Macrocyclic compounds with trk, ros1 and alk multitargeted degradation activity and methods of making and medical uses thereof

Abstract

This invention discloses a macrocyclic compound with multi-target degradation activities of TRK, ROS1, and ALK, its preparation method, and its pharmaceutical uses. The general structural formula of the macrocyclic compound with multi-target degradation activities of TRK, ROS1, and ALK is: [Insert structural formula here]. This invention also specifically discloses the preparation method of this macrocyclic compound and its use in the preparation of drugs for treating various cancers or coronavirus infections. The macrocyclic compound prepared by this invention simultaneously possesses multi-target degradation activities of TRK, ROS1, and ALK, and can serve as a multi-target degrader for TRK, ROS1, and ALK proteins, and can be used to treat various cancers or coronavirus infections.

Description

Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to a macrocyclic compound with multi-target degradation activities of TRK, ROS1 and ALK, its preparation method and pharmaceutical applications. Background Technology

[0004] The cumulative genetic and epigenetic modifications of kinases (such as site mutations, deletions, insertions, amplifications, and translocations) lead to abnormal activation of cell signaling pathways, thereby driving cancer cell growth, proliferation, metastasis, and survival (Can. Lett. 2009, 281, 117-127; Nat. Rev. Gen. 2010, 11, 60-74). TRK, ROS1, and ALK fusion genes are closely related to the occurrence and development of various tumors.

[0005] Neurotrophic tyrosine receptor kinase (NTRK) genes encode tropomyosin receptor kinase (TRK), which can be activated through fusion events, thus becoming oncogenes. The NTRK family, including NTRK1, NTRK2, and NTRK3, is a pan-tumor oncogenic driver found in various cancer types. These mutations are relatively rare in common cancers such as breast, colorectal, and lung cancer, but are highly prevalent in some rare cancers, such as secretory-like breast cancer and infantile fibrosarcoma, with an incidence exceeding 90%. These oncogenes promote the synthesis of TRK fusion chimeric proteins, which are aberrantly expressed and persistently activated in a ligand-independent manner (Nat. Rev. Clin. Oncol. 2018, 15, 731-747). The FDA-approved TRK inhibitors larotrectinib, entrectinib, and ripretinib have demonstrated significant treatment response rates in NTRK fusion-positive cancers, independent of histological classification (J. Med. Chem. 2019, 62, 1731−1760; Eur. J. Med. Chem. 2023, 258, 115618; Cancer Discovery 2018, 8, 1227-1236). Despite the significant efficacy of these first-generation drugs, acquired resistance inevitably occurs, ultimately limiting the duration of drug response. The main mechanisms of acquired resistance to TRK inhibitors involve specific mutations in the kinase domains, such as SF mutations, GK mutations, and xDFG motif mutations. Among these, TRKA... G667C (xDFG mutation) and TRKA G595R (SF mutation) is the most common (J. Med. Chem. 2025, 68, 15233-15259).

[0006] ROS1 is a receptor tyrosine kinase encoded by the ROS1 proto-oncogene (N. Engl. J. Med. 2014, 371 1963-1971). Chromosomal rearrangements involving ROS1 and its fusion partner can produce persistently activated ROS1 fusion kinases, which act as oncogenic drivers in human cancers. In advanced non-small cell lung cancer (NSCLC), approximately 2% of patients have ROS1 fusions, with CD74-ROS1 and SLC34A2-ROS1 being the most common rearrangements (JTO Clin. Res. Rep. 2020, 1, 100048). The ROS1 kinase domain shares 70% sequence homology with anaplastic lymphoma kinase, and ROS1 gene rearrangements are generally mutually exclusive with ALK rearrangements. Due to their high homology, NSCLC with ROS1 rearrangements and ALK rearrangements are often treated with the same tyrosine kinase inhibitors. Crizotinib, an ALK / ROS1 / cMET inhibitor, achieved an objective response rate of 72% and a median progression-free survival of 19.2 months in treatment. It was the first tyrosine kinase inhibitor approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of advanced ROS1 fusion-positive lung cancer (Ann. Oncol. 2019, 30 (7), 1121-1126). The second multi-kinase inhibitor, entrectinib, has intracranial activity against ROS1, ALK, and TRK. Based on a meta-analysis of efficacy data from the STARTRK-1, STARTRK-2, and ALKA-372-001 clinical trials, it has been approved for the treatment of metastatic ROS1-positive non-small cell lung cancer (Lancet Oncol. 2020, 21, 261-270). Although ROS1 inhibitors show significant initial efficacy, most patients eventually develop resistance, which has become a major challenge in clinical treatment, ultimately limiting the duration of their efficacy. Targeting mutations of the ROS1 kinase domain have been found in patient samples, the most common being the G2032R mutation (approximately 33%-41%). Other common mutations include D2033N (2.4%-6%), L2026M (1%), L1951R (1%), L2086F (3.6%), and S1986F / Y (2.4%-6%) (N. Engl. J. Med. 2013, 368, 2395−2401; Clin. Cancer Res. 2016, 22, 2351−2358; Clin. Cancer Res. 2021, 27(10), 2899−2909). In more than one-third of acquired resistance cases, some ROS1 mutations lead to resistance to crizotinib and lorlatinib, for which there are currently no effective treatment options.

