H),8.50-8.34(m,3H),8.29(s,1H),8.06(s,1H ),8.02-7.98(m,1H),7.88-7.83(m,1H),7.73(d,J=7.38Hz,2H),7.61-7.59(m,1H),7. 49(t,J=7.50Hz,1H),7.07-6.96(m,1H),6.90(d,J=8.52Hz,1H),5.17(dd,J=13.26,5. 04Hz,1H),4.69(s,2H),4.59(s,2H),4.50(q,J=17.40Hz,2H),4.29(brs,1H),4.24(d, J=13.26Hz,2H),4.05(d,J=12.36Hz,1H),3.95(s,3H),3.78(d,J=12.18Hz,2H),3.60 -3.53(m,1H),3.22-3.19(m,3H),2.94-2.88(m,1H),2.81-2.78(m,1H),2.57-2.51(m, 3H),2.40-2.37(m,4H),2.20-2.18(m,2H),1.88-1.83(m,2H),1.73(p,J=7.32Hz,2H), 1.61(p,J=7.20Hz,2H),1.37(t,J=6.12Hz,1H),1.31(t,J=7.32Hz,1H).MS(ESI,[M+H] + )m / z 923.4.

[0216] Example 18

[0217] Synthesis of 6-(1-(1-(9-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)non-8-yn-1-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S18)

[0218]

[0219] The preparation was carried out by replacing 9-decyn-1-ol with 8-nonyn-1-ol, as described in Example 12. 1H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.51-8.42(m,2H),8.37-8.28(m,2 H),8.06(s,1H),8.02-7.98(m,1H),7.87-7.83(m,1H),7.75-7.72(m,2H),7.6 0(d,J=7.62Hz,1H),7.50(t,J=7.62Hz,1H),7.07-6.96(m,1H),6.90(d,J=6. 78Hz,1H),5.20-5.17(m,1H),4.69(s,2H),4.59(s,2H),4.54-4.45(m,2H),4. 29(brs,1H),4.25-4.23(m,2H),4.05-4.03(m,1H),3.95(s,3H),3.75-3.73( m,2H),3.61-3.51(m,1H),3.19-3.17(m,4H),2.94-2.88(m,1H),2.81-2.78(m ,1H),2.55-2.51(m,4H),2.40-2.37(m,4H),2.21-2.19(m,2H),1.82-1.76(m ,2H),1.68-1.65(m,2H),1.58-1.53(m,2H),1.50-1.43(m,4H).MS(ESI,[M+H] + )m / z 951.3.

[0220] Example 19

[0221] Synthesis of 6-(1-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S19).

[0222]

[0223] Compound 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione 19a (300 mg, 1.09 mmol, 1.0 equiv) was dissolved in DMSO (5 mL), followed by the addition of DIPEA (567 μL, 3.26 mmol, 3.0 equiv), and then piperazine-1-carboxylic acid tert-butyl ester (304 mg, 1.63 mmol, 1.5 equiv). The mixture was stirred overnight at 110 °C. The reaction solution was concentrated and purified by silica gel column chromatography with petroleum ether:ethyl acetate (5:1) to give a bright yellow oily compound 19b (350 mg, yield: 73%). 1 H NMR(400MHz,Chloroform-d)δ8.23(s,1H),7.70(d,J=8.44Hz,1H),7.04(d,J=8.44Hz,1H),4.94(dd,J=11.80,4.88H z,1H),3.61-3.59(m,4H),3.42-3.40(m,4H),2.14-2.09(m,1H),2.06-2.04(m,1H),2.01-1.97(m,2H),1.48(s,9H).

[0224] Compound 19b (100 mg, 0.22 mmol, 1.0 equiv) was dissolved in DCM (2 mL), and trifluoroacetic acid (0.6 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was concentrated by rotary evaporation to give a yellow oily compound 19c (74 mg, yield: 97%).

[0225] Compound 19c (50 mg, 0.15 mmol, 1.0 equiv) was dissolved in ACN (2 mL), followed by the addition of DIPEA (76 μL, 0.44 mmol, 3.0 equiv), and then tert-butyl bromoacetate (33 μL, 0.22 mmol, 1.5 equiv). The reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (1:1) to give a yellow-green oily compound 19d (40 mg, yield: 60%). 1 H NMR(400MHz,Chloroform-d)δ7.66(d,J=8.48Hz,1H),7.26(s,1H),7.03(dd,J=8.56,2.16Hz,1H),4.92(dd,J=12.16 ,5.24Hz,1H),3.46-3.45(m,6H),3.17(s,2H),2.87-2.76(m,2H),2.74-2.71(m,4H),2.11-2.09(m,1H),1.45(s,9H).

[0226] Compound 19d (40 mg, 0.09 mmol, 1.0 equiv) was dissolved in DCM (1 mL), and trifluoroacetic acid (0.3 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was evaporated to dryness and concentrated to give a yellow oily compound 19e (34 mg, yield: 97%).

[0227] Compound 19e (20 mg, 0.05 mmol, 1.0 equiv) was dissolved in DMF (1 mL), followed by the addition of DIPEA (88 μL, 0.5 mmol, 10.0 equiv), then HATU (19 mg, 0.05 mmol, 1 equiv), and finally 19f (29 mg, 0.05 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 1 h before being stopped. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S19 (15 mg, 42% yield).