[0007] Anaplastic lymphoma kinase (ALK) has been a promising therapeutic target in non-small cell lung cancer since its discovery in an anaplastic large cell lymphoma cell line in 1994 (Science 1994, 263, 1281-1284; Mol. Cancer 2018, 17, 52). Significant efforts have been invested in the research and development of small molecule ALK inhibitors (J. Med. Chem. 2013, 56, 5675-5690; J. Med. Chem. 2016, 59, 4948-4964). However, drug resistance issues limit the efficacy of kinase inhibitor-based therapies, particularly the compensatory upregulation of ALK observed when using ALK inhibitor antagonists (Pharmacol. Ther. 2017, 174, 138-144).

[0008] PROTAC, a protein degradation-targeting chimeric technology, can overcome acquired resistance by inducing the degradation of an entire protein through brief, low-dose drug exposure (J. Med. Chem. 2022, 65, 2313-2328; J. Med. Chem. 2025, 68, 17303-17322). PROTAC is a drug development technology that utilizes the ubiquitin-proteasome system to degrade target proteins. PROTAC consists of three parts: an E3 ubiquitin ligase ligand and a target protein ligand. The two active ligands are linked together by a specially designed linker structure, ultimately forming the active form of the triplet PROTAC. In the patient's body, the target protein ligand of the PROTAC binds to the target protein, and the E3 ubiquitin ligase ligand binds to the substrate-binding region of the intracellular E3 ubiquitin ligase. This, through the linker, pulls the target protein closer to the E3 ubiquitin ligase, enabling the UPS system to degrade the target protein, reducing protein levels rather than inhibiting protein function, thus achieving the therapeutic goal. Current research on degradative agents mainly focuses on degrading single proteins such as TRK, ROS1, or ALK, with relatively few reports on multi-target degradative agents. Summary of the Invention

[0010] The purpose of this invention is to provide a macrocyclic compound with multi-target degradation activities of TRK, ROS1 and ALK, its preparation method and pharmaceutical uses, which can be used to treat a variety of cancers.

[0011] In a first aspect, the present invention provides a macrocyclic compound with multi-target degradation activities of TRK, ROS1, and ALK, the general structural formula of which is shown below:

[0012]

[0013] The linker group is independently selected from: oxygen-containing alkyl chains, alkyl chains, etc. , or .

[0014] Furthermore, the macrocyclic compounds possessing multi-target degradation activities of TRK, ROS1, and ALK include the following specific compounds:

[0015] .

[0016] In a second aspect, the present invention provides a method for preparing macrocyclic compounds with multi-target degradation activities of TRK, ROS1, and ALK, the synthetic route of which is as follows:

[0017]

[0018] By replacing different raw materials, compounds with other structures can be synthesized.

[0019] In a third aspect, the present invention provides a multi-target degrader of TRK, ROS1 and ALK, comprising the above-mentioned macrocyclic compounds having multi-target degradation activities of TRK, ROS1 and ALK or pharmaceutically acceptable salts thereof.

[0020] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-described macrocyclic compound having multi-target degradation activities of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0021] Further, the pharmaceutical composition is in a form suitable for administration, including but not limited to oral administration, parenteral administration, topical administration, and rectal administration. In further or other embodiments, the pharmaceutical composition is in the following forms: tablets, capsules, pills, powders, sustained-release formulations, solutions, suspensions, emulsions, ointments, or suppositories; sterile solutions, suspensions, or emulsions for parenteral injection; ointments or creams for topical administration; or suppositories for rectal administration. In further or other embodiments, the pharmaceutical composition is a unit dosage form suitable for a single, precise dose. In further or other embodiments, the amount of the macrocyclic compound is in the range of about 0.001 mg / kg body weight / day to about 1000 mg / kg body weight / day. In further or other embodiments, the range of the amount of the macrocyclic compound is preferably from about 0.001 g / day to about 7 g / day. In further or other embodiments, dose levels below the lower limit of the above range may be sufficient. In further or other embodiments, dose levels above the upper limit of the above range may be required. In further or other embodiments, the macrocyclic compound is administered as a single dose once daily. In further or other embodiments, the macrocyclic compound is administered as multiple doses more than once daily. In further or other embodiments, the pharmaceutical composition further comprises at least one therapeutic agent.

[0022] In a fifth aspect, the present invention provides a medicament for treating and / or preventing coronavirus-related diseases, said medicament comprising an effective amount of the above-described macrocyclic compound having multi-target degradation activity of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition.

[0023] In a sixth aspect, the present invention provides a medicament for treating and / or preventing cancer, said medicament comprising an effective amount of the above-described macrocyclic compound having multi-target degradation activities of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition.

[0024] In a seventh aspect, the present invention provides macrocyclic compounds or pharmaceutically acceptable salts thereof having multi-target degradation activities of TRK, ROS1, and ALK in the preparation of compounds for inhibiting M pro Uses in active pharmaceutical ingredients.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: the macrocyclic compounds prepared by the present invention have multi-target degradation activities of TRK, ROS1 and ALK, and can be used as multi-target degradative agents of TRK, ROS1 and ALK for multi-target degradation of TRK, ROS1 and ALK proteins, and can be used to treat a variety of cancers or coronavirus infections. Detailed Implementation

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent applications, and publications cited in this document are incorporated herein in their entirety. Where multiple definitions exist for terms herein, the definitions in this chapter shall prevail.

[0028] It should be understood that the above overview and the detailed description below are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this application, the singular is used to include the plural unless specifically stated otherwise. It should also be noted that, unless the context clearly specifies otherwise, as used in the specification and claims, the singular forms “a” and “the” or “the” include the plural referent. It should also be noted that, unless specifically stated otherwise, “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms is not limiting. Similarly, the use of the term “including” and other forms is not limiting.

[0029] See the references (including Carey and Sundberg, "Advanced Organic Chemistry 4") TH Definitions of standard chemical terms can be found in *ED. Vols. A (2000) and B (2001), Plenum Press, New York*. Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, HPLC, IR and UV / Vis spectroscopy, and pharmacological methods, are employed. Unless specifically defined, the nomenclature, experimental procedures, and techniques used in relation to the analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry described herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reaction and purification techniques can be performed using the manufacturer's instructions for use of kits, or in a manner known in the art or as described in this invention. The techniques and methods described herein are generally performed according to conventional methods well known in the art, based on the descriptions in the various summary and more specific literatures cited and discussed in this specification.

[0030] Unless otherwise stated, the commonly used chemical terms, such as, but not limited to, “alkyl”, “aryl”, are equivalent to their optionally substituted forms. For example, “alkyl” as used herein includes optionally substituted alkyl groups.

[0031] The term "alkyl" as used alone or in combination herein refers to a monovalent saturated hydrocarbon with optional substituted straight or optional substituted branched chains having 1 to about 18 carbon atoms, or 1 to about 10 carbon atoms, or 1 to about 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl.

[0032] This invention also includes isotope-labeled compounds. Common isotopic atoms include, but are not limited to, those listed below. 2 H, 3 H, 13 C 14 C 17 O、 18 O、 15 N, etc. These atoms are the same as their most abundant atoms in nature but have different mass numbers. The application of isotopic labeling in drug discovery has been reported (Elmore, Charles S., Annual Report of Medicinal Chemistry, 2009, 44, 515-534).

[0033] As used herein, the term “pharmaceutically acceptable” means a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compound described herein and is relatively non-toxic, i.e., that the substance can be administered to an individual without causing adverse biological effects or interacting with any component contained in the composition in a harmful manner.

[0034] As used herein, the term "pharmaceutical composition" refers to a bioactive compound optionally mixed with at least one pharmaceutically acceptable chemical component, including but not limited to carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients.