[0228]

[0229] 1 H NMR(400MHz,Methanol-d4)δ9.02(s,1H),8.50-8.42(m,2H),8.37-8.30(m, 2H),8.06(s,1H),8.00-7.96(m,1H),7.88-7.85(m,1H),7.78-7.74(m,2H),7 .49(d,J=1.96Hz,1H),7.37(dd,J=8.40,2.04Hz,1H),7.05-6.95(m,1H),6.9 1(d,J=8.56Hz,1H),5.10(dd,J=12.80,5.68Hz,1H),4.69-4.66(m,3H),4.59 -4.57(m,2H),4.45(q,J=15.24Hz,2H),4.28(brs,1H),4.24(d,J=13.16Hz, 2H),4.04(d,J=12.32Hz,1H),3.95(s,3H),3.88(d,J=14.16Hz,2H),3.62-3. 57(m,5H),3.35(s,4H),3.04-2.98(m,1H),2.92-2.82(m,1H),2.77-2.70(m, 2H),2.26-2.19(m,4H),2.14-2.11(m,2H),2.06-2.02(m,1H).MS(ESI,[M+H] + )m / z969.7.

[0230] Example 20

[0231] Synthesis of 6-(1-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)-2-oxoethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S20).

[0232]

[0233] Compound 20a (100 mg, 0.17 mmol, 1.0 equiv) was dissolved in ACN (2 mL), followed by the addition of DIPEA (89 μL, 0.51 mmol, 3.0 equiv), and then tert-butyl bromoacetate (37 μL, 0.26 mmol, 1.5 equiv). The reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by silica gel rapid column chromatography with dichloromethane:methanol (40:1) to give a yellow oily compound 20b (72 mg, yield: 60%).

[0234] Compound 20b (72 mg, 0.10 mmol, 1.0 equiv) was dissolved in DCM (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was concentrated by rotary evaporation to give a yellow oily compound 20c (64 mg, yield: 97%).

[0235] Compound 20c (20 mg, 0.03 mmol, 1.0 equiv) was dissolved in DMF (1 mL), followed by the addition of DIPEA (52 μL, 0.3 mmol, 10.0 equiv), then HATU (12 mg, 0.03 mmol, 1 equiv), and finally 20d (11 mg, 0.03 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 1 h before being stopped. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S20 (15 mg, 50% yield).

[0236]

[0237] 1H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.50-8.42(m,2H),8.37-8.28(m,2H ),8.09(s,1H),8.02-7.97(m,1H),7.88-7.82(m,1H),7.74(brs,1H),7.71(d,J =8.40Hz,1H),7.38(s,1H),7.26(d,J=8.46Hz,1H),7.07-6.96(m,1H),6.90(d, J=8.34Hz,1H),5.08(dd,J=12.72,5.46Hz,1H),4.72-4.69(m,4H),4.59(brs,1 H),4.42(s,2H),4.29(brs,1H),4.24(d,J=13.20Hz,2H),4.06-4.03(m,1H),3 .95(s,3H),3.86-3.82(m,3H),3.66-3.65(m,2H),3.60-3.59(m,3H),3.56-3.5 4(m,3H),3.38-3.35(m,2H),2.89-2.83(m,1H),2.76-2.68(m,2H),2.54-2.52( m,2H),2.46-2.44(m,2H),2.21-2.20(m,1H),2.13-2.10(m,1H).MS(ESI,[M+H] + )m / z 969.6.

[0238] Example 21

[0239] Synthesis of 6-(1-(1-(2-(6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S21).

[0240]

[0241] The compound piperazine-1-carboxylic acid tert-butyl ester was replaced with 2-(4-hydroxypiperidin-4-yl)acetic acid tert-butyl ester for synthesis. 1HNMR(600MHz,Methanol-d4)δ9.01(s,1H),8.50-8.41(m,2H),8.37(s,1H), 8.30(s,1H),8.03(s,1H),7.97(brs,1H),7.87–7.81(m,1H),7.73(s,1H),7. 6(d,J=8.40Hz,1H),7.3(s,1H),7.22(d,J=8.64Hz,1H),7.05–6.94(m,1H), 6.91(d,J=7.62Hz,1H),5.05(dd,J=12.78,5.10Hz,1H),4.73-4.68(m,4H),4 .59(s,1H),4.52(brs,1H),4.28(s,1H),4.23(d,J=13.44Hz,3H),4.04-4.0 1(m,1H),3.95(s,3H),3.79(d,J=12.90Hz,2H),3.60(s,1H),3.44-3.40(m,3 H),2.89-2.82(m,2H),2.75-2.68(m,3H),2.64-2.61(m,1H),2.23-2.18(m, 3H),2.11-2.03(m,2H),1.97-1.95(m,1H),1.86-1.79(m,4H).MS(ESI,[M+H] + )m / z 926.4.

[0242] Example 22

[0243] Synthesis of 6-(1-(3-(6-(2,6-dioxopiperidin-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)-3-oxopropyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S22).

[0244]

[0245] Compound 22a (50 mg, 0.09 mmol, 1.0 equiv) and tert-butyl 3-bromopropionate (37 mg, 0.18 mmol, 2 equiv) were dissolved in DMF (1 mL), followed by the addition of K2CO3 (38 mg, 0.27 mmol, 3.0 equiv), and the reaction was carried out overnight at 80 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (20:1) to give a yellow oily liquid compound 22b (35 mg, yield 57%).1 H NMR(400MHz,Chloroform-d)δ8.64(s,1H),8.42(d,J=2.28Hz,1H),8.26(s,1H),8.09(s,1H),7.82-7.79(m,2H ),7.74(s,1H),7.62(dd,J=10.76,2.28Hz,1H),7.42(s,1H),6.70(dd,J=8.48,3.96Hz,2H),4.20-4.15(m,1H), 3.91(s,3H),3.85-3.82(m,2H),3.77(d,J=5.72Hz,2H),3.63-3.60(m,1H),3.57(s,2H),3.05(d,J=11.68Hz,2 H),2.73-2.69(m,2H),2.43(t,J=7.32Hz,2H),2.25-2.19(m,4H),2.10-2.03(m,1H),1.85(s,4H),1.45(s,9H).

[0246] Compound 22b (35 mg, 0.05 mmol, 1.0 equiv) was dissolved in DCM (1 mL), and trifluoroacetic acid (0.3 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was concentrated by rotary evaporation to give a yellow oily compound 22c (31 mg, yield: 96%).

[0247] Compound 22c (20 mg, 0.03 mmol, 1.0 equiv) was dissolved in DMF (1 mL), followed by the addition of DIPEA (52 μL, 0.3 mmol, 10.0 equiv), then HATU (12 mg, 0.03 mmol, 1 equiv), and finally 22d (10 mg, 0.03 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 1 h before being stopped. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S22 (14 mg, 49% yield).

[0248]

[0249] 1H NMR (400MHz, Methanol-d4) δ9.02(s,1H),8.50-8.42(m,2H),8.37-8.31(m,2H),8.08(s,1H),8.00-7.94(m,1H),7.88(d,J=9 .56Hz,2H),7.85-7.84(m,1H),7.74(brs,1H),7.05-6.94(m,1H),5.16(dd,J=12.80,5.60Hz,1H),5.07(s,2H),4.94(s,2H),4 .72-4.65(m,3H),4.59(s,2H),4.28(s,1H),4.23(d,J=13.24,3H),4.04-4.01(m,1H),3.95(s,3H),3.92-3.87(m,2H),3.62- 3.30(m,4H),3.05(t,J=6.12,2H),2.93-2.84(m,1H),2.78-2.72(m,2H),2.45-2.42(m,4H),2.21-2.14(m,3H).MS(ESI,[M+H] + )m / z 940.4.

[0250] Example 23

[0251] Synthesis of 6-(1-(1-(2-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-4-hydroxypiperidin-4-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S23)

[0252]

[0253] The compound piperazine-1-carboxylic acid tert-butyl ester was replaced with 2-(4-hydroxypiperidin-4-yl)acetic acid tert-butyl ester for synthesis. 1HNMR(600MHz,Methanol-d4)δ9.01(s,1H),8.50-8.41(m,2H),8.37(s,1H), 8.30(s,1H),8.03(s,1H),7.97(brs,1H),7.87–7.81(m,1H),7.73(s,1H),7. 6(d,J=8.40Hz,1H),7.3(s,1H),7.22(d,J=8.64Hz,1H),7.05–6.94(m,1H), 6.91(d,J=7.62Hz,1H),5.05(dd,J=12.78,5.10Hz,1H),4.73-4.68(m,4H),4 .59(s,1H),4.52(brs,1H),4.28(s,1H),4.23(d,J=13.44Hz,3H),4.04-4.0 1(m,1H),3.95(s,3H),3.79(d,J=12.90Hz,2H),3.60(s,1H),3.44-3.40(m,3 H),2.89-2.82(m,2H),2.75-2.68(m,3H),2.64-2.61(m,1H),2.23-2.18(m, 3H),2.11-2.03(m,2H),1.97-1.95(m,1H),1.86-1.79(m,4H).MS(ESI,[M+H] + )m / z 984.4.

[0254] Example 24

[0255] Synthesis of 6-(1-(1-(2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S24).

[0256]

[0257] The compound piperazine-1-carboxylic acid tert-butyl ester was replaced with 4-piperidine acetate tert-butyl ester for synthesis. 1H NMR(400MHz,Methanol-d4)δ9.02(s,1H),8.50-8.41(m,2H),8.37-8.31(m,2 H),8.04(s,1H),8.00-7.97(m,1H),7.87-7.81(m,1H),7.74(s,1H),7.64(d,J =8.52Hz,1H),7.32(s,1H),7.20(d,J=8.56Hz,1H),7.07-6.96(m,1H),6.91( d,J=8.72Hz,1H),5.06(dd,J=12.44,5.48Hz,1H),4.72-4.69(m,3H),4.60-4. 58(m,2H),4.53-4.50(m,1H),4.29(brs,1H),4.24(d,J=12.96Hz,2H),4.19- 4.15(m,1H),4.06-4.03(m,3H),3.95(s,3H),3.05-2.99(m,3H),2.90-2.81(m ,2H),2.76-2.68(m,2H),2.45-2.43(m,2H),2.28-2.19(m,3H),2.11-2.07(m ,2H),2.03-1.95(m,2H),1.91-1.88(m,3H),1.44-1.34(m,2H).MS(ESI,[M+H] + )m / z 968.5.

[0258] Example 25

[0259] Synthesis of 6-(1-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-1-yl)-2-oxoethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S25).