[0035] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of the free acid and free base of the specified compound and has no adverse effects in a biological or other respect. The compounds described herein may have acidic or basic groups and thus can react with any number of inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts. These salts can be prepared either in situ during the final separation and purification of the compounds of the invention, or by reacting the purified compound, in its free base form, with a suitable organic or inorganic acid alone, and then separating the resulting salt. Examples of pharmaceutically acceptable salts include those prepared by reaction of the compounds described herein with inorganic or organic acids or inorganic or organic bases.

[0036] As used herein, the term "isomer" refers to an isomer that is readily interconvertible with the compounds of the present invention through, for example, hydrogen atom migration or proton migration.

[0037] IC 50 This refers to the concentration at which a specific compound inhibits a particular measured activity by 50%.

[0038] The claims of this invention specifically set forth the novel features of the invention. Exemplary embodiments utilizing the principles of the invention are set forth in the following detailed description of the invention, and the features and advantages of the invention can be better understood by referring to this detailed description.

[0039] This document has shown and described some embodiments of the invention, but these embodiments are provided by way of example only. It should be understood that variations of the embodiments of the invention described herein can also be used to implement the invention. It will be understood by those skilled in the art that many variations, changes, and substitutions may occur without departing from the scope of the invention. It should be understood that the scope of protection of each aspect of the invention is determined by the claims, and the methods and structures within the scope of these claims, as well as their equivalents, are all within the scope of the claims of this invention.

[0040] Example 1:

[0041] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-3-(2-(((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2 ,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)ethoxy)propanamide

[0042]

[0043]

[0044] Preparation of compound 2

[0045]

[0046] Compound 1 (60.00 g, 1.0 eq), (S)-tert-butyl-2-hydroxypropylcarbamate (102.30 g, 1.5 eq), and triphenylphosphine (153.20 g, 1.5 eq) were added to 600 mL of tetrahydrofuran under nitrogen protection and cooled to -10 °C with stirring for 10 min. Diethyl azodicarbonate (108.50 g, 1.6 eq) was added dropwise, and the reaction was stirred at 25 °C for 18 h. 900 mL of petroleum ether was added to the reaction mixture, and the mixture was stirred for 30 min. The mixture was filtered, and the filter cake was purified by column chromatography at a volume ratio of petroleum ether:ethyl acetate of 20:1 to 5:1 to obtain target compound 2 (105.72 g, yield 87.23%).

[0047] Preparation of compound 3

[0048]

[0049] Compound 2 (105.72 g, 1.0 eq), ethyl titanate (154.84 g, 2.0 eq), and tert-butylsulfinamide (61.73 g, 1.5 eq) were added to a reaction flask and stirred at 75 °C for 18 hours. 1 L of water and 530 mL of ethyl acetate were added, and the mixture was stirred and filtered. The solution was washed with ethyl acetate. The filtrate was separated, and the aqueous phase was extracted once with ethyl acetate. The organic phases were combined, dried, and concentrated to dryness. The mixture was purified by column chromatography using a petroleum ether:ethyl acetate mixture to obtain target compound 3 (77.84 g, purity 93.24%, yield 55.30%).

[0050] Preparation of compound 4

[0051]

[0052] Compound 3 (77.84 g, 1.0 eq) was added to 390 mL of tetrahydrofuran and stirred at -10 °C. Sodium borohydride (9.95 g, 1.4 eq) was added in batches, and the mixture was stirred for 2 hours after the addition was complete. 389 mL of water and 389 mL of ethyl acetate were added, and the mixture was stirred and separated. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, dried and concentrated to obtain target compound 4 (61.08 g, yield 78%).

[0053] Preparation of compound 5

[0054]

[0055] Compound 4 (82.22 g, 1.0 eq) was added to 500 mL of tetrahydrofuran, along with elemental iodine (10.02 g, 0.2 eq) and 160 mL of water. The mixture was stirred at 60 °C for 18 hours. After the mixture was concentrated to dryness, 400 mL of ethyl acetate and 400 mL of saturated sodium bicarbonate solution were added. The mixture was stirred and separated. The organic phase was dried and concentrated to dryness to obtain the target compound 5 (49.90 g, yield 81%).