[0260]

[0261] Compound 25a, 5-bromo-2-(2,6-dioxopiperidin-3-yl)isodihydroindole-1,3-dione (200 mg, 0.59 mmol, 1.0 equiv), and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (367 mg, 1.19 mmol, 2.0 equiv) were dissolved in DMF (3 mL). Then, CsF (269 mg, 1.77 mmol, 3.0 equiv) was added, and the mixture was purged three times under argon protection. Pd(dppf)Cl2 (86 mg, 0.12 mmol, 0.2 equiv) was added, and the mixture was stirred overnight at 90 °C. The reaction mixture was concentrated and purified by rapid silica gel column chromatography with petroleum ether:ethyl acetate (1:1) to give a yellow oily compound 25b (150 mg, 58% yield). 1 H NMR(400MHz,Chloroform-d)δ7.81(s,1H),7.76(d,J=7.88Hz,1H),7.67(d,J=7.84Hz,1H),6.21(s,1H),4.87-4.92(m,1H) ,4.07(brs,2H),3.60(t,J=5.44Hz,1H),2.74-2.69(m,2H),2.50(brs,2H),2.33(brs,1H),2.09-2.06(m,1H),1.43(s,9H).

[0262] Compound 25b (150 mg, 0.34 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (3 mL), followed by the addition of Pd(OH)₂ (24 mg, 0.17 mmol, 0.5 equiv), and the mixture was purged twice under H₂ protection. The reaction was stirred overnight at room temperature. After filtration through diatomaceous earth, the solution was concentrated to give a colorless oily liquid 25c (110 mg, 73% yield). 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.72Hz,1H),7.70(s,1H),7.56(d,J=7.72Hz,1H),4.98-4.94(m,1H),2. 86-2.72(m,4H),2.11-2.08(m,1H),1.82(d,J=12.56Hz,1H),1.66-1.60(m,2H),1.45(s,9H),1.30-1.22(m,4H).

[0263] Compound 25c (30 mg, 0.07 mmol, 1.0 equiv) was dissolved in DCM (1 mL), and trifluoroacetic acid (0.3 mL) was added. The reaction was stirred for 1 hour and then stopped. The reaction solution was evaporated to dryness and concentrated to give a yellow oily compound 25d (22 mg, yield: 96%).

[0264] 25 g (20 mg, 0.03 mmol, 1.0 equiv) of the compound was dissolved in DMF (1 mL), followed by the addition of DIPEA (52 μL, 0.3 mmol, 10.0 equiv), then HATU (11 mg, 0.03 mmol, 1 equiv), and finally 25 d (10 mg, 0.03 mmol, 1.0 equiv). The reaction was stirred at room temperature for 1 h before stopping. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S25 (15 mg, 45% yield).

[0265]

[0266] 1 H NMR(400MHz, Methanol-d4)δ9.03(s,1H),8.50-8.43(m,2H),8.37-8.27(m,2H),8.10(s,1H),8.00-7.95(m,1H),7.85-7.81(m,3H),7.76(d, J=8.08Hz,2H),7.04-6.95(m,1H),6.91(d,J=8.64Hz,1H),5.14(dd,J=12.80,5.64Hz,1H),4.73-4.69(m,4H),4.58(brs,1H),4.46-4.38(m, 2H),4.28-4.25(m,2H),4.22(s,1H),4.03(d,J=12.88Hz,1H),3.95(s ,3H),3.88-3.86(m,2H),3.14-3.08(m,1H),2.93-2.83(m,3H),2.79-2 .71(m,2H),2.54-2.43(m,5H),2.22-2.13(m,3H),2.02-1.99(m,2H),1 .89-1.82(m,1H),1.74-1.62(m,1H),1.44-1.27(m,1H).MS(ESI,[M+H] + )m / z 968.5.

[0267] Example 26

[0268] Synthesis of 6-(1-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-1-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S26).

[0269]

[0270] 1 H NMR(400MHz,Methanol-d4)δ9.01(s,1H),8.49-8.41(m,2H),8.37-8.30(m, 2H),8.05(s,1H),8.00(d,J=8.96Hz,1H),7.87-7.84(m,3H),7.80-7.78(m,1 H),7.74(brs,1H),7.08-6.98(m,1H),6.90(d,J=8.60Hz,1H),5.14(dd,J=12 .80,5.68Hz,1H),4.69-4.67(m,2H),4.65(brs,1H),4.60-4.54(m,2H),4.45 -4.33(m,2H),4.31-4.26(m,2H),4.23(s,1H),4.06(d,J=12.68Hz,1H),3.94 (s,3H),3.91-3.88(m,1H),3.83-3.80(m,2H),3.60(brs,1H),3.40-3.33(m, 2H),3.20-3.14(m,1H),3.03-2.97(m,2H),2.93-2.84(m,1H),2.78-2.71(m, 2H),2.27-2.13(m,9H),2.08-1.98(m,2H),1.65-1.62(m,1H).MS(ESI,[M+H] + )m / z968.5.

[0271] Example 27

[0272] Synthesis of 6-(1-((1R,4R)-4-((6-(2,6-dioxopiridine-3-yl)-5,7-dioxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S27).

[0273]

[0274] Compound cis-4-hydroxycyclohexylcarboxylate methyl ester 27a (300 mg, 1.90 mmol, 1.0 equiv) was dissolved in DCM (4 mL), and pyridine (457 μL, 5.70 mmol, 3.0 equiv) was added. Then, p-toluenesulfonyl chloride (542 mg, 2.84 mmol, 1.5 equiv) was added in an ice bath at 0 °C. The reaction was stopped after stirring at room temperature for 2 h. The reaction solution was concentrated and purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (20:1) to give colorless oily compound 27b (300 mg, yield 51%).