[0056] Preparation of compound 6

[0057]

[0058] Compound 5 (75.50 g, 1.0 eq) was added to 378 mL of N,N-dimethylformamide, followed by 2-amino-3-nitro-6-chloropyridine (41.86 g, 1.0 eq) and triethylamine (73.32 g, 3.0 eq). The mixture was stirred at 110 °C for 3 hours. 378 mL of ethyl acetate and 378 mL of water were added, the mixture was separated, the organic phase was washed once with brine, dried, and concentrated to dryness. 378 mL of methyl tert-butyl ether and 150 mL of water were added, and the mixture was stirred for 18 hours. The mixture was filtered, the filtrate was separated, the organic phase was dried, concentrated to dryness, and purified by column chromatography at a volume ratio of petroleum ether:ethyl acetate = 1:0~20:1. The target compound 6 (76.30 g, yield 70.24%) was obtained.

[0059] Preparation of compound 7

[0060]

[0061] Compound 6 (76.30 g, 1.0 eq) was added to 380 mL of ethanol, followed by wet Raney nickel (7.63 g, 0.1 wt%). The mixture was stirred at 25 °C, and 152 mL of hydrazine hydrate was added dropwise. After the addition was complete, the mixture was stirred at 25 °C for 2 hours. The mixture was filtered, and the filtrate was concentrated to dryness to obtain the target compound 7 (66.23 g, 93% yield).

[0062] Preparation of compound 8

[0063]

[0064] Compound 7 (73.00 g, 1.0 eq) was added to 350 mL of toluene, followed by p-toluenesulfonic acid (6.62 g, 0.2 eq) and triethyl orthoformate (128.00 g, 5.0 eq). The mixture was stirred at 120 °C for 2 hours. 210 mL of sodium bicarbonate solution and 210 mL of ethyl acetate were added, and the mixture was stirred and separated. The aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, dried, and concentrated to dryness. The mixture was purified by column chromatography at a volume ratio of petroleum ether to ethyl acetate of 1:1 to 1:3 to obtain the target compound 8 (26.34 g, yield 35.24%).

[0065] Preparation of compound 9

[0066]

[0067] Compound 8 (26.34 g, 1.0 eq) was added to 263 mL of N,N-dimethylformamide, cooled to -10 °C, and under nitrogen protection, 1 mol / L LiHMDS (64 mL, 1.1 eq) was added dropwise, and the mixture was stirred for 30 minutes. Then, diphenylphosphohydroxylamine (17.05 g, 1.25 eq) was added, and the mixture was stirred and reacted at -10 °C for 1 hour. 263 mL of water and 263 mL of ethyl acetate were added, and the mixture was stirred and separated. The aqueous phase was extracted once with ethyl acetate, and the organic phases were combined, dried, and concentrated to dryness to obtain 30.94 g of crude product. The crude product was purified by column chromatography at a volume ratio of dichloromethane:methanol = 100:1~30:1 to obtain the target compound 9 (14.02 g, yield 51.43%).

[0068] Preparation of compound 10

[0069]

[0070] Compound 9 (14.02 g, 1.0 eq) was added to 28 mL of ethanol, and 70 mL of 4% hydrochloric acid-ethanol solution was added dropwise at a temperature below 30 °C. The mixture was stirred at 25 °C for 1 hour. The mixture was concentrated to dryness, and 28 mL of methanol and 14 mL of 7 mol / L ammonia-methanol solution were added. The mixture was stirred for 30 minutes, filtered, and the filtrate was concentrated to dryness. The filtrate was purified by column chromatography at a volume ratio of dichloromethane to methanol of 50:1 to 10:1 to obtain the target compound 10 (7.56 g, yield 69.60%).

[0071] Preparation of compound 11

[0072]

[0073] Compound 10 (7.56 g, 1.0 eq) was added to 1.13 L of tetrahydrofuran, followed by triethylamine (6.66 g, 3.0 eq). Under nitrogen protection, the mixture was cooled to 0 °C, and a tetrahydrofuran (75 mL) solution of thiocarbonyl diimidazole (4.69 g, 1.2 eq) was slowly added dropwise. After the addition was complete, the mixture was heated to 60 °C and stirred for 20 hours. The mixture was concentrated to dryness, and then 38 mL of ethyl acetate and 38 mL of sodium bicarbonate solution were added. The mixture was stirred and separated. The aqueous phase was extracted once with ethyl acetate, and the organic phases were combined, washed once with brine, dried, and concentrated to dryness to obtain the crude product (8.49 g, purity 57.28%). The crude product was purified by column chromatography at a volume ratio of petroleum ether:ethyl acetate = 30:1 to 1:1 to obtain the target compound 11 (1.89 g, yield 22.28%).