[0275] 1 H NMR(400MHz,Chloroform-d)δ7.78(d,J=8.24Hz,2H),7.32(d,J=8.16Hz,2H),4.70(s,1H),3.65( s,3H),2.43(s,3H),2.35-2.28(m,1H),1.89-1.81(m,4H),1.73-1.66(m,2H),1.57-1.49(m,2H).

[0276] Compound 27b (300 mg, 0.96 mmol, 1.0 equiv) was dissolved in DMF (4 mL), Cs₂CO₃ (940 mg, 2.88 mmol, 3.0 equiv) was added, and finally 4-bromopyrazole (212 mg, 1.44 mmol, 1.5 equiv) was added. The mixture was stirred at 80 °C for 16 h. The reaction solution was extracted and concentrated, and then purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (10:1) to give a colorless oily compound 27c (300 mg, yield 44%).

[0277] 1H NMR(600MHz,Chloroform-d)δ7.44-7.42(m,1H),7.41(s,1H),4.09-4.05(m,1H),3.68(s,3H),2.38-2.34(m,1H),2.22-2.14(m,4H),1.76-1.72(m,4H).

[0278] Compound 27c (150 mg, 0.52 mmol, 1.0 equiv) was dissolved in DCM (3 mL), and DIBAL-H (42 μL, 0.63 mmol, 1.2 equiv) was slowly added at -78 °C. The reaction was stirred for 2 h until the reaction was complete. The reaction solution was extracted and concentrated, and then purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (6:1) to give a colorless oily compound 27d (80 mg, yield 60%).

[0279] 1 H NMR(400MHz,Chloroform-d)δ9.65(s,1H),7.42(s,1H),7.41(s,1H),4.05-3.99(m ,1H),2.32-2.28(m,1H),2.26-2.14(m,4H),1.83-1.74(m,2H),1.48-1.41(m,2H).

[0280] Compound 27d (120 mg, 0.39 mmol, 1.0 equiv) and pinacol diboronate (367 mg, 0.59 mmol, 1.5 equiv) were dissolved in 1,4-dioxane (3 mL), followed by the addition of KoAC (115 mg, 1.17 mmol, 3.0 equiv). The mixture was purged three times under argon protection, and then PdCl2(PCy3)2 (29 mg, 0.039 mmol, 0.1 equiv) was added. The mixture was stirred overnight at 90 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (1:1) to give a yellow oily compound 27e (60 mg, 42% yield).

[0281] 1 H NMR(400MHz,Chloroform-d)δ9.65(s,1H),7.77(s,1H),7.72(s,1H),4.12-4.06 (m,1H),2.35-2.33(m,1H),2.29-1.14(m,4H),1.86-1.76(m,4H),1.29(s,12H).

[0282] Compounds 27e (60 mg, 0.20 mmol, 1.2 equiv) and 27f (85 mg, 0.17 mmol, 1.0 equiv) were dissolved in 1,4-dioxane (1.5 mL) and H₂O (0.2 mL), followed by the addition of Na₂CO₃ (54 mg, 0.51 mmol, 3.0 equiv). The mixture was purged three times under argon protection, and then Pd(dppf)Cl₂ (12 mg, 0.017 mmol, 0.1 equiv) was added. The mixture was stirred overnight at 90 °C. The reaction solution was concentrated and purified by silica gel rapid column chromatography using dichloromethane:methanol (20:1) to give 27 g (45 mg, 44% yield) of a yellow oily compound.

[0283] 1 H NMR(400MHz,Chloroform-d)δ9.70(s,1H),8.65-8.64(m,1H),8.42(d,J=2.40Hz,1H),8.27(s,1H),8.08(s ,1H),7.83-7.79(m,2H),7.75(s,1H),7.64-7.61(m,1H),7.43-7.42(m,1H),6.72-6.69(m,2H),4.14-4.08 (m,1H),3.91(s,3H),3.85-3.82(m,1H),3.78(d,J=5.80Hz,1H),3.61(brs,1H),3.67(s,2H),2.71-2.66(m ,1H),2.38-2.33(m,3H),2.24(d,J=13.52Hz,2H),1.96-1.85(m,2H),1.66-1.64(m,2H),1.54-1.45(m,2H).

[0284] 27 g (20 mg, 0.03 mmol, 1.0 equiv) and 27 h (10 mg, 0.17 mmol, 1.0 equiv) of the compound were dissolved in MeOH (1 mL). The mixture was stirred at room temperature for 0.5 h, and then NaBH3CN was added under an ice bath at 0 °C. The reaction was stirred at room temperature for 1 h until complete. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S27 (13 mg, yield 45%).

[0285]

[0286] 1H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.51-8.42(m,2H),8.37-8.29(m,2H),8.05(s,1H),7.95(s,3H),7.86-7.82(m,1H),7 .74(brs,1H),7.04-6.94(m,1H),6.91(d,J=9.00Hz,1H),5.18(dd,J=12.78,5.34Hz,1H),4.95(brs,4H),4.68(s,2H),4.58(brs ,1H),4.31-4.28(m,3H),4.23(d,J=13.20Hz,2H),4.03(d,J=11.76Hz,1H),3.95(s,3H),3.60(s,1H),3.48(d,J=6.90Hz,2H),2 .91-2.85(m,1H),2.78-2.73(m,2H),2.29-2.27(m,2H),2.19-2.11(m,4H),2.07-1.97(m,3H),1.45-1.38(m,2H).MS(ESI,[M+H] + )m / z 897.4.