[0074] Preparation of compound 13

[0075]

[0076] Compound 11 (50.0 mg, 1.0 eq) was added to 0.5 mL of N,N-dimethylformamide, followed by compound 12 (77.7 mg, 2 eq) and sodium carbonate (27.4 mg, 2 eq). The mixture was stirred at 40 °C for 18 hours. 0.5 mL of water and 0.5 mL of ethyl acetate were added, and the mixture was separated. The aqueous phase was extracted once with ethyl acetate. The organic phases were combined, washed once with brine, dried, and concentrated to obtain the target compound 13, which was used directly in the next reaction without purification.

[0077] Preparation of compound 14

[0078]

[0079] Compound 13 (132.7 mg, 1.0 eq) was added to 1.5 mL of dichloromethane, followed by 0.8 mL of trifluoroacetic acid. The mixture was stirred at 25 °C for 1 hour. The mixture was then concentrated to dryness and purified by column chromatography at a volume ratio of dichloromethane to methanol of 15:1 to obtain target compound 14 (40.0 mg, yield 33.51%).

[0080] Preparation of compound I

[0081]

[0082] Compound 14 (40.0 mg, 1.0 eq) was added to 5 mL of N,N-dimethylformamide, followed by diisopropylethylamine (41.2 mg, 4 eq) and lenadoamine (compound 15) (41.3 mg, 2 eq). The mixture was stirred at 25 °C for 5 minutes, then HATU (45.4 mg, 1.5 eq) was added, and the reaction was stirred for 1 hour. 5 mL of water and 5 mL of ethyl acetate were added, and the mixture was stirred and separated. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, washed once with brine, dried, concentrated to dryness, and purified by column chromatography at a volume ratio of dichloromethane:methanol = 15:1 to give compound I (10.0 mg, yield 16.89%). The compound was a white solid. LC-MS Calcd for C 36 H 39 FN9O6S + [M+ H] + : m / z 744.3, Found: 744.4.

[0083] Example 2

[0084] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)-3-(2-(2-(((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2,8 ,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)ethoxy)ethoxy)propanamide

[0085]

[0086] Compound J was prepared according to the method for the compound in Example 1. 60%; white solid; LC-MS Calcd for C 38 H 43 FN9O7S + [M + H] + : m / z 788.3, ​​Found: 788.1.

[0087] Example 3

[0088] N- (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-2-(2-(((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2 ,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)ethoxy)acetamide

[0089]

[0090] Compound K was prepared according to the method for the compound in Example 1. 50%; white solid; LC-MS Calcd for C 35 H 37 FN9O6S + [M + H] + : m / z 730.3, Found: 730.1.

[0091] Example 4

[0092] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)-6-(((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-ox a-2,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)hexanamide

[0093]

[0094] Compound L was prepared according to the method for compound in Example 1. 44%; yellow solid; LC-MS Calcd for C 37 H 41 FN9O5S + [M + H] + : m / z 742.3, Found: 742.2.

[0095] Example 5

[0096] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-1-((((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2,8,10-tr iaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)methyl)cyclopropane-1-carboxamide

[0097]

[0098] Compound M was prepared according to the method for the compound in Example 1. 45%; white solid; LC-MS Calcd for C 36 H 37 FN9O5S + [M + H] + : m / z 726.3, Found: 726.6.

[0099] Example 6

[0100] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-2-(2-(2-(((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2, 8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)ethoxy)phenyl)acetamide

[0101]

[0102] Compound N was prepared according to the method for the compound in Example 1. 30%; white solid; LC-MS Calcd for C 41 H 41 FN9O6S + [M + H] + : m / z 806.3, Found: 806.0.

[0103] Example 7

[0104] N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-3-((((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2 ,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodecaphan-9-en-9-yl)thio)methyl)benzamide

[0105]

[0106] Compound O was prepared according to the method for the compound in Example 1. 52%; white solid; LC-MS Calcd for C 39 H 37 FN9O5S + [M + H] + : m / z 762.3, Found: 762.1.