[0287] Example 28

[0288] Synthesis of 6-(1-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)ethyl)piperidin-4-alkyl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (S28).

[0289]

[0290] Compound 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione 28a (214 mg) was used.

[0291] 0.77 mmol (1.0 equiv) was dissolved in DMSO (2 mL), DIPEA (410 μL, 2.31 mmol, 3.0 equiv) was added, followed by 4-piperidineethanol (100 mg, 0.77 mmol, 1.0 equiv), and the mixture was stirred overnight at 110 °C. The reaction solution was extracted and concentrated, and then purified by silica gel rapid column chromatography with petroleum ether:ethyl acetate (2:1) to give a bright yellow solid compound 28b (268 mg, yield: 90%).

[0292] 1 H NMR(400MHz,Chloroform-d)δ8.16(s,1H),7.66(d,J=8.56Hz,1H),7.03(dd,J=8.56,2.24Hz,1H),7.00(dd,J=12.12,5.12Hz,1H),3.94(d,J=13.08Hz,1 H),3.74(t,J=6.48Hz,1H),3.00-2.93(m,2H),2.90-2.71(m,3H),2.14-2.09 (m,1H),1.84(d,J=13.36Hz,2H),1.55(q,J=6.56Hz,2H),1.37-1.29(m,3H).

[0293] Compound 28b (120 mg, 0.31 mmol, 1.0 equiv) was dissolved in DCM (3 mL), and DMP (158 mg, 0.37 mmol, 1.2 equiv) was added under ice bath conditions at 0 °C. The reaction mixture was stirred at room temperature for 2 h before the reaction was stopped. The reaction solution was concentrated and purified by silica gel rapid column chromatography using petroleum ether:ethyl acetate (1:1) to give a bright yellow oily compound 28c (68 mg, yield 57%).

[0294] 1 H NMR(400MHz,Chloroform-d)δ9.80(s,1H),8.08(s,1H),7.67(d,J=8.56Hz,1H),7.6 7(d,J=8.56Hz,1H),7.04(d,J=8.60Hz,1H),4.93(dd,J=12.08,5.04Hz,1H),3.94(d ,J=13.32Hz,1H),3.02(t,J=11.32Hz,2H),2.91-2.71(m,3H),2.44(d,J=6.68Hz,2H ),2.24-2.16(m,1H),2.14-2.10(m,1H),1.86(d,J=12.72Hz,2H),1.42-1.31(m,2H).

[0295] Compounds 28c (16 mg, 0.04 mmol, 1.2 equiv) and 28d (20 mg, 0.03 mmol, 1.0 equiv) were dissolved in DCM (1 mL). After stirring at room temperature for 0.5 h, NaBH3CN was added under an ice bath at 0 °C, and the reaction was stirred at room temperature for 1 h until complete. The reaction solution was concentrated and separated by reverse-phase column chromatography to obtain compound S28 (16 mg, yield 49%).

[0296]

[0297] 1 H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.50-8.42(m,2H),8.37-8.29(m,2H ),8.07(s,1H),8.02-7.98(m,1H),7.86-7.82(m,1H),7.74(s,1H),7.65(d,J=8 .46Hz,1H),7.33(s,1H),7.21(d,J=8.52Hz,1H),7.07-6.97(m,1H),6.90(d,J =8.64Hz,1H),5.06(dd,J=12.78,5.34Hz,1H),4.69(s,2H),4.59(s,2H),4.29( s,1H),4.24(d,J=13.20Hz,2H),4.05(d,J=12.00Hz,3H),3.95(s,3H),3.80(d ,J=12.42Hz,2H),3.60-3.51(m,1H),3.29-3.20(m,4H),3.00(t,J=12.60Hz,2H ),2.89-2.82(m,1H),2.76-2.67(m,2H),3.43-2.36(m,4H),2.21-2.09(m,2H) ,1.88(d,J=12.72Hz,2H),1.78-1.71(m,3H),1.42-1.36(m,2H).MS(ESI,[M+H] + )m / z 954.5.

[0298] Example 29

[0299] Synthesis of 6-(1-(1-((1-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxynitrile (S29).

[0300]

[0301] 1H NMR(600MHz,Methanol-d4)δ9.02(s,1H),8.51-8.42(m,2H),8.37-8.30(m,2H), 8.08(s,1H),8.04-8.02(m,1H),7.87-7.83(m,1H),7.75(s,1H),7.66(d,J=8.52H z,1H),7.33(s,1H),7.22(d,J=8.52Hz,1H),7.10-7.00(m,1H),6.90(d,J=8.64H z,1H),5.06(dd,J=12.66,5.46Hz,1H),4.69(s,2H),4.63-4.59(m,2H),4.30(s,1 H),4.25(d,J=13.14Hz,2H),4.08(d,J=12.84Hz,3H),3.95(s,3H),3.84(d,J=12 .60Hz,2H),3.60-3.54(m,2H),3.28-3.23(m,2H),3.15-3.14(m,2H),3.05(t,J=1 2.36Hz,2H),2.89-2.82(m,1H),2.76-2.67(m,2H),2.49-2.42(m,4H),2.22-2.1 9(m,2H),2.11-2.09(m,1H),1.97-1.95(m,2H),1.47-1.41(m,2H).MS(ESI,[M+H] + )m / z940.5.