[0107] Example 8

[0108] (2S,4R)-1-((S)-2-(6-((3R,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacycl odecaphan-9-en-9-yl)thio)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0109]

[0110] Compound P was prepared according to the method for the compound in Example 1. 44%; yellow solid; LC-MS Calcd for C 46 H 58 FN 10 O5S2 + [M + H] + : m / z 913.4, Found: 913.5.

[0111] Example 9

[0112] (2S,4R)-1-((S)-2-(2-(2-(2-(((3S,6S,Z)-45-fluoro-3,6-dimethyl-13H-5-oxa-2,8,10-triaza-1(5,3)-imidazo[4,5-b]pyridina-4(1,2)-benzenacyclodec aphan-9-en-9-yl)thio)ethoxy)phenyl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0113]

[0114] Compound Q was prepared according to the method of Compound 1 in Example 1. 36%; white solid; LC-MS Calcd for C 50 H 58 FN 10 O6S2 + [M + H] + : m / z 977.4, Found: 977.1.

[0115] Example 10

[0116] (2S,4R)-1-((2S)-2-(3-(2-((5S,E)-35-fluoro-5-methyl-13H-4-oxa-7,9-diaza-1(5,3)-imidazo[4,5-b]pyridina-3(3,2)-pyridina-2(1,2)-pyrrolidina cyclononaphan-8-en-7-yl)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0117]

[0118] Compound R was prepared according to the method for compounds in Example TM. 45%; white solid; LC-MS Calcd for C 46 H 57 FN 11 O6S + [M + H]+ : m / z 910.4, Found: 910.3.

[0119] Example 11

[0120] (2S,4R)-1-((2S)-2-(2-(2-(2-((5S,E)-35-fluoro-5-methyl-13H-4-oxa-7,9-diaza-1(5,3)-imidazo[4,5-b]pyridina-3(3,2)-pyridina-2(1,2)-pyrrolidinac yclononaphan-8-en-7-yl)ethoxy)phenyl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0121]

[0122] Compound S was prepared according to the method for compound in Example 1. 49%; white solid; LC-MS Calcd for C 51 H 59 FN 11 O6S + [M + H] + : m / z 972.4, Found: 972.3.

[0123] Example 12

[0124] (2S,4R)-1-((2S)-2-(2-(2-((5S,E)-35-fluoro-5-methyl-13H-4-oxa-7,9-diaza-1(5,3)-imidazo[4,5-b]pyridina-3(3,2)-pyridina-2(1,2)-pyrrolidin acyclononaphan-8-en-7-yl)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0125]

[0126] Compound T was prepared according to the method for the compound in Example 1. 47%; white solid; LC-MS Calcd for C 45 H 55 FN 11 O6S + [M + H] + : m / z 896.4, Found: 896.8.

[0127] Example 13

[0128] TRKA kinase activity assay method:

[0129] ELISA method

[0130] The TRKA kinase assay of the compound was evaluated using an enzyme-linked immunosorbent assay (ELISA). PY713 antibody (abcam, Cambridge Science Park, UK) was coated onto 96-well ELISA plates (10 ng / mL). TRKA kinase and the compound were added to wells containing 20 μmol / L substrate (NH2-ETVYSEVRK-biotin) in 1×KR buffer (50 mmol / L HEPES pH 7.4, 20 mmol / L MgCl2, 0.1 mmol / L MnCl2, 1 mmol / L DTT) and incubated at 25°C for 1 hour. Then, ATP (3 μmol / L) was added, and the reaction proceeded for 2 hours. The product was transferred to 96-well ELISA plates and incubated at 25°C for 30 minutes. The plates were then washed with PBST, and 100 μL of horseradish peroxidase (HRP)-conjugated streptavidin was added, followed by incubation at 25°C for 30 minutes. Wash the plate and add TMB (3,3',5,5'-tetramethylbenzidine) for color development. Stop the color development reaction with 2 mol / L H₂SO₄ and read the OD values ​​using a multi-functional microplate reader. 450 (Perkinlemer, USA). IC calculation using GraphPad Prism 6.0 software. 50 The values ​​are used to fit a 4-parameter equation to generate a concentration response curve.