[0302] Example 30: Degradation activity test of the compound against BaF3-KIF5B-RET wild-type protein (Western Blot)

[0303] To ensure cells were in logarithmic growth phase, they were seeded in six-well plates and incubated overnight before treatment. BaF3-KIF5B-RET cells were treated with gradually increasing doses of the compound for 6 hours. After treatment, cells were collected and washed once with ice-cold phosphate-buffered saline (PBS) before Western blotting analysis. Following the specified treatment, cells were lysed by dissolving them in 1× sodium dodecyl sulfate (SDS) sample lysis buffer containing protease and phosphatase inhibitors. Cell lysates were loaded onto a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel, proteins were separated by electrophoresis, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 1×TBS (0.5% Tween 20 and 5% skim milk), the membrane was incubated overnight at 4°C with the corresponding primary antibody. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 1–2 hours. Finally, the protein lanes were visualized using the ELC Western blot assay kit (Therm Scientific, Waltham, MA). Degradation rate <30%: +; 30% < degradation rate <60%: ++; 60% < degradation rate <100%: +++.

[0304] Table 1

[0305]

[0306]

[0307] The results are shown in Table 1, which presents the quantitative results of the degradation of RET protein by the compounds of the present invention in BaF3-RET-WT cells. The results show that most of the compounds of the present invention exhibit good degradation effects on RET protein in the BaF3-RET-WT cell line.

[0308] Example 31: Degradation activity test of compound S17 against BaF3-KIF5B-RET protein

[0309] This embodiment evaluated the degradation ability of compound S17 on wild-type and mutant BaF3-KIF5B-RET proteins. The results showed that the representative compound in this invention has a strong degradation ability on both wild-type and mutant RET proteins.

[0310] The results are attached. Figure 1 As shown, compound S17, when used in BaF3-KIF5B-RET-WT cells for 6 hours, exhibited a very significant degradation effect on RET protein, achieving almost complete degradation at a concentration of 33.3 nM.

[0311] As attached Figure 2As shown, when compound S17 was co-incubated with BaF3-KIF5B-RET-WT cells at a concentration of 30 nM for different times, RET protein showed significant degradation in 0.5 hours and more than 80% of the protein was degraded in 12 hours.

[0312] As attached Figure 3 As shown, when compound S17 was used in mutant cell lines BaF3-KIF5B-RET-G810C, BaF3-KIF5B-RET-G810R, and BaF3-KIF5B-RET-V804M for 6 hours, it showed a very significant degradation effect on RET protein. At a concentration of 300 nM, compound S17 could degrade more than 60% of the protein.

[0313] Example 32: Test of the inhibitory activity of the compound on the proliferation of BaF3-KIF5B-RET cell line

[0314] Three cell lines, BaF3-KIF5B-RET-WT, BaF3-KIF5B-RET-G810C, and BaF3-KIF5B-RET-G810R, were seeded at 8000-10000 cells per well in 96-well plates containing complete culture medium. After overnight seeding, the cells were cultured for 72 hours with gradient concentrations (0-10 μM) of the test compound. After treatment, 10 μL of Cell Counting Kit-8 (CCK8 reagent) was added to each well, and incubation continued for 1-3 hours. The absorbance at 450 nm and 650 nm was then measured using a microplate reader, and the absorbance value (A) of each well was calculated as OD450-OD650. The half-maximal inhibitory concentration (IC50) was determined. 50 The concentration-response curves were fitted using GraphPadPrism 8.0 software to calculate the IC50. The data are the average of at least three independent experiments. 50 >10μM:+; 1μM <IC 50 <10μM:++;100nM <IC 50 <1μM:++; IC 50 <100nM:++++

[0315] Table 2

[0316]

[0317]

[0318] The results are shown in Table 2, which presents the inhibitory activity of the compounds of the present invention against Ba / F3-RET-KIF5B-WT cells and against the mutant cell lines Ba / F3-RET-KIF5B-G810C and Ba / F3-RET-KIF5B-G810R. The results show that most of the compounds of the present invention exhibit good inhibitory activity against both wild-type and mutant Ba / F3-RET-KIF5B cell lines.

[0319] Example 33 Compound S17 induces apoptosis

[0320] Apoptosis analysis was performed using the PE Annexin V Apoptosis Detection Kit (#559763, BD Bioscience) following the procedures provided by BD Bioscience (Fisher Scientific, USA).

[0321] Cells were treated with the specified compound for 48 hours. Cells were then collected and washed twice with ice-cold phosphate-buffered saline (PBS). Cells were resuspended in 1×BD binding buffer (#556454, BD Bioscience) and stained in the dark with 7ADD (#559925, BD Bioscience) and Annexin V-PE (#556422, BD) for 15 minutes. 1×BD binding buffer was added to stop staining. Cells were then analyzed using a Guava EeasyCyte flow cytometer (MerckUSA).

[0322] The results are attached. Figure 4 As shown, the representative compound S17 of this invention can induce early apoptosis in Ba / F3-RET-KIF5B-WT and Ba / F3-RET-KIF5B-G810C cells in a concentration-dependent manner.

[0323] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, or an optical isomer thereof: in, RET inhibitors are the inhibitory portion of the RET protein, and they have the following structure: Wherein, A is selected from the group consisting of: benzene ring; 5-7 membered nitrogen-containing heterocyclic group; chemical bond; wherein, when A is a benzene ring or a 5-7 membered nitrogen-containing heterocyclic group, A may be substituted by a substituent selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -O C1-C4 haloalkyl, -CN; Y is selected from CH or N; M is a divalent linker with -(L) x - The structure shown, wherein each L is independently selected from the group consisting of -O-, -S-, -NH-, -C(O)-, -CH2-; x is 0, 1, 2, 3 or 4 (when x is 0, the M is a chemical bond); The B ring is a 5-12 member nitrogen-containing heterocyclic group; wherein the B ring can be composed of 0, 1, 2, 3, or 4 R groups. 1b Substituent substitution; R 1a Selected from the following group: H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -OC1-C4 haloalkyl, -CN, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or unsaturated 3-10 membered heterocyclic groups containing 1-3 heteroatoms selected from O, N and S, C6-C 10 Aryl, a 5-10 membered heteroaryl group containing 1-3 heteroatoms selected from O, N, and S; and the R 1a It can be 0, 1, 2, 3 or 4 Rs 2a Substituent substitution; R 1b and R 2a Each is independently selected from the following group: H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterated alkyl, -OC1-C4 alkyl, -O C1-C4 haloalkyl and -CN; The linkers mentioned are selected from the following group (unless otherwise specified, the following linkers can be connected to RETinhibitors and E3 ligase ligand in any order): Wherein, D is independently -CH2-, -CH2CH2-, -C(O)- or a chemical bond; Each E is independently -C(O)-, vinyl, Or chemical bonds; each J is independently -C(O)- or a chemical bond; Each of F, G, L, and M is independently CH, C(OH), C(CF3), CF, or N; Each P bond is independently a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -C(O)NH-, -C(O)CH2- or -C(O)CH2CH2-, -(CH2CH2O). p -、-NHC(O)NH-、-S(O)-、-S(O)2-、Vinyl、 Each Q is independently -C(O)-, -C(O)NH-, vinyl, -NH- or chemical bonds; Each Cy is independently a substituted or unsubstituted benzene ring or a substituted or unsubstituted 5-10 membered heteroaryl ring containing 1-3 members selected from O, N and S; wherein the substitution refers to the H on the group being substituted by one or more groups selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated C1-C4 alkyl; Each n and r is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; Each of m, j, s, and t is independently 0, 1, or 2; p is 1, 2, 3, 4, 5; The E3 ligase ligand is the ligand portion of the E3 ubiquitin ligase; Unless otherwise specified, the terms "heteroaryl" or "heterocyclic" include 1 to 3 heteroatoms selected from the group consisting of N, S, or O; the cycloalkyl or heterocyclic group may be saturated or partially unsaturated, and may be monocyclic, fused, bridged, or spirocyclic structures, but does not include aromatic structures.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, The E3 ligase ligand is selected from the following group: Or the E3 ligase ligand described herein has the structure shown in the following formula: W is selected from the following group: -CH2-, -C(O)-, -SO2-; R1 is independently 0, 1 or 2, and each R1 is independently selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated-C1-C4 alkyl. Z is selected from the following group: -O-, -NH-, Chemical bond, Linker-C(O)NH-, Or a 6-10 spirocyclic group containing 1-2 N atoms.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, The E3 ligase ligand is selected from the following structures: Among them, W is selected from the following groups: -CH2-, -C(O)-; R1 is 0, 1 or 2, and each R1 is independently selected from the following group: D, halogen, C1-C4 alkyl and halo-C1-C4 alkyl; Z is selected from the following group: -O-, -NH-, Chemical bond, Linker-C(O)NH-, 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, In Formula I, ring B is selected from the following group:

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, In Formula I, the RET inhibitors are In another preferred embodiment, the E3 ligase ligand is Among them, W is selected from the following groups: -CH2-, -C(O)-. In another preferred embodiment, R1 is 0 or 1, and each R1 is independently selected from the group consisting of: D, halogens, C1-C4 alkyl groups and halogenated C1-C4 alkyl groups. In another preferred embodiment, Z is Or chemical bonds.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, The Linker mentioned is selected from the following group: In another preferred embodiment, the Linker is a -C6-C12 alkyl-CO-, a -C2-C13 alkyl-(ethyl, vinyl, or ethynyl)-, or a -C2-C6 alkyl-(ethyl, vinyl, or ethynyl)-substituted or unsubstituted phenyl-CO-, The substitution refers to the H on the group being replaced by one or more groups selected from the group consisting of: H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halo-C1-C4 alkyl, deuterated C1-C4 alkyl; In another preferred embodiment, n1 is 6, 7, 8, 9, 10, 11, or 12. In another preferred embodiment, n2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. In another preferred embodiment, n3 is 1, 2, 3, 4, 5, or 6.

7. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof or an optical isomer thereof, characterized in that, The Linker mentioned is selected from the following group:

8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, characterized in that, The compounds are selected from the following group:

9. A pharmaceutical composition comprising one or more compounds as described in any one of claims 1-7, or a pharmaceutically acceptable salt thereof or an optical isomer thereof, and a pharmaceutically acceptable carrier.

10. Use of the compound of any one of claims 1-8, or a pharmaceutically acceptable salt thereof or an optical isomer thereof, or the pharmaceutical composition of claim 9, in the preparation of a medicament for the prevention or treatment of cancer, or diseases related to RET activity or expression levels; preferably, the cancer is selected from the group consisting of: non-small cell lung cancer, medullary thyroid carcinoma, thyroid cancer, ovarian epithelial carcinoma, salivary gland adenocarcinoma, pancreatic ductal carcinoma, breast cancer, prostate cancer, congenital megacolon cancer, neuroblastoma, and irritable bowel syndrome.

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