[0131]

[0132] Example 14

[0133] Cell anti-proliferation activity assay:

[0134] Cell antiproliferative activity was assessed using the CellTiter-Glo assay (Promega, USA). The compound solution was diluted 1000-fold with DMSO, and 1 µL of the 1000-fold compound was added to 49 µL of growth medium to prepare a 20-fold compound solution. The cell suspension was diluted to the desired density with growth medium, and 95 µL was seeded into 96-well plates. 5 µL of the 20-fold compound was added to each well according to the plate plot, with a final DMSO concentration of 0.1% per well. The cells were then incubated at 37°C with 5% CO2 for 72 hours. The assay plate was equilibrated to room temperature before measurement. 20 µL of CellTiter-Glo was added to each well. ® The reagents were mixed in a track shaker for 2 minutes to induce cell lysis. Cells were incubated at room temperature for 10 minutes to stabilize the luminescence signal, and the luminescence intensity was recorded using an EnVision multi-label reader (PerkinElmer). Cell viability (CV%) was calculated relative to the solvent (DMSO) treated control wells using the following formula: Cell viability (%) = (RLU) / (CV%) 化合物 - RLU 空白 ) / (RLU 对照 - RLU 空白 The half-maximum inhibitory concentration (WMC) was calculated as 100%. Concentration response curves were generated using GraphPad Prism 6.0 software by fitting a four-parameter equation. All assays were performed with three parallel samples and three replicates.

[0135] Example 15

[0136] ELISA method for detecting TRKA protein degradation rate:

[0137] Ba / F3-LMNA-NTRK1 cells were treated with TRKA-PROTACs at a final concentration of 1 μM for 24 h. The control group was treated with DMSO at a final volume concentration of 0.1%. Cells were collected after centrifugation at 1000 rpm for 5 min. Cells were washed twice with PBS, ensuring that any residual PBS was removed after the second wash. 7 Cell lysis buffer was added at a concentration of [cell / mL], and samples were collected after incubation on ice for 15 min. The samples were centrifuged at 2000×g for 5 min, and the supernatant was transferred to clean test tubes. The concentration of TRKA in each sample was detected using a human TRKA enzyme-linked immunosorbent assay kit (Hangzhou Linke Biotechnology Co., Ltd., China). TRKA protein degradation rate was calculated as follows: TRKA protein degradation rate = (1-C [cell / mL]) / [[cell / mL]] 样品 / C0)*100%, where C 样品 C0 represents the concentration of TRKA protein in the sample, and C0 represents the concentration of TRKA protein in the control group.

[0138]

[0139] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A macrocyclic compound with multi-target degradation activity against TRK, ROS1, and ALK, characterized in that... The general structural formula of the macrocyclic compound is shown below: The linker group is independently selected from: oxygen-containing alkyl chains, alkyl chains, etc. , or .

2. The macrocyclic compound with multi-target degradation activity of TRK, ROS1, and ALK according to claim 1, characterized in that... The macrocyclic compounds include the following specific compounds: 。 3. A method for preparing a macrocyclic compound with multi-target degradation activities of TRK, ROS1, and ALK, characterized in that... The synthetic route is as follows: By replacing different raw materials, compounds with other structures can be synthesized.

4. A multi-target degrader of TRK, ROS1, and ALK, characterized in that... Includes macrocyclic compounds with multi-target degradation activities of TRK, ROS1 and ALK as described in claim 1 or 2, or pharmaceutically acceptable salts thereof.

5. A pharmaceutical composition, characterized in that... This includes macrocyclic compounds with multi-target degradation activities of TRK, ROS1 and ALK as described in claim 1 or 2, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to claim 5, characterized in that: The drug composition is administered orally, parenterally, topically, or rectally; the dosage form of the drug composition is tablets, capsules, pills, powders, sustained-release preparations, solutions, suspensions, emulsions, ointments, or suppositories; and the dosage of the drug compound is such that the amount of the macrocyclic compound is between 0.001 mg / kg body weight / day and 1000 mg / kg body weight / day.

7. A drug for treating and / or preventing coronavirus-related diseases, characterized in that: The drug comprises an effective amount of the macrocyclic compound of claim 1 or 2 having multi-target degradation activity of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 5.

8. A drug for treating and / or preventing cancer, characterized in that: The drug comprises an effective amount of the macrocyclic compound of claim 1 or 2 having multi-target degradation activity of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 5.

9. The macrocyclic compound of claim 1 or 2 having multi-target degradation activity of TRK, ROS1 and ALK, or a pharmaceutically acceptable salt thereof, in the preparation of a compound for inhibiting M pro Uses in active pharmaceutical ingredients.

